Protozoa

Chapter 9 

Protozoa

General information on parasites

• A parasite (from the Greek word parasite's) is defined as an organism that lives in a more or less close association with another organism of a different species (the host), derives sustenance from it and is pathogenic to the host, although this potential is not always expressed. In the wider sense, the term parasite refers to all organisms with such characteristics. In medicine the term is used in a narrower sense and designates eukaryotic pathogens, which belong to the protozoa (unicellular organisms Chapter 9) and metazoan, including helminths (parasitic “worms,” Chapter 10), arthropods, and some other groups of lower medical significance (Annelida, Pentatomidae, not covered in this book). Parasites cause numerous diseases (parasitosis) in humans, some being of extraordinary significance (e.g., malaria). Of practical concern in central Europe are both autochthonous and imported (tropical and travelers’) parasitic infections. A uniform disease nomenclature has been adopted in this book with the sole use of the suffix –Osis (plural –Oses)—for example trypanosomiasis and not trypanosomiasis. This system, based on the Standardized Nomenclature of Parasitic Diseases (SNOPAD) (originally published in 1988 and recommended by the International Society of Parasitologists) avoids the inconsistent usage of disease names, such as leishmaniasis on the one hand and toxoplasmosis on the other. A selection of the most important parasitosis is presented in the following chapters. In Table 1.10 (p. 28) zoonoses caused by parasites are listed.

Parasitic protozoa

• are eukaryotic, single-celled microorganisms about 1–150 lm in size and enclosed by a trilaminate cell membrane. They possess one, rarely two nuclei (and multinuclear reproductive forms). Reproduction is asexual by binary or multiple fission of the cell, or sexual. The cellular construction of the protozoa is generally the same as in other eukaryotes, but they also exhibit some special features. For example, during the course of evolution some protozoa (Giardia, Entamoeba) have lost the mitochondria secondarily, except several genomic traits that were laterally transferred to the nuclei. The apoplast present in some species of Apicomplexa (see Toxoplasma) is a residual of a former plastid typical for their ancestors. Some protozoa contain specialized organelles, such as glycosides (exclusively in trypanosomiasis), hydrogenases (trichomonads and protozoa of other groups), and metasomas (Entamoeba) (see under specific protozoan groups). Motile stages of the parasitic protozoa mostly move by means of flagella, cilia, or pseudopodia. Some species produce resistant stages (cysts, oocysts) in which the parasites can survive outside of their hosts for longer periods. According to current theories, the protozoa are a heterogeneous group consisting of different phyla within the regnum of Eukaryotes. The term protozoa has no phylogenetic significance but is still used as a collective name for the various eukaryotic unicellular organisms. The classification of the protozoa is highly controversial. Therefore, all classification systems have to be regarded as provisional (Table 9.1).

Giardia intestinalis

• Giardia intestinalis (syn. Giardia lamblia, G. duodenales), a parasite of worldwide distribution, occurs also in Europe with relatively high frequency. It is a parasite of the small intestine of humans that can cause enteritis. Infection occurs by peroral ingestion of Giardia cysts. Various species of mammalian animals are reservoir hosts.

• Occurrence. G. intestinalis has a worldwide distribution with prevalence rates of 2–5% in industrialized countries and very high rates, up to 50%, in developing countries. Children up to the age of five are frequently infected. Parasite and life cycle. Giardia exists in two morphological forms: a motile vegetative stage, the trophozoite, and a cyst stage. The trophozoites live on the small intestine mucosa (less frequently on the gallbladder mucosa as well). They resemble a pear split lengthwise, are 9–21 lm long and 5–12 lm wide and possess eight flagella, two nuclei—one on each side of the longitudinal axis—and two claw-shaped median bodies (Figs. 9.1 and 9.11a). Their dorsal side is convex, the anterior part of the ventral side forms a concave adhesive disk. Reproduction is by means of longitudinal binary fission of the trophozoites, which are able to produce variant specific surface proteins. G. intestinalis produces oval cysts (8–18 ! 7–10 lm) with four nuclei, flagella, and claw-shaped median bodies. The cysts (and, less frequently, trophozoites) are excreted in stool. Fig. 9.1 illustrates the life cycle of G. intestinalis. Epidemiology. The genus Giardia includes several species (G. intestinalis, G. murus, G. Agilis, etc.) that show morphological, biological, and genetic differences. Giardia isolates obtained from humans and various species of.

• were found in both humans and domestic animals (e.g., cattle, sheep, dogs). These and other facts support the conclusion that some strains of G. duodenales can be transmitted from vertebrate animals to humans and that giardiasis is a zoonosis. Humans are apparently the most important reservoir hosts and certain mammalian animal species are considered additional sources of infection. The cysts excreted in stool are responsible for spreading the infection. They remain viable for up to three weeks in moist surroundings at 21 8C and up to about three months in cool water. The trophozoites, by contrast, die off soon outside the host. Infection is per os, whereby cysts are transmitted by the fecal-oral route from person to person (within families, kindergartens, between homosexuals, etc.) or in food and drinking water. Numerous epidemic outbreaks of giardiasis' due to contaminated drinking water have been described in the US and other countries with up to 7000 persons locally involved.

Pathogenesis and clinical manifestations. 

• In the small intestine, G. intestinalis can cause inflammation as well as other morphological changes and malabsorption. Gallbladder infections have also been described. The pathogenesis is unclear; new data provide evidence that Giardia produce toxinlike proteins. The course of infection is frequently asymptomatic. The parasite can be eliminated spontaneously within a few weeks; on the other hand, it may persist for years. The ability to produce variable surface proteins may influence elimination and persistence. Patients with symptomatic infections experience chronic and recurrent diarrhea, steatorrhea, and signs of malabsorption as well as upper abdominal pains, vomiting, occasionally fever, and weight loss.

Diagnosis, therapy, and prevention

• The standard diagnostic method is stool examination using the SAFC technique to detect cysts and (more rarely) trophozoites. Trophozoites can also be found in duodenal aspirate. IFAT and ELISA kits are now also available to detect Giardia-specific structural and soluble antigens in stool samples. Nitroimidazole compounds are used for chemotherapy of infections, for instance metronidazole, ornidazole, and tinidazol, as well as the benzimidazole compound albendazole and the recently introduced nitazoxanide (nitro thiazole compound). Prophylactic measures are the same as for amitosis (p. 499). A vaccine induces a reliably protective effect in dogs and cats.

Trichomonas vaginalis

• Trichomonas vaginalis is a frequent flagellate species that occurs worldwide and is transmitted mainly by sexual intercourse. It causes vaginitis in women and urethritis in men.

• Occurrence. The number of new cases is estimated at 170 million annually (WHO, 1998). In average populations of developed countries, infection rates are about 5–20% in women and usually below 5% in men. Parasite, life cycle, and epidemiology. Trichomonas vaginalis is a pear-shaped protozoon about 10–20 lm long and 2–14 lm wide. Five flagella emerge from a basal body at the anterior pole, four freely extend forwards and one extends backwards, forming the outer edge of the undulating membrane, which reaches back only just beyond the middle of the cell. An axial rod made up of microtubules (the ax style) protrudes with its free tip from the posterior end of the cell. The oval cell nucleus lies near the upper pole of the protozoon. Trichomonads are anaerobic protozoa that possess hydrogenases, which are specialized organelles producing H2 as a metabolite. T. vaginalis colonizes the mucosa of the urogenital tract and reproduces by longitudinal binary fission. Trichomonads do not encyst, although rounded, nonmotile forms are observed which are degenerated stages without epidemiological significance.

• Clinical manifestations. In women, T. vaginalis primarily colonizes the vaginal mucosa, more rarely that of the cervix. In about 20–50% of cases the infection is asymptomatic, but vaginitis can develop after an incubation period of two to 24 days. The infection results in production of a purulent, thin, yellowish discharge in which trichomonads, pus cells, and bacteria are found. The parasites also enter the urethra in about 75–90% of cases, where they can also cause an inflammation, but only rarely infect the urinary bladder or uterus. Infections in men are for the most part asymptomatic (50–90%), but they may also cause a symptomatic urethritis, more rarely involving the prostate gland and seminal vesicles as well. Infection does not confer effective immunity.

• Diagnosis. A fresh specimen of vaginal or urethral secretion is mixed with physiological saline solution and examined under a microscope for trichomonads. The trichomonads are readily recognized by their typical tumbling movements. The round trichomonad forms, by contrast, are hardly distinguishable from leukocytes. Trichomonads can also be identified in smear preparations following Giemsa staining or in an immunofluorescence test with monoclonal antibodies. The most reliable diagnostic results are obtained by culturing specimens in special liquid media. The “In-Pouch Test System” (BioMed Diagnostics) has proved useful: two flexible plastic chambers containing culture medium for combined microscopic and cultural analysis. Other special methods are based on detection of antigen (ELISA) or DNA (PCR).

• Therapy and prevention. It is always necessary for both sexual partners to receive treatment. Effective nitroimidazoles preparations for oral application— in women vaginal application—include metronidazole, tinidazole and ornidazole. These substances are contraindicated in early pregnancy. Preventive measures are the same as for other venereal diseases.

Trypanosoma

• Trypanosoma brucei ambiens and Trypanosoma brucei rhodesiense cause African trypanosomiasis (sleeping sickness) in humans, which presents inter alia as fever and meningoencephalitis. In a chronic form (T. Gambians) the disease occurs mainly in western and central Africa, whereas the acute form (T. rhodesiense) is predominately distributed in eastern and southeastern Africa. The trypanosomes are transmitted by the bites of tsetse flies (Glossina). Antelopes and other wild or domestic animals serve as reservoir hosts of varying significance. Trypanosoma cruse, the causative agent of American trypanosomiasis (Chagas disease) occurs in humans and many vertebrate animals in Central and South America. It is transmitted in the feces of bloodsucking reduviid bugs. In recent years, considerable progress has been made in the control of Chagas disease.

• General. The genus Trypanosoma (from trypanin: borer and soma: body) belongs to the family Trypanosomiases (subphylum Kinetoplast). One feature of this family is that various forms develop during the life cycle in vertebrates and vectors (insect) involved. The morphologically differentiated forms include spindly, uniflagellate stages (trypomastigote, amastigote, promastigote) and a rounded, amastigote form (Fig. 9.3b). The trypomastigote form of the genus Trypanosoma has the following characteristic features: a central nucleus, an elongated mitochondrion containing the kinetoplast in its posterior section, an area free of cristae with especially densely packed DNA (Fig. 9.3a). Close to, but outside of the mitochondrion is the base of the flagellum, which originates in the plasmatic basal body. The flagellum is at first enclosed by the flagellar pocket, and then emerges onto the surface of the organism and runs to the anterior end of the organism as a pulling flagellum. The flagellar adheres locally to the cell surface so that an “undulating membrane” is folded out during movement—visible under a light microscope. Special organelles of the kinetoplast ds are the membrane-enclosed glycosomal, which contain glycolytic enzymes. The cell is enclosed by an elementary membrane, which in the bloodstream stages is covered by a surface coat or glycocalyx (see below). Spiral microtubules forming a cytoskeleton are arranged along the inner cell membrane a for more cell organelles). In the amastigote and promastigote forms, the kinetoplast and base of the flagellum are near the nucleus or more toward the anterior end. In the amastigote form, a reduced flagellum is visible by electron microscopy, but it does not emerge onto the cell surface (Fig. 9.3b).

Parasite species and occurrence

• The causative agents of sleeping sickness are considered to be a subspecies of Trypanosoma brucei and therefore their taxonomically correct designations are Trypanosoma brucei ambience and Trypanosoma brucei rhodesiense. In the following text they will be designated as T. ambience and T. rhodesiense. Morphologically, these two subspecies differ neither from one another nor from Trypanosoma brucei brucei, one of the causative agents of the nagana in domestic animals that does not infect humans. These subspecies can be differentiated by means of biological criteria (e.g., host specificity, sensitivity to human serum) as well as Iso enzymatic and DNA analysis. Sleeping sickness occurs only in sub-Saharan Africa in regions between 14! north and 20! south latitude where the vectors (tsetse flies) are endemic. Currently between 300 000 and 500 000 persons are infected in the heterogeneously distributed endemic areas in 36 African countries. The official numbers of new cases diagnosed annually (1999: 45 000) are unrealistically low because of the large numbers of unregistered cases (WHO, 2000). In some areas, sleeping sickness has occurred in increased.

• Life cycle. T. gambiense and T. rhodesiense parasitize extracellular in the blood plasma or in other body fluids of vertebrates. The trypomastigote forms are pleomorphic in human blood: with increasing parasitemia they transform to slender, 25–40 lm-long forms with the flagellar tip extending beyond the anterior end which reproduce by longitudinal binary fission. With decreasing parasitemia, they appear as short, “stumpy” approximately 12–25 lm long forms without a free flagellar end. These forms do not divide in blood but are infective for Glossina (tsetse flies). Under a light microscope in a Giemsa-stained blood smear the trypanosomes present as spindly organisms with a central nucleus, a kinetoplast at the posterior end (both stained violet) and an undulating membrane. The cell surface of the bloodstream forms is covered with a uniform layer (about 10–15 nm thick) of a specific glycoprotein, which can be replaced by another glycoprotein. These glycoproteins are denominated as variant specific surface antigens (VSSA), the expression of which is coded by about 1000 genes; they form the basis of the organisms’ antigen variation (see below).

• Epidemiology. There are epidemiological differences between T. Gambians and T. rhodesiense, the main one being that T. rhodesiense persists in a latent enzootic cycle in wild and domestic animals and is normally transmitted by Glossina from animal to animal, more rarely to humans. T. Gambians, on the other hand, is transmitted mainly from human to human by the tsetse flies, although various animal species have also been identified as reservoir hosts for T. ambience strains.

• Clinical manifestations. Sleeping sickness is, in the initial phase, a febrile, generalized disease with lymphadenopathy and is later characterized by meningoencephalitis symptoms. The infection runs a two-stage course: the febrile-glandular or hemolymph tic stage 1 and the meningoencephalitis stage 2. The difference is therapeutically significant. In stage 1, the trypanosomes multiply in the tissue fluid at the inoculation site. Within 2–4 days an inflammatory, edematous swelling can develop—the primary lesion or “trypanosome chancre,” which then disappears within about three weeks. Within a period of approximately two weeks the trypanosomes enter the bloodstream and lymphatic system. Later, in the second stage, they also invade the central nervous system. Table 9.3 summarizes further details of the disease.

• Pathogenesis and immunology. The course of the infection is characterized by successive waves of parasitemia caused by antigenic variation in successive trypanosome populations (see above). Parallel to an increasing parasitemia, IgM antibodies are produced that are directed against a certain variant specific surface antigen (VSSA) of the trypanosomes, whereupon they eliminate the segment of the parasite population bearing this VSSA. The parasitemia then declines, but the trypanosomes with a different VSSA multiply, whereupon specific antibodies are once again produced. Antigen variation is one of a number of strategies to circumvent host defenses (immunoreactions). About the time when one VSSA variant of trypanosomes is being eliminated from the body, the concentrations of IgG antibodies rise and immune complexes form.

• Diagnosis. Important diagnostic tools include direct detection of the trypanosomes in the blood, lymph node aspirate and, in cerebral forms, in the cerebrospinal fluid (Fig. 9.5). Trypanosomes can be detected in native blood preparations, in Giemsa-stained thin smears or in thick blood films (p. 622). Since low-level parasitemia's are often present, concentration methods may be required, e.g., microhematocrit centrifugation, anion exchange chromatography, or the QBC technique (p. 531). Other methods are cultivation and mouse inoculation tests (suitable for T. rhodesiense). Analysis of lymph node aspirate has a high diagnostic value in infections with T. Gambians. To confirm or exclude CNS infections obtain a cerebrospinal fluid sample, centrifuge it, and examine the sediment for trypanosomes. Antibodies in the bloodstream can be detected using various techniques. The card agglutination trypanosomiases test (CATT) has proved valuable in epidemiological surveys. Indicators of a stage 2 infection include presence of trypanosomes and/or raised leukocyte numbers and elevated concentrations of protein and IgM in cerebrospinal fluid.

• Therapy. Medical treatment of sleeping sickness is highly problematical, since only a small number of effective drugs are available, serious side effects are fairly frequent and drug-resistant trypanosomes are to be expected. In stage 1, T. Gambians infections are mainly treated with pentamidine, whereas T. rhodesiense infections are treated with suramin. These drugs are not effective in the second stage (cerebrospinal fluid-positive cases), so that the arsenic compound megaspore, a relatively toxic substance, must be used in these cases. The worst side effect of this substance is a potentially lethal encephalopathy observed in 1–10% of patients treated with megaspore. Eflornithine is used for treating the late stage of the T. gamifies infection. Treatment of sleeping sickness victims should be entrusted to specialists if possible.

• Prevention and control. Use individual prophylactic measures to protect against the diurnally active (!) Glossina flies. It is very important that tourists wear clothing that covers the skin as much as possible and treat uncovered skin with repellents (see Malaria, p. 535). They should also inspect the into rigor of cars for tsetse flies and spray with insecticides. Glossina flies are targeted by insecticide sprayings in preventive programs. More recently, the flies are also being caught in insecticide-charged traps using attractant colors and odors.

• Occurrence. Human Chagas disease is endemic in Central and South America and is caused by Trypanosoma cursi (discovered in 1908 by Chagas). This parasite circulates in endemic sylvatic foci between vertebrates and insects (reduviid bugs), the latter transmitting it to humans. Until a few years ago, the endemic area of Chagas disease extended from Mexico to southern Argentina. In recent years parasite transmission to humans has been reduced or prevented in some countries (Argentina, Brazil, Chile, Paraguay, Uruguay) by control measures. The number of infected persons is currently estimated to be 16–18 million (WHO, 2000).

• Causative agent and life cycle. In the natural cycle, the reduviid bugs ingest trypomastigote forms of T. cruzi in bloodmeals from infected hosts (vertebrate animals, humans). In the intestine of the vector, the parasites convert into intensively multiplying amastigotes stages, and later into trypomastigote forms that are excreted in feces after six to seven days. At subsequent bloodmeals, infected reduviids excrete droppings from which the trypomastigotes infect the host through skin lesions (e.g., lesions of bug bites) or the mucosa (e.g., conjunctiva). Once in the human body, the parasites are phagocytosed by macrophages or invade other cells, mainly muscle cells (heart, skeletal, or smooth musculature) as well as neuroglial cells. Within the cells, they transform into amastigote forms (1.5–4.0 lm) and multiply by binary fission. Cells filled with up to 500 parasites are called “pseudocysts." After about five days the parasites develop into to the amastigote form and then the trypomastigote form and return to the bloodstream, whereupon the cell infection cycle is repeated. The T. cursi organism in the blood of infected hosts (vertebrate animals, humans) is 16–22 lm long. It has a pointed posterior end.

• Epidemiology. The bloodsucking bugs of the family Reduviid Ae find a hiding place for the day and quest for food at night. The natural habitats of these insects are nests, animal dens, and other places frequented by vertebrate animals whose blood provides their sustenance. Some species of reduviids (e.g., Triatoma infesting, Rhodium prolix us, Petrongolo's meniscus) have invaded domestic habitats (also in urban areas!) and are typically found in simple human domiciles. Potential carriers of T. cursi include over 150 species of and domestic mammals. The most important in epidemiological terms are dogs, cats, rodents, chickens, opossums, and armadillos. Aside from the reduviid vector, T. cursi can be transmitted between humans by blood transfusions, implacental infection, or organ transplants.

• Clinical manifestations and pathogenesis. Some infected persons react to entry of the parasite into the skin or conjunctiva with a local, inflammatory dermal reaction (caeoma) or conjunctivitis with eyelid edema (Romanda sign). The following symptoms are observed in the acute phase, which follows an incubation period of seven to 30 days: fever, edema, lymph node swelling, hepatomegaly, splenomegaly, myocarditis, and, less frequently, meningoencephalitis. Beginning about eight to 10 weeks after the acute phase the infection turns to an inapparent phase: serum antibodies are detectable, as are parasites in 20–60% of cases (by means of xenodiagnoses). Clinical manifestations of the chronic phase, often starting 10–20 years after the acute phase, are cardiopathy (cardiomegaly, 30% of cases), digestive tract damage (megaesophagus, megacolon, etc., 6%), and neuropathies (3%). The important pathogenic processes include immunologically induced destruction of ganglia cells in the autonomic nervous system that have adsorbed T. cruzi antigen (resulting in dysfunction and organomegaly in various organs) and inflammatory processes, especially in the myocardial tissues, probably the result of autoimmune reactions. Inapparent T. cursi infections can be reactivated by AIDS.

• Diagnosis. In the acute phase, trypanosomes are detectable in peripheral blood at the earliest one to two weeks after infection (thick blood films, centrifugation in hematocrit tubes, blood cultures; sensitivity 60–100%). In the chronic phase, detection of the parasites by conventional means is no longer reliable (sensitivity <10%). Tools for detection of low-level parasitemia include xenodiagnoses (from xenon, foreign: reduviids free of trypanosomes are allowed to suck the blood of persons in whom an infection is suspected or suck patient blood through a membrane; after a few weeks, the reduviids are examined for trypanosomes) or specific DNA detection by PCR. The apathogenic species Trypanosoma rangoli must be taken into consideration in differential diagnosis. Serological methods are also available that can be diagnostically useful in the chronic phase in particular (Table 11.5, p. 625).

• Therapy and prevention. In the early phase of an infection, cure rates of 80% have been achieved with nifurtimox and benznidazole. Both of these preparations frequently cause side effects. Preventive measures concentrate mainly on vector eradication with insecticides, improvement of living conditions, individual protection from reduviid bites with mosquito nets (see Malaria, p. 535), and measures to prevent transfusion and transplantation infections.

Leishmania

• Leishmanial are transmitted by sandflies (Phlebotomid Al) and cause the following main forms of leishmaniases in warm regions: visceral leishmaniosis (VL), cutaneous leishmaniosis (oriental sore) (CL), and mucocutaneous leishmaniases (MCL). In Central Europe, leishmaniosis is of significance as an imported disease and as an HIV-associated infection.`

• Occurrence. Various forms of leishmaniosis occur in the warmer regions of 88 countries in Asia, Africa, Europe (Mediterranean countries!), and Latin America (Fig. 9.7). The annual number of new cases is estimated at 1.5–2 million (0.5 million VL, 1–1.5 million CL and MCL). Both geographic distribution and case numbers are reported to be on the increase (WHO, 2000)

• Parasites and life cycle. The many (about 15) species of the genus Leishmania pathogenic to humans do not show morphological differences. They can be differentiated on the basis of biological criteria, laboratory analyzes (mainly isoenzyme patterns and DNA analysis),the different clinical pictures, and epidemiological facts. In humans and other vertebrates, leishmania's parasitize in mononuclear phagocytic cells (macrophages, monocytes, Langerhans cells) in the amastigote form. The Giemsa-stained organisms are recognizable under a light microscope as round-to-oval cells 2–5 lm in diameter with a nucleus and a small, rod-shaped kinetoplast. A rudimentary flagellum, a single mitochondrion and other cell organelles are also rendered visible on the electron microscopic level (see also Trypanosoma). The leishmania species are transmitted by female mosquitoes of the genera Phlebotomus (Old World) and Lutzomyia (New World) known as “sandflies”. The amastigote stages of the parasite ingested by the insect with a blood meal are transformed in its intestine into slender, flagellate promastigote forms 10–15 lm long, which multiply and migrate back into the proboscis. At tropical temperatures this process takes five to eight days. When infected sandflies take another bloodmeal the promastigote forms are inoculated into a new host (humans or other vertebrates)

• Clinical manifestations and immunology. The most important human forms of leishmaniosis are summarized in. It is important to note that in CL and MCL the parasites generally remain restricted to the skin or skin and mucosa. CL lesions may persist for long periods, but tend to heal spontaneously, whereas a greater tendency to destructive changes is seen in MCL infections. By contrast, in VL the leishmania organisms can invade the entire.