Chapter outline

1. Chapter outline A-Ameabiasis • Entamoebahistolytica • Other intestinal Amoebae B-Flagellates • Intestinal flagellates Giardialamblia and Trichomonasvaginalis • HemoflagellatesTrypanosomaand Leishmania C-Sporozoa • Toxoplasmagondii • Cryptosporidium parvum • Plasmodium species D- Ciliates • Balantidium coli

2. Sporozoa These protozoa have no locomotory extensions of the body and all species are parasitic. Their motile stages move by bending, creeping and gliding and the Apicomplexa have an APICAL COMPLEX formed by micronemes, rhoptries, dense granules and microtubules at their anterior end playing a role in the invasion Coccidia constitute a very large group called Apicomplexa, some members of which are intestinal parasites and others are blood and tissue parasites. All coccidia demonstrate typical characteristics, especially the existence of asexual (schizogony) and sexual (gametogony) reproduction. Most members of the group also share alternative hosts; for example, in malaria, mosquitoes harbor the sexual cycle and humans the asexual cycle.

3. Micronemes Rhoptries Dense Granules Cliché JF Dubremetz

4. Micronemes: adhesion and motility Rhoptry neck proteins RONs+ MICs: Moving junction formation Rhoptry Bulb Content: Parasitophorous vacuole formation Dense Granules: Parasitophorous vacuole remodeling

6. Most sporozoans are intracellular parasites or at least part of their life-cycle takes place inside a host cell. • The Apicomplexa are a large group of protists, characterized by the presence of a unique organelle called an apical complex. Some diseases caused by apicomplexan organisms include: • Malaria (Plasmodium) • Coccidian diseases including: • Cryptosporidiosis (Cryptosporidium parvum) • Toxoplasmosis (Toxoplasma gondii)

8. Sporozoa 1- TOXOPLASMA GONDII T. gondii is a typical coccidian parasite related to Plasmodium, Isospora, and other members of the phylum Apicomplexa. T. gondii is an intracellular parasite, and it is found in a wide variety of animals, including birds and humans. The essential reservoir host of T. gondii is the common house cat and other felines.

9. Sporozoa 1- TOXOPLASMA GONDII Physiology and Structure Organisms develop in the intestinal cells of the cat, as well as during an extraintestinal cycle with passage to the tissues via the bloodstream. The organisms from the intestinal cycle are passed in cat feces and mature into infective cysts within 3 to 4 days in the external environment.

10. Infection in cats is established when the tissues of infected rodents are eaten. Some infective forms, or trophozoites, from the oocyst develop as slender, crescentic types, called tachyzoites. These rapidly multiplying forms are responsible for the initial infection and the tissue damage. Slow-growing, shorter forms called bradyzoites also develop and form cysts in chronic infections.

11. Life cycle of Toxoplasma gondii.

12. a b 10 µm OOCYSTES a- non sporulated b- sporulated 20 µm BRADYZOITES Slow multiplication Cyst formation 20 µm TACHYZOITES Fast multiplication bradyzoite Reactivation tachyzoite carnivorism Intermediate host sporozoite sporogony schizogony merozoite zygote Final host gamogony

13. Toxoplasma gondii • Obligate Intracellular Parasite  Responsible of toxoplasmosis Chorioretinitis Neurotoxoplasmosis Congenital Toxoplasmosis Immuno-compromised individuals

15. Cliché JF Dubremetz

16. Sporozoa 1- TOXOPLASMA GONDII Epidemiology Human infection with T. gondii is ubiquitous; however, it is increasingly apparent that certain immunocompromised individuals (patients with acquired immune deficiency syndrome [AIDS]) are more likely to have severe manifestations. The wide variety of animals that harbor the organism-carnivores and herbivores, as well as birds-accounts for the widespread transmission.

17. Humans become infected from two sources: (1) ingestion of improperly cooked meat from animals that serve as intermediate hosts and (2) the ingestion of infective oocysts from contaminated cat feces. Serologic studies show an increased prevalence in human populations where the consumption of uncooked meat or meat juices is popular. It is noteworthy that serologic tests of human and rodent populations are negative in the few geographic areas where cats have not existed. Outbreaks of toxoplasmosis in the United States are usually traced to poorly cooked meat (e.g., hamburgers) and contact with cat feces.

18. Transplacental infection can occur in pregnancy, either from infection acquired from meat and meat juices or from contact with cat feces. Transfusion infection via contaminated blood can occur but is not common. Transplacental infection from an infected mother has a devastating effect on the fetus. Although the rate of seroconversion is similar for individuals within a geographic location, the rate of severe infection is dramatically affected by the immune status of the individual.

19. Patients with defects in cell-mediated immunity, especially those who are infected with the human immunodeficiency virus (HIV) or who have had an organ transplant or immunosuppressive therapy, are most likely to have disseminated or central nervous system (CNS) disease. It is believed that illness in this setting is caused by reactivation of previously latent infection rather than new exposure to the organism.

20. Sporozoa 1- TOXOPLASMA GONDII Clinical syndromes Most T. gondii infections are benign and asymptomatic, with symptoms occurring as the parasite moves from the blood to tissues, where it becomes an intracellular parasite. When symptomatic disease occurs, the infection is characterized by cell destruction, reproduction of more organisms, and eventual cyst formation. Many tissues may be affected; however, the organism has a particular predilection for cells of the lung, heart, lymphoid organs, and CNS, including the eye.

21. Symptoms of acute disease include chills, fever, headaches, myalgia, lymphadenitis, and fatigue; the symptoms occasionally resemble those of infectious mononucleosis. In chronic disease the signs and symptoms include lymphadenitis, occasionally a rash, evidence of hepatitis, encephalomyelitis, and myocarditis. In some of the cases, chorioretinitis appears and may lead to blindness. Congenital infection with T. gondii also occurs in infants born to mothers infected during pregnancy. If infection occurs in the first trimester, the result is spontaneous abortion, stillbirth, or severe disease.

22. Manifestations in the infant infected after the first trimester include epilepsy, encephalitis, microcephaly (abnormal smallness of the head), intracranial calcifications, hydrocephalus (Hydrocephalus is an abnormal expansion of cavities (ventricles) within the brain that is caused by the accumulation of cerebrospinal fluid), psychomotor or mental retardation, chorioretinitis, blindness, anemia, jaundice, rash, pneumonia, diarrhea, and hypothermia (Hypothermia, a potentially fatal condition, occurs when body temperature falls below 35°C). Infants may be asymptomatic at birth, only to develop disease months to years later. Most often these children develop chorioretinitis with or without blindness or other neurologic problems, including retardation, seizures, microcephaly, and hearing loss.

23. A different spectrum of disease is seen in immunocompromised older patients. Reactivation of latent toxoplasmosis is a special problem for these people. The presenting symptoms of Toxoplasma infection in immunocompromised patients are usually neurologic, most frequently consistent with diffuse encephalopathy, meningoencephalitis, or cerebral mass lesions. Reactivation of cerebral toxoplasmosis has emerged as a major cause of encephalitis in patients with AIDS.

24. Sporozoa 1- TOXOPLASMA GONDII Laboratory Diagnosis Serologic testing is required for the diagnosis of acute active infection; the diagnosis is established by the finding of increasing antibody titers documented in serially collected blood specimens. Because contact with the organism is common, attention to increasing titers is essential to differentiate acute, active infection from previous asymptomatic or chronic infection. Currently the enzyme-linked immunosorbent assay (ELISA) for detecting immunoglobulin (Ig) M antibodies appears to be the most reliable procedure because of its simplicity and rapidity in documenting acute infections.

25. The test is not generally satisfactory in AIDS patients with latent or reactivated infections, because they fail to produce an IgM response or increasing IgG titer. Demonstration of these organisms as trophozoites and cysts in tissue and body fluids is the definitive method of diagnosis. Biopsy specimens from lymph nodes, brain, myocardium, or other suspected tissue, as well as body fluids, including cerebrospinal fluid, amniotic fluid, or bronchoalveolar lavage fluid, can be directly examined for the organisms. Newer monoclonal antibody-based fluorescent stains may facilitate direct detection of T. gondii in tissue. Culture methods for T. gondii are largely experimental and not usually available in clinical laboratories.

26. Cyst of T. gondii in tissue. Hundreds of organisms may be present in the cyst, which may become active and initiate disease with decreased host immunity (e.g., immunosuppression in transplant patients and in diseases such as AIDS).

27. The two methods available are to inoculate potentially infected material into either mouse peritoneum or tissue culture. Advances in developing polymerase chain reaction-based detection methods are promising and may provide rapid and sensitive approaches for detecting the organism in blood, cerebrospinal fluid, amniotic fluid, and other clinical specimens.

28. Sporozoa 2- CRYPTOSORIDIUM SPECIES Physiology and Structure The life cycle of Cryptosporidium species is typical of coccidians, as is the intestinal disease, but this species differs in the intracellular location in the epithelial cells. Cryptosporidium organisms are found just within the brush border of the intestinal epithelium. The coccidia attach to the surface of the cells and replicate by a process that involves schizogony.

31. 2- CRYPTOSORIDIUM SPECIES Epidemiology Cryptosporidium species are distributed worldwide. Infection is reported in a wide variety of animals, including mammals, reptiles, and fish. Waterborne transmission of cryptosporidiosis is well documented as an important route of infection. The massive outbreak of cryptosporidiosis in Milwaukee (approximately 300,000 individuals infected) was linked to contamination of the municipal water supply.

32. Cryptosporidia are resistant to the usual water-purification procedures (chlorination and ozone), and it is believed that runoff of local waste and surface water into municipal water supplies is an important source of contamination. Zoonotic spread from animal reservoirs to humans, as well as person-to-person spread by fecal-oral and oral-anal routes, is also common means of infection. Veterinary personnel, animal handlers, and homosexuals are at particularly high risk for infection. Many outbreaks have now been described in daycare centers where fecal-oral transmission is common.

33. CLINICAL SYNDROMES As with other protozoan infections, exposure to Cryptosporidium organisms may result in asymptomatic carriage. Disease in previously healthy individuals is usually a mild, self-limiting enterocolitis characterized by watery diarrhea without blood. Spontaneous remission after an average of 10 days is characteristic. In contrast, disease in immunocompromised patients (e.g., patients with AIDS), characterized by 50 or more stools per day and tremendous fluid loss, can be severe and last for months to years. In some patients with AIDS, disseminated Cryptosporidium infections have been reported.

34. LABORATORY DIAGNOSIS Cryptosporidium may be detected in large numbers in unconcentrated stool specimens obtained from immunocompromised individuals with diarrhea. Specimens may be stained using the modified acid-fast method or by an indirect immunofluorescence assay. The number of oocysts shed in stool may fluctuate; therefore a minimum of three specimens should be examined. Serologic procedures for diagnosing and monitoring infections are under investigation but are not yet widely available.

36. Sporozoa 3- PLASMODIUM SPECIES Plasmodia are coccidian or sporozoan parasites of blood cells, and as seen with other coccidia, they require two hosts: the mosquito for the sexual reproductive stages and humans and other animals for the asexual reproductive stages. Infection with Plasmodium spp. (i.e., malaria) accounts for 1 to 5 billion febrile episodes and 1 to 3 million deaths annually, 85% of which are in Africa.

37. The four species of plasmodia that infect humans are Plasmodium vivax, Plasmodium ovale, Plasmodium malariae, and Plasmodium falciparum. These species share a common life cycle. Human infection is initiated by the bite of an Anopheles mosquito, which introduces infectious plasmodia sporozoites via its saliva into the circulatory system.

39. Asexual reproduction (Human Liver and Red Blood Cells) Sexual reproduction (Anopheles midgut) Salivary glands Liver Red Blood Cells Midgut

41. Plasmodium falciparum:Blood Stage Parasites A: Stages of P. falciparum in thin blood smears.  Fig. 1: Normal red cell; Figs. 2-18: Trophozoites (among these, Figs. 2-10 correspond to ring-stage trophozoites); Figs. 19-26: Schizonts (Fig. 26 is a ruptured schizont); Figs.27, 28: Mature macrogametocytes (female); Figs. 29, 30: Mature microgametocytes (male).Illustrations from: Coatney GR, Collins WE, Warren M, Contacos PG. The Primate Malarias. Bethesda: U.S. Department of Health, Education and Welfare; 1971

42. The sporozoites are carried to the parenchymal cells of the liver, where asexual reproduction (schizogony) occurs. This phase of growth is termed the exoerythrocytic cycle and lasts 8 to 25 days, depending on the plasmodial species. Some species (e.g., P. vivax, P. ovale) can establish a dormant hepatic phase in which the sporozoites (called hypnozoites or sleeping forms) do not divide. The presence of these viable plasmodia can lead to the relapse of infections months to years after the initial clinical disease (relapsing malaria). The hepatocytes eventually rupture, liberating the plasmodia (termed merozoites at this stage), which in turn attach to specific receptors on the surface of erythrocytes and enter the cells, thus initiating the erythrocytic cycle.

43. Asexual replication progresses through a series of stages (ring, trophozoite, schizont) that culminates in the rupture of the erythrocyte, releasing up to 24 merozoites, which initiates another cycle of replication by infecting other erythrocytes. Some merozoites also develop within erythrocytes into male and female gametocytes. If a mosquito ingests mature male and female gametocytes during a blood meal, the sexual reproductive cycle of malaria can be initiated, with the eventual production of sporozoites infectious for humans. This sexual reproductive stage within the mosquito is necessary for the maintenance of malaria within a population.

44. Most malaria seen in the United States is acquired by visitors or residents of countries with endemic disease (imported malaria). However, the appropriate vector Anopheles mosquito is found in several sections of the United States, and domestic transmission of disease has been observed (introduced malaria). In addition to transmission by mosquitoes, malaria can also be acquired by blood transfusions from an infected donor (transfusion malaria). This type of transmission can also occur among narcotic addicts who share needles and syringes ("mainline" malaria). Congenital acquisition, although rare, is also a possible mode of transmission (congenital malaria)..

45. Sporozoa 3-1-PLASMODIUM VIVAX P. vivax is selective in that it invades only young, immature erythrocytes. In infections caused by P. vivax, infected red blood cells are usually enlarged and contain numerous pink granules or Schüffner's dots, the trophozoite is ring shaped but amoeboid in appearance, more mature trophozoites and erythrocytic schizonts containing up to 24 merozoites are present, and the gametocytes are round. These characteristics are helpful in identifying the specific plasmodial species, which is important for the treatment of malaria.

46. Plasmodium vivax ring forms and young trophozoites. Note the multiple stages of the parasite (rings and trophozoite) seen in the peripheral blood smear, the enlarged parasitized erythrocytes, and the presence of Schüffner's dots with the trophozoite form. These are characteristic of P. vivax infections.

47. Epidemiology P. vivax is the most prevalent of the human plasmodia, with the widest geographic distribution, including the tropics, subtropics, and temperate regions. Clinical Syndromes After an incubation period (usually 10 to 17 days) the patient experiences vague influenza-like symptoms with headache, muscle pains, photophobia, anorexia, nausea, and vomiting.

48. Clinical Syndromes As the infection progresses, increased numbers of rupturing erythrocytes liberate merozoites and toxic cellular debris and hemoglobin into the circulation. Together, these produce the typical pattern of chills, fever, and malarial rigors. These paroxysms usually reappear periodically (generally every 48 hours) as the cycle of infection, replication, and cell lysis progresses. The paroxysms may remain relatively mild or progress to severe attacks, with hours of sweating, chills, shaking, persistently high temperatures (103° to 106°F [39° to 41°C]), and exhaustion.

49. Clinical Syndromes P. vivax causes "benign tertian malaria," which refers to the cycle of paroxysms every 48 hours (in untreated patients) and the fact that most patients tolerate the attacks and can survive for years without treatment. If left untreated, however, chronic P. vivax infections can lead to brain, kidney, and liver damage as a result of the malarial pigment, cellular debris, and capillary plugging of these organs by masses of adherent erythrocytes.

50. Laboratory Diagnosis Microscopic examination of thick and thin films of blood is the method of choice for confirming the clinical diagnosis of malaria and identifying the specific species responsible for disease. The thick film is a concentration method and may be used to detect the presence of organisms. With training, thick films may be used to diagnose the species as well. The thin film is most useful for establishing species identification. Blood films can be taken at any time over the course of the infection, but the best time is midway between paroxysms of chills and fever, when the greatest numbers of intracellular organisms are present.