Miscellaneous Protozoa PDF

CHAPTER 7 Miscellaneous Protozoa Jill Dennis and Elizabeth Zeibig...

CHAPTER 7 Miscellaneous Protozoa Jill Dennis and Elizabeth Zeibig WHAT’S AHEAD Focusing In Sarcocystis species Toxoplasma gondii Miscellaneous Protozoa Cryptosporidium parvum Pneumocystis jiroveci Classification Blastocystis hominis (Pneumocystis carinii) Balantidium coli Cyclospora cayetanensis Looking Back Isospora belli Microsporidia LEARNING OBJECTIVES On completion of this chapter and review of significant disease processes associated with its figures and corresponding photomicrographs, each of the pathogenic organisms discussed. the successful learner will be able to: 7-7. Identify and describe each of the following 7-1. Define the following key terms: as they relate to the organisms discussed: Bradyzoite (pl., bradyzoites) A. Disease, condition, and prognosis Cilia B. Treatment options Ciliate (pl., ciliates) C. Prevention and control measures Coccidia 7-8. Select the specimen(s) of choice, collection Gametogony and processing protocol(s), and laboratory Macronucleus diagnostic technique for the recovery of Micronucleus each of the miscellaneous Protozoa. Sporoblast (pl., sporoblasts) 7-9. Given a description, photomicrograph, Sporocyst (pl., sporocysts) and/or diagram of one of the organisms Tachyzoite (pl., tachyzoites) described, correctly: 7-2. State the geographic distribution of each of A. Identify and/or label the designated the protozoa discussed. characteristic structure(s). 7-3. Given a list of parasites, select the B. State the purpose of the designated organism(s) belonging to the class Ciliata, characteristic structure(s). Sporozoa, or Blastocystea. Discuss any C. Identify the parasite by scientific name controversy surrounding classification, if and morphologic form. applicable. D. State the common name for associated 7-4. Classify each of these protozoa as intestinal conditions and diseases, if applicable. or extraintestinal. 7-10. Analyze case studies that include pertinent 7-5. Briefly summarize the life cycle of each information and laboratory data and do the organism discussed. following: 7-6. Identify and describe the populations prone A. Identify and differentiate each to contracting symptoms and clinically responsible organism by scientific name, Copyright © 2013 by Saunders, an imprint of Elsevier Inc. 159 160 CHAPTER 7 Miscellaneous Protozoa common name, and morphologic form, G. Interpret laboratory data, determine with justification. specific follow-up tests to be done, and B. Identify the diseases and conditions predict the results of those identified associated with the responsible parasite tests. C. Construct a life cycle associated with H. Determine additional morphologic each organism present that includes forms, where appropriate, that may also corresponding epidemiology, route of be detected in clinical specimens. transmission, infective stage, and 7-11 Identify, compare, and contrast the diagnostic stage. similarities and differences among the D. Propose each of the following related to parasites discussed in this and the other stopping and preventing further parasite chapters in this text. infections: 7-12. Describe standard, immunologic, and new 1. Treatment options laboratory diagnostic approaches for the 2. Prevention and control plan recovery of the miscellaneous Protozoa in E. Determine the specimen(s) of choice and clinical specimens. alternative specimen types, when 7-13. Given prepared laboratory specimens, and appropriate, as well as appropriate with the assistance of this manual, the laboratory diagnostic techniques for the learner will be able to: recovery of each responsible parasite. A. Differentiate the protozoan organisms F. Recognize sources of error including from artifacts and other parasites. but not limited to those involved in B. Correctly name each protozoan parasite specimen collection, processing, and based on its key characteristic testing and propose solutions to remedy structure(s). them. CAS E STUDY 7-1 UNDER THE MICROSCOPE Joseph, a 29-year old man, went to a local health clinic Questions and Issues for Consideration with complaints of intermittent diarrhea for a few months 1. What parasite do you suspect, and why? (Objectives and trouble sleeping. A stool specimen was collected and 7-10A) preserved in 10% formalin and PVA. An O&P examination 2. Why do false-negative results sometime occur with this was performed, along with a permanent trichrome stain. organism? (Objective 7-10F) The stain revealed the organism shown below that mea- 3. How does the organism reproduce? (Objective 7-10C) sured 10 μm in diameter, with a large, centrally located 4. Describe the controversy surrounding the classification vacuole and small red nuclei within the peripheral ring of of this organism. (Objective 7-3) cytoplasm. 5. Discuss treatment and prevention measures. (Objective 7-10D) Size range: 5-32 !m Average size: 7-10 !m  CHAPTER 7 Miscellaneous Protozoa 161 protozoan organisms, their exact classification FOCUSING IN has not been well described. The remaining protozoa of human clinical sig- nificance are described in this chapter. This group of organisms is similar in that each of its members MISCELLANEOUS PROTOZOA is unicellular. However, the specific morphologic CLASSIFICATION forms, methods of laboratory diagnosis, life cycle The remaining members of the protozoa are clas- notes, epidemiology, clinical symptoms treat- sified in four groups. The first group, the ciliates, ment protocols, and prevention and control mea- parasites that move by means of hairlike cyto- sures vary among the organisms in this group. plasmic extensions called cilia, contains one Because of these variations, the specific informa- human pathogen known as Balantidium coli tion associated with each of the protozoa is (Fig. 7-1). The second group consists of select described on an individual basis. In addition to sporozoa (Fig. 7-2), excluding Plasmodium and the laboratory diagnosis information in this Babesis spp., which are discussed in Chapter 6. chapter, representative diagnostic methodologies These parasites, which are intestinal and tissue- are discussed in Chapter 2. dwelling in nature, belong to the subclass Coc- In addition to a concise yet comprehensive cidia, a group of protozoal parasites in which discussion of the well-known miscellaneous pro- asexual replication occurs outside a human host tozoa, two relatively new genera, Cyclospora and sexual replication occurs inside a human and Microsporidia, are briefly mentioned in this host, and are often referred to as coccidian pro- chapter. These organisms are known to produce tozoans. Blastocystis hominis (Fig. 7-3), initially human intestinal disease. Because of their rela- considered as a yeast, makes up the third group tively recent discovery and the fact that much and is now classified as a Protozoa. This organ- is still to be learned about these genera of ism is the sole member of the class Blastocystea. Phylum Class Intestinal Species Ciliophora Kinetofragminophorea Balantidium coli FIGURE 7-1 Parasite Classification: The Cilliates. Intestinal Species Isospora belli Sarcocystis species Cryptosporidium parvum Phylum Class Apicomplexa Sporozoa Tissue Species Toxoplasma gondii FIGURE 7-2 Parasite Classification: The Sporozoa.  162 CHAPTER 7 Miscellaneous Protozoa Order Class Intestinal Species Blastocystida Blastocystea Blastocystis hominis FIGURE 7-3 Parasite Classification: Blastocystis hominis. Now classified as a fungus Pneumocystis jiroveci FIGURE 7-4 Parasite Classification: Pneumocystis jirvoeci. Cytostome Vacuole Ingested Micronucleus microbes (bacteria) Cilia Macronucleus A Size range: 28-152 !m by 22-123 !m B Average size: 35-50 !m by 40 !m FIGURE 7-5 A, Balantidium coli trophozoite. B, Balantidium coli trophozoite. (B, from Mahon CR, Lehmann DC, Manuselis G: Textbook of diagnostic microbiology, ed 4, St Louis, 2011, Saunders.) Pneumocystis jiroveci (formerly known as Pneu- Balantidium coli mocystis carinii) is the only member of the fourth (bal’an-tid’ee-um/ko’ly) group (Fig. 7-4). This organism was traditionally included with the Protozoa, even though it has Common associated disease and condition been recently reclassified as a fungus. names: Balantidiasis. Morphology Trophozoites. Considered as the largest pro- Quick Quiz! 7-1 tozoan known to humans, the typical Balantid- ium coli trophozoite may measure from 28 to What makes each member of the the parasites dis- 152 μm in length, with an average length of 35 cussed in this chapter similar? (Objective 7-11) to 50 μm (Fig. 7-5; Table 7-1). The average tro- A. Presence of cilia phozoite width is approximately 40 μm but may B. They are all unicellular. range from 22 to 123 μm. The ovoid to sac- C. All are intestinal protozoa. shaped B. coli trophozoite tapers at the anterior D. Presence of tachyzoites end. The organism typically exhibits rotary,  CHAPTER 7 Miscellaneous Protozoa 163 Cilia Contractile vacuole Cyst wall Macronucleus Micronucleus A Size range: 43-66 !m B Average size: 52-55 !m FIGURE 7-6 A, Balantidium coli cyst. B, Balantidium coli cyst. (B, from Mahon CR, Lehmann DC, Manuselis G: Textbook of diagnostic microbiology, ed 4, St Louis, 2011, Saunders.) TAB LE 7-1 Balantidium coli TA B L E 7 - 2 Balantidium coli Cyst: Trophozoite: Typical Typical Characteristics at Characteristics at a Glance a Glance* Parameter Description Parameter Description Size range 28-152 μm in length, 22-123 μm Size range 43-66 μm wide Number and Two Motility Rotary, boring appearance of Kidney-shaped macronucleus Number of nuclei Two nuclei usually present Kidney-shaped macronucleus Small spherical micrcnucleus; Small spherical micronucleus may not be observable Other features One or two visible contractile Other features One or two visible contractile vacuoles vacuoles in young cysts Cytoplasm may contain food Double cyst wall vacuoles and/or bacteria Row of cilia visible in between Small cytostome present cyst wall layers of young Layer of cilia around organism cysts *Mature cysts typically only reveal macronucleus on examina- tion. The other structures are usually not apparent. boring motility. The trophozoite contains two nuclei. A small dotlike nucleus (micronucleus) is (bacteria). The trophozoite is equipped with a located adjacent to a large, often kidney bean– small cytostome. A layer of cilia surrounds shaped nucleus known as a macronucleus. The the organism, which serves as its means of micronucleus is often not readily visible, even in locomotion. stained preparations, whereas the macronucleus Cysts. Averaging in size from 52 to 55 μm, may often appear as a hyaline mass, especially in the subspherical to oval B. coli cyst may measure unstained preparations. Two contractile vacuoles from 43 to 66 μm (Fig. 7-6; Table 7-2). Although are located in the granular cytoplasm, although the cyst technically contains the macronucleus sometimes only one is readily visible, as in Figure and micronucleus, the micronucleus may not 7-5A. In addition, the cytoplasm may also contain be observed in wet or permanent preparations. food vacuoles, as well as ingested microbes One or two contractile vacuoles may be visible, 164 CHAPTER 7 Miscellaneous Protozoa particularly in young unstained cysts. A double- cysts may survive for weeks in the outside protective cyst wall surrounds the organism. A environment. row of cilia may be visible between the two cyst wall layers in unstained young cysts. Mature Epidemiology cysts tend to lose their cilia. Stained cysts typi- cally reveal only the macronucleus; the other Although B. coli is distributed worldwide and structures are not usually apparent. outbreaks have been known to occur, the typical incidence of human infection is very low. The documented frequency of infections in the general Laboratory Diagnosis population is considered rare. However, epidem- Laboratory diagnosis of B. coli is accomplished ics caused by infections with B. coli have been by examining stool specimens for the presence noted in psychiatric facilities in the United States. of trophozoites and cysts. Stools from infected B. coli infections are transmitted by ingesting patients experiencing diarrhea are more likely to contaminated food and water by the oral-fecal contain B. coli trophozoites. Although it does as well as person-to-person routes. Recently, it not occur frequently, suspicious formed stools has been presumed that water contaminated with may contain cysts. Sigmoidoscopy material may feces (the oral-fecal route) from a pig, which is a also reveal B. coli organisms when collected from known reservoir host, may be a significant source patients suffering from sigmoidorectal infection. of infection. There is now considerable evidence As with any sample submitted for parasitic study, to support the theory that the pig may not be thorough screening of the wet preparations and the primary infection source, because the docu- the permanent stain is crucial to ensure an accu- mented incidence of infection among humans rate laboratory test report. In addition, the study with high pig contact is relatively low. Infected of multiple samples may be required to deter- food handlers appear to be the culprit in person- mine the presence or absence of the parasite to-person spread of the disease. correctly. Clinical Symptoms Life Cycle Notes Asymptomatic Carrier State. Similar to that The B. coli life cycle is similar to that of Ent- seen in certain patients infected with E. histo- amoeba histolytica. Human infection with B. lytica, some patients are just carriers of B. coli coli is initiated on ingestion of infective cysts in and remain asymptomatic. contaminated food or water. Unlike that of E. Balantidiasis. Symptomatic patients may histolytica, multiplication of the B. coli nuclei experience a variety of discomforts, ranging from does not occur in the cyst phase. Following mild colitis and diarrhea to full-blown clinical excystation in the small intestine, the resulting balantidiasis, which may often resemble amebic trophozoites take up residence and feed primar- dysentery. In this case, abscesses and ulcers may ily in the cecal region and terminal portion of form in the mucosa and submucosa of the large the ileum, as well as in the lumen, mucosa, and intestine, followed by secondary bacterial infec- submucosa of the large intestine. The multiplica- tion. Acute infections are characterized by up to tion of each trophozoite occurs by transverse 15 liquid stools daily containing pus, mucus, and binary fission, from which two young trophozo- blood. Patients who suffer from chronic infec- ites emerge. The B. coli trophozoites are delicate tions may develop a tender colon, anemia, and do not survive in the outside environment. cachexia, and occasional diarrhea, alternating Encystation occurs in the lumen. The resulting with constipation. B. coli has been known to cysts mature and ultimately become the infective invade areas other than the intestine, such as form for transmission into a new host. These the liver, lungs, pleura, mesenteric nodes, and  CHAPTER 7 Miscellaneous Protozoa 165 urogenital tract. However, the incidence of such Quick Quiz! 7-2 extraintestinal infections is rare. Which structure is always visible in the stained cyst and troph of Balantidium coli? (Objective 7-9A) Treatment A. Macronucleus B. Micronucleus Two factors play an important role in determin- C. Cilia ing the prognosis of patients infected with B. D. Ingested bacteria coli, the severity of the infection and the patient’s response to treatment. Asymptomatic patients and those suffering from chronic disease typi- cally have a good chance of recovery. There are Quick Quiz! 7-3 two medication choices for the effective treat- ment of B. coli infections, oxytetracycline (Ter- The life cycle of Balantidium coli and clinical symp- ramycin) and iodoquinol. Metronidazole (Flagyl) toms are similar to that of which of the following? may also be used to treat infected patients. (Objectives 7-11) A. Isospora belli B. Entamoeba histolytica C. Crytosporidium parvum Prevention and Control D. Giardia intestinalis Personal hygiene and proper sanitary conditions are effective measures for B. coli prevention and control. Until the questions surrounding the pig’s Quick Quiz! 7-4 role in transmitting B. coli are completely under- stood, the pig should be considered as a possible Which two factors play an important role in the prog- source of infection and proper precautions should nosis of a Balantidum coli infection? (Objective 7-7A) be exercised when handling and dealing with A. How infection occurred and duration of the pigs and their feces. infection B. Presence of coinfection and duration of the infection C. Severity of infection and response to treatment Notes of Interest and New Trends D. Immunocompetent status and severity of The B. coli trophozoite is often referred to as infection resembling a sac in shape. As a reflection of this shape, the organism was named Balantidium, which means “little bag.” Isospora belli It is estimated that 63% to 91% of pigs harbor (eye”sos’puh-ruh/bell-eye) B. coli. In addition, pigs also carry Balantidium suis, a parasite that is morphologically identical Common associated disease and condition to B. coli but does not appear to cause human names: Isosporiasis. infections. Unsuccessful attempts have been made to infect humans purposely with B. suis. Morphology Because the incidence of B. coli is low in the population as a whole and in those who have Oocysts. The oval transparent oocyst of Isos- regular contact with pigs, it has been suggested pora belli ranges in size from 25 to 35 μm long that humans have a relatively high natural resis- by 10 to 15 μm wide, with an average of 30 by tance to this organism. 12 μm (Fig. 7-7; Table 7-3). The developing 166 CHAPTER 7 Miscellaneous Protozoa Double Laboratory Diagnosis layered cell wall The specimens of choice for recovery of the I. belli oocysts are fresh feces and duodenal con- tents. Stool samples may contain oocysts that are immature, partially mature, and/or fully mature. In addition, material collected via an Enterotest Two mature sporocysts may also be used to obtain the oocysts. Intestinal (each contains biopsies collected from infected patients may 4 sporozoites) reveal the intracellular morphologic stages of the organism. It is interesting to note that a biopsy Size range: 25-35 !m by 10-15 !m from an infected patient may contain I. belli Average size: 30 !m by 12 !m oocysts, whereas a stool specimen from the same FIGURE 7-7 Isospora belli oocyst. patient may be free of the parasites. This occurs particularly in patients who have only small numbers of organisms present. I. belli oocysts may be visible in direct wet TABLE 7-3 Isospora belli Oocyst: preparations and in those made following the con- Typical Characteristics at centration or flotation procedures. Promising a Glance results have been obtained on stool specimens pro- Parameter Description cessed using the Sheather’s sugar flotation proce- Size range 25-35 μm long, 10-15 μm dure. It is important to note that I. belli oocysts wide appear transparent and may be difficult to recog- Appearance Transparent nize when present in saline wet preparations. The Shape Oval oocysts are more readily discernible in iodine prep- Cell wall Two layered, colorless and arations. It is therefore important to include an smooth iodine wet preparation in the standard processing Developing sporoblast Unicellular with granular of samples for parasite study, particularly those in cytoplasm which I. belli is suspected. In addition, a decreased Young oocyst Two sporoblasts microscope light level and proper contrast are nec- Mature oocyst Two sporocysts, each essary when screening suspicious slides to achieve containing four the most favorable conditions for parasite recov- sausage-shaped sporozoites ery. This is particularly true when screening samples that have been tested by the zinc sulfate technique or another concentration procedure fol- morphologic form within the oocyst, known as lowing polyvinyl alcohol (PVA) preservation. a sporoblast, consists of a roundish immature sac A tentative diagnosis may be made following that contains a small discrete nucleus and granu- preparation and examination of an auramine- lar cytoplasm. As it matures, the young oocyst rhodamine permanent stain. However, the rec- divides into two sporoblasts. Each sporoblast ommended permanent stain for successful I. belli continues to mature and eventually becomes a oocyst confirmatory identification is a modified sporocyst, which consists of a mature roundish acid-fast stain. This clearly shows the organism’s sac containing four sausage-shaped sporozoites. characteristics, as well as those of Cryptospo- This stage is known as the mature oocyst (Fig. ridium oocysts, another important member of 7-7). Throughout its development, the sporo- the sporozoa (see later). Wet preparations for blast and sporocysts are surrounded by a smooth, Isospora may also serve as adequate confirma- colorless, two-layered cell wall. tory tests when necessary.  CHAPTER 7 Miscellaneous Protozoa 167 noted in patients suffering from AIDS. Unpro- Life Cycle Notes tected oral-anal sexual contact has been sug- I. belli was initially thought to be a typical coc- gested as the mode of parasite transmission in cidial parasite. Pigs and cattle appeared to be these patients. The resulting infections with I. intermediate hosts for this organism. It has now belli are now considered opportunistic. been determined that I. belli is the only known coccidial parasite that does not have intermedi- Clinical Symptoms ate hosts. Humans serve as the definitive host, in whom both sexual and asexual reproduction Asymptomatic. A number of infected indi- take place. viduals remain asymptomatic. In such cases, the It is presumed that infection with I. belli is infection is self-limited. initiated following the ingestion of infective Isosporiasis. Infected patients may complain mature (also known as sporulated) oocysts in of a number of symptoms, ranging from mild contaminated food or water. The sporozoites gastrointestinal discomfort to severe dysentery. emerge after excystation of the oocyst in the The more commonly noted clinical symptoms small intestine. Asexual reproduction (schizog- include weight loss, chronic diarrhea, abdominal ony), resulting in merozoites, occurs in the cells pain, anorexia, weakness, and malaise. In addi- of the intestinal mucosa. The formation of mac- tion, eosinophilia may occur in asymptomatic rogametocytes and microgametocytes (gametog- and symptomatic patients. Charcot-Leyden crys- ony) takes place in the same intestinal area. The tals (Chapter 12) may form in response to the resulting gametocytes develop and ultimately eosinophilia and may be visible in corresponding unite to form oocysts, the form that is excreted stool samples. Patients experiencing severe infec- in the stool. Immature oocysts typically complete tion typically develop a malabsorption syndrome. their development in the outside environment. It In these cases, patients produce foul-smelling is the mature sporulated oocyst that is capable stools that are pale yellow and of a loose consis- of initiating another infection. When this occurs, tency. Fecal fat levels of these stool samples may the life cycle repeats itself. There is evidence sug- be increased. Infected patients may shed oocysts gesting that I. belli may also be transmitted in their stools for as long as 120 days. Death may through oral-anal sexual contact. result from such severe infections. Treatment Epidemiology The treatment of choice for asymptomatic or The frequency of contracting I. belli has tradi- mild infections consists of consuming a bland tionally been considered rare, even though the diet and obtaining plenty of rest. Patients suffer- organism has worldwide geographic distribu- ing from more severe infections respond best to tion. The difficulty often experienced in organ- chemotherapy, consisting of a combination of ism recognition may have led to potentially trimethoprim and sulfamethoxazole or pyrimeth- false-negative results, which thus may have been amine and sulfadiazine. It is interesting to note the major contributing factor to the documented that chemotherapy at a lower dosage for a longer rare frequency of infection. An increase in period may be necessary for AIDS patients reported cases began to occur during and follow- infected with I. belli. ing World War II. Specifically, cases were reported in Africa, Southeast Asia, and Central America. Prevention and Control In addition, countries in South America, particularly in Chile, have reported I. belli infec- The prevention and control measures for I. belli tions. An increase in frequency was particularly are similar to those of E. histolytica. They include 168 CHAPTER 7 Miscellaneous Protozoa proper personal hygiene, adequate sanitation known as Sarcocystis hominis. Similarly, Sarco- practices, and avoidance of unprotected sex, par- cystis suihominis may be found in pigs. In addi- ticularly among homosexual men. tion to these typical farm animals, a variety of wild animals may also harbor members of the Sarcocystis group. Sarcocystis lindemanni Quick Quiz! 7-5 has been designated as the umbrella term for those organisms that may potentially parasitize All the following are highly recommended when pro- humans. cessing samples for the identification of Isospora belli to ensure identification except: (Objective 7-8) A. Iodine wet prep Morphology B. Decreased microscope light level Mature Oocysts. Members of the genus Sar- C. Modified acid-fast stain cocystis were originally classified and considered D. Saline wet prep as members of the genus Isospora, in part because of the striking morphologic similarities of these parasites (Fig. 7-8; Table 7-4). The oval transpar- Quick Quiz! 7-6 ent organism consists of two mature sporocysts that each average from 10 to 18 μm in length. Which stage of reproduction is considered capable of Each sporocyst is equipped with four sausage- initiating another infection of Isospora belli? (Objec- shaped sporozoites. A double-layered clear and tives 7-5) colorless cell wall surrounds the sporocysts. A. Sporozoites B. Immature oocysts Laboratory Diagnosis C. Merozoites D. Mature oocysts Stool is the specimen of choice for the recovery of Sarcocystis organisms. The oocysts are usually passed into the feces fully developed. When present, these mature oocysts are typically seen Quick Quiz! 7-7 Which of the following patients would be more likely to contract an infection with Isospora belli? (Objective 7-6) Double layered A. HIV-positive man Ruptured sporocyst wall sporocysts are B. Female leukemia patient most often C. Pig farmer seen singly D. Nursing home resident or in pairs “cemented together” in clinical specimens. Sarcocystis species Two mature (sahr”ko-sis-tis) sporocysts (each contains Common associated disease and condition 4 sporozoites) names: Sarcocystis infection. There are a number of species of parasites that fall within the group known as Sarcocystis. Average sporocyst length: 10-18 !m Cattle may harbor Sarcocystis hovihominis, also FIGURE 7-8 Sarcocystis species oocyst.  CHAPTER 7 Miscellaneous Protozoa 169 TAB LE 7-4 Sarcocystis spp. Mature take up residence in human striated muscle. Oocyst: Typical Under these circumstances, the human serves as Characteristics at a Glance* the intermediate host. It is interesting to note that Sarcocystis oocysts do not infect the host of their Parameter Description origin. Shape Oval Appearance Transparent Number of sporocysts Two Epidemiology Size of each sporocyst 10-18 μm long The frequency of Sarcocystis infections is rela- Contents of each sporocyst Four sausage-shaped tively low, even though its distribution is world- sporozoites Oocyst cell wall appearance Clear, colorless, double wide. In addition to its presence in cattle and layered pigs, Sarcocystis spp. may also be found in a variety of wild animals. *In many cases, only single or double sporocysts cemented together may be visible in stool samples. Clinical Symptoms in wet preparations. However, in many cases, the Sarcocystis Infection. There have only been a oocysts have already ruptured and only the spo- few documented symptomatic cases of Sarcocys- rocysts are visible on examination of the stool tis infections in compromised patients. These specimen. The sporocysts may be seen singly or persons experienced fever, severe diarrhea, weight in pairs that appear to be cemented together. loss, and abdominal pain. It is presumed that Routine histologic methods may be used to iden- patients suffering from muscle tenderness and tify the Sarcocystis cyst stage, known as the sar- other local symptoms are exhibiting symptoms cocyst, from human muscle samples. An in-depth caused by Sarcocystis invasion of the striated discussion of these histologic methods is beyond muscle. the scope of this text. Treatment Life Cycle Notes The treatment protocol for infections with Sar- Although the morphology of the oocysts of Sar- cocystis spp. when humans are the definitive host cocystis resembles that of Isospora, the life cycles is similar to that for Isospora belli. The com- of these two genera are different—hence, the bined medications of trimethoprim plus sulfa- current organism classification. Asexual repro- methoxazole or pyrimethamine plus sulfadiazine duction of Sarcocystis occurs in the intermediate are typically given to treat these infections. There host. Human infection of Sarcocystis species may is no known specific chemotherapy to treat Sar- be initiated in one of two ways. The first trans- cocystis infections of the striated muscle when mission route occurs when uncooked pig or humans are the intermediate host. cattle meat infected with Sarcocystis sarcocysts is ingested. Humans are the definitive host. Gam- Prevention and Control etogony usually occurs in the human intestinal cells. The development of oocysts and subse- The primary prevention and control measures of quent release of sporocysts thus follow. This sets Sarcocystis infections in which humans are the the stage for continuation of the life cycle in a definitive host consist of adequate cooking of new intermediate host. The second transmission beef and pork. Prevention of those infections in route occurs when humans accidentally swallow which humans are the intermediate host includes oocysts from stool sources of animals other than the proper care and disposal of animal stool that cattle or pigs. In this case, the ingested sarcocysts may be potentially infected with Sarcocystis. 170 CHAPTER 7 Miscellaneous Protozoa Dark granule Quick Quiz! 7-8 (may contain 1 to 6 granules) Which genus of parasite is most similar to Sarcocystis Sporozoites based on morphologic similarities? (Objective 7-11) A. Isospora B. Blastocystis C. Entamoeba D. Toxoplasma Average size: 4-6 !m FIGURE 7-9 Cryptosporidium parvum oocyst. Quick Quiz! 7-9 How do humans become infected with Sarcocystis? (Objectives 7-5) A. Ingestion of uncooked or undercooked beef or pork B. Inhalation of oocysts C. Ingestion of animal fecal contaminated food D. More than one of the above: ________________ (specify) Quick Quiz! 7-10 FIGURE 7-10 Modified acid-fast stain, ×1000). Arrows indicate Cryptosporidium oocysts, each containing four In addition to oocysts, these Sarcocystis morphologic undefined sporozoites. Note dark-staining granules. forms may be seen in human samples: (Objective 7-5) A. Packets of eggs B. Single or double sporocysts TA B L E 7 - 5 Cryptosporidium parvum C. Clusters of cysts Oocyst: Typical D. Groups of sporoblasts Characteristics at a Glance Parameter Description Size 4-6 μm Cryptosporidium parvum Shape Roundish (krip”toe-spor-i’dee-um/par-voom) Number of sporocysts None Number of sporozoites Four (small) Common associated disease and condition Other features Thick cell wall names: Cryptosporidosis. One to six dark granules may be visible Morphology Oocysts. Measuring only 4 to 6 μm, the surrounded by a thick cell wall. Contrary to roundish Cryptosporidium oocysts are often other members of the sporozoa, such as Isos- confused with yeast (Figs. 7-9 and 7-10; Table pora, Cryptosporidium oocysts do not contain 7-5). Although not always visible, the mature sporocysts. One to six dark granules may also oocyst consists of four small sporozoites be seen.  CHAPTER 7 Miscellaneous Protozoa 171 Schizonts and Gametocytes. The other mor- most likely responsible for autoinfections because phologic forms required to complete the life cycle it always seems to rupture while still inside the of Cryptosporidium include schizonts containing host. The thick-shelled oocyst usually remains four to eight merozoites, microgametocytes, and intact and is passed out of the body. This form macrogametocytes. The average size of these is believed to initiate autoinfections only forms is a mere 2 to 4 μm. It is important to note occasionally. that these morphologic forms are not routinely seen in patient samples. Epidemiology Cryptosporidium has worldwide distribution. Of Laboratory Diagnosis the 20 species known to exist, only C. parvum is The specimen of choice for the recovery of Cryp- known to infect humans. Infection appears to tosporidium oocysts is stool. Several methods primarily occur by water or food contaminated have been found to identify these organisms suc- with infected feces, as well as by person-to- cessfully. The oocysts may be seen using iodine or person transmission. Immunocompromised per- modified acid-fast stain. In addition, formalin- sons, such as those infected with the AIDS virus, fixed smears stained with Giemsa may also yield are at risk of contracting this parasite. Other the desired oocysts. As noted, it is important to populations potentially at risk include immuno- distinguish yeast (Chapter 12) from true oocysts. competent children in tropical areas, children in Oocysts have also been detected using the follow- day care centers, animal handlers, and those who ing methods: the Enterotest, enzyme-linked travel abroad. immunosorbent assay (ELISA), and indirect immunofluorescence. Concentration via modified Clinical Symptoms zinc sulfate flotation or by Sheather’s sugar flota- tion have also proven successful, especially when Cryptosporidiosis. Otherwise healthy persons the treated sample is examined under phase con- infected with Cryptosporidium typically com- trast microscopy. It is important to note that plain of diarrhea, which is self-limiting and lasts merozoites and gametocytes are usually only approximately 2 weeks. Episodes of diarrhea recovered in intestinal biopsy material. lasting 1 to 4 weeks have been reported in some day care centers. Fever, nausea, vomiting, weight loss, and abdominal pain may also be Life Cycle Notes present. When fluid loss is great because of the Cryptosporidium infection typically occurs fol- diarrhea and/or severe vomiting, this condition lowing ingestion of the mature oocyst. Sporozo- may be fatal, particularly in young children. ites emerge after excystation in the upper Infected immunocompromised individuals, gastrointestinal tract, where they take up resi- particularly AIDS patients, usually suffer from dence in the cell membrane of epithelial cells. severe diarrhea and one or more of the symptoms Asexual and sexual multiplication may then described earlier. Malabsorption may also accom- occur. Sporozoites rupture from the resulting pany infection in these patients. In addition, oocysts and are capable of initiating an autoin- infection may migrate to other body areas, such fection by invading new epithelial cells. A number as the stomach and respiratory tract. A debilitat- of the resulting oocysts remain intact, pass ing condition that leads to death may result in through the feces, and serve as the infective stage these patients. Estimated infection rates in AIDS for a new host. patients range from 3 to 20% in the United It is interesting to note that two forms of States and 50 to 60% in Africa and Haiti. Cryp- oocysts are believed to be involved in the Cryp- tosporidium infection is considered to be a cause tosporidium life cycle. The thin-shelled version is of morbidity and mortality. 172 CHAPTER 7 Miscellaneous Protozoa contaminated with Cryptosporidium from calf Treatment feces. Numerous experiments to treat Cryptosporid- A modification of the standard stool process- ium using a wide variety of medications have ing technique (see Chapter 2), which includes been conducted. Unfortunately, most of these layering and flotation of the sample over a hyper- potential treatments have proven ineffective. tonic sodium chloride solution, has successfully However, the use of spiramycin, even though separated Cryptosporidium oocysts from fecal still in the experimental stage, has preliminarily debris. proven helpful in ridding the host of Cryptospo- ridium. More research on this treatment and on Quick Quiz! 7-11 the newer antiparasitic medications are necessary to develop effective medications. Which stage of reproduction is considered capable of autoinfection of Cryptosporidium? (Objectives 7-5) A. Intact oocysts Prevention and Control B. Merozoites Proper treatment of water supplies, handling C. Gametocytes known infected material by using gloves and D. Sporozoites wearing a gown (when appropriate), proper hand washing, and properly disinfecting poten- Quick Quiz! 7-12 tially infected equipment with full-strength commercial bleach or 5% to 10% household The permanent stain of choice for the recovery of ammonia are crucial to the prevention and Cryptosporidium parvum is: (Objective 7-8) control of Cryptosporidium. In addition, enteric A. Iron hematoxylin precautions should be observed when working B. Modifed acid-fast with known infected persons. C. Gram D. Trichrome Notes of Interest and New Trends Quick Quiz! 7-13 Cryptosporidium spp. were first associated with poultry and cattle. C. parvum is now recognized All the following are recommended to prevent and as the agent responsible for neonatal diarrhea in control an outbreak of Cryptosporidium except: calves and lambs, a life-threatening condition. (Objective 7-7C) Human Cryptosporidium infection was first A. Proper treatment of water supplies reported in 1976. The first cases were isolated B. Sterlize equipment using high heat. from persons with compromised immune systems C. Sterilize equipment using full-strength bleach. and were considered infrequent in occurrence. D. Sterilize equipment using 5% to 10% household Several outbreaks in the public water supply ammonia. were attributed to contamination of Cryptospo- ridium oocysts. This occurred in Carroll County, Blastocystis hominis Georgia in 1987. More recently in June 2011, (blas’toe-sis-tis/hom’i-nis) and Indiana fire station reported gastrointestinal illness in a substantial percentage of their workers Common associated disease and condition who had extinguished a barn fire on a nearby names: Blastocystis hominis infection. Michigan farm. An investigation by the Michi- gan Department of Community Health revealed Morphology that the firefighters used local hydrant water and on site swimming pond water to extinguish the Although a number of different morphologic fire. The pond water was discovered to be forms of B. hominis are known to exist, the most  CHAPTER 7 Miscellaneous Protozoa 173 TA B L E 7 - 6 Blastocystis hominis Vacuolated Form: Typical Vacuole Characteristics at a Glance Nucleii Parameter Description Size 5-32 μm Vacuole Centrally located Fluid-filled structure Consumes almost 90% of organism Cytoplasm Appears as ring around periphery of organism Nuclei Two to four located in cytoplasm Size range: 5-32 !m Average size: 7-10 !m FIGURE 7-11 Blastocystis hominis vacuolated form. Laboratory Diagnosis Stool is the specimen of choice for the recovery of Blastocystis. In iodine wet preparations, the peripheral cytoplasm containing one or more nuclei appears a light yellow in color, whereas the central vacuole does not stain and appears clear and transparent. In permanent stain prepa- rations, the central vacuole may vary in its ability to stain from not at all to very apparent. The nuclei located in the peripheral cytoplasm in these preparations typically stain dark. It is important to note here that saline, like water, usually lyses this organism and may lead to a false-negative result. Therefore, it is important to screen suspicious samples with an iodine wet preparation and to use a permanent stain to confirm the presence of the parasite. FIGURE 7-12 Trichrome stain, 1000×. Typical Blastocystis hominis vacuolated form. Life Cycle Notes B. hominis reproduces by sporulation or binary fission. The organism passes through a number common form seen and the easiest to recognize of morphologic forms during these processes. B. is the vacuolated form. Therefore, only this form hominis participates in sexual and asexual repro- will be described here. duction, and exhibits pseudopod extension and Vacuolated Forms. Although the vacuolated retraction. A detailed discussion of the B. hominis form of B. hominis may range in size from 5 to life cycle has not been widely presented. 32 μm, the average form measures only 7 to 10 μm (Figs. 7-11 and 7-12; Table 7-6). This Epidemiology morphologic form is characterized by a large, central, fluid-filled vacuole that consumes almost Early nonscientific documentation of B. hominis 90% of the cell. The remaining 10% assumes the infections indicated that they occurred as epi- periphery of the organism; it consists of a ring of demics in subtropical countries. Select articles on cytoplasm in which two to four nuclei are typi- B. hominis over the past 10 to 25 years or so cally present. suggest that this organism may be found in a 174 CHAPTER 7 Miscellaneous Protozoa number of climates worldwide, ranging from Since its discovery, B. hominis has been the Saudi Arabia to British Columbia. The results of subject of controversy. Initially, the organism was one study conducted in Saudi Arabia were incon- considered as an algae, then as a harmless intes- clusive regarding whether travel is a risk factor tinal yeast, and as a protozoan parasite since the in contracting this parasite. Infection of B. 1970s. Genetic analyses in 1996 showed that hominis is initiated by ingestion of fecally con- Blastocystis is not fungal or protozoan. Since taminated food or water. then, its classification has undergone major reviews which definitely place it into Strameno- piles, a major line of eukaryotes. Clinical Symptoms Blastocystis hominis Infection. The pathoge- Quick Quiz! 7-14 nicity of B. hominis is not totally clear, although the symptoms have been defined. Patients who Which is the best screening method for the identifica- suffer from infection with B. hominis in the tion of Blastocystis hominis? (Objective 7-8) absence of other intestinal pathogens (including A. Saline wet prep parasites, bacteria, and viruses) may experience B. Modified acid-fast stain diarrhea, vomiting, nausea, and fever, as well as C. Iodine wet prep abdominal pain and cramping. Thus, B. hominis D. Iron hematoxylin stain might be considered a pathogen. However, it has Quick Quiz! 7-15 also been suggested that these patients may have an additional undetected pathogen that is ulti- Blastocystis hominis is always considered as being mately responsible for the discomfort. responsible for clinical symptoms when present in In persons infected with B. hominis in addi- human samples. (Objective 7-6) tion to another pathogenic organism (e.g., E. A. True histolytica, Giardia intestinalis), it is this under- B. False lying agent that is thought to be the pathogen. These patients usually experience severe symp- Quick Quiz! 7-16 toms, as described earlier. Which of the following measures that when taken Treatment can prevent the spread of Blastocystis hominis? (Objective 7-7C) Iodoquinol or metronidazole is recommended for A. Avoid swimming in potentially contaminated the treatment of B. hominis. This has been sug- water. gested for patients infected with Blastocystis who B. Proper sewage treatment have no other obvious reason for their diarrhea. C. Use insect repellent. D. Avoid unprotected sex. Prevention and Control Cyclospora cayetanensis Proper treatment of fecal material, thorough (si’klō-spor-uh) hand washing, and subsequent proper handling of food and water are critical to halt the spread Common associated disease and condition of Blastocystis. names: Cyclospora cayetanensis infection. Notes of Interest and New Trends Morphology Blastocystis hominis was given its current name Oocysts. Cyclospora cayetanensis infection is in 1912 by Emile Brumpt. similar to cryptosporidiosis (Table 7-7). It is an  CHAPTER 7 Miscellaneous Protozoa 175 TAB LE 7-7 Cyclospora cayetanensis a process that may take 1 or more weeks to Mature Oocyst: Typical complete. Once the maturation process is com- Characteristics at a Glance plete, the resultant oocysts are capable of initiat- ing a new cycle. No animal reservoir exists. Parameter Description Size 7-10 μm in diameter Number of sporocysts Two Epidemiology Contents of sporocysts Each sporocyst contains C. cayetanensis infections are known to occur two sporozoites in many countries, including the United States and Canada. Furthermore, cases of infection intestinal coccidial organism. Infected patients caused by C. cayetanensisa have been reported shed oocysts that measure 7 to 10 μm in diam- in children living in unsanitary conditions in eter and, on maturation, form two sporocysts, Lima, Peru, as well as in travelers and foreigners each containing only two sporozoites. residing in Nepal and parts of Asia. Contami- nated water in Chicago presumably was the source of a minioutbreak in 1990 that occurred Laboratory Diagnosis in a physician’s dormitory. Contaminated lettuce Diagnosis of C. cayetanensis may be accom- and fresh fruit (raspberries have been known plished when stool samples are concentrated to be a source of infection), often imported, nontraditionally without the use of formalin have also been associated with C. cayetanensis fixative. C. cayetanensis oocysts sporulate best at infections. room temperature. The addition of 5% potas- sium dichromate allows the sporocysts to become Clinical Symptoms visible. Flotation methods followed by examina- tion using the preferred phase contrast or bright Cyclospora cayetanensis Infection. The clini- field microscopy have also proven successful cal symptoms associated with C. cayetanensis in isolating C. cayetanensis. Modified acid-fast infections in children are similar to those seen in stain may also be used to detect the oocysts. cases of cryptosporidiosis. The notable difference Oocysts autofluoresce under ultraviolet light among infections caused by these two organisms microscopy. in adults is that C. cayetanensis produces a longer duration of diarrhea. There is no known con- nection between C. cayetanensis infection and Life Cycle Notes immunocompromised patients. The life-cycle of C. cayetanensis, like that of Isospora, begins with ingestion of an oocyst. The Prevention and Control oocyst contains two sporocysts, each enclosing two sporozoites. Once inside a human host, cells Prevention and control measures associated with in the small intestine provide a suitable environ- C. cayetanensis consist of properly treating water ment for the emergence of sporozoites. The prior to use and only using treated water when sporozoites undergo asexual reproduction, pro- handling and processing food. ducing numerous merozoites, as well as sexual development, resulting in macrogametocyte and Notes of Interest and New Trends microgametocyte production. Male and female gametocytes unite and form oocysts. Infected It appears that this parasite may not be recovered humans pass immature oocysts in the stool. using standard or traditional specimen process- Under optimal conditions, these oocyts continue ing techniques. The alternative techniques dis- to develop and mature outside the human body, cussed in the laboratory diagnosis section may 176 CHAPTER 7 Miscellaneous Protozoa be necessary for samples suspected of containing human disease have been reported in patients C. cayetanensis in the future. suffering from AIDS. The most well-known member is Enterocytozoon bieneusi, which causes enteritis in these patients. Species of Quick Quiz! 7-17 Encephulitozoon and Pleistophora have also been described as infecting AIDS patients and Diagnosis of Cyclospora can be accomplished by all causing severe tissue infections. Of the remaining the following except: (Objective 7-8) two genera, Microsporidium is noted for corneal A. Concentration with formalin fixative infections, as well as Nosema. In addition, B. Flotation methods Nosema produced a fatal infection in a severely C. Modified acid-fast stain immunocompromised infant. D. Addition of 5% potassium dichromate Morphology Quick Quiz! 7-18 Spores. Although it has been documented that there are a number of different morphologic The clinical symptoms associated with Cyclospora forms, spores are the only ones that have been infections in children are similar to those seen in well described (Table 7-8). These spores are very cases of infection by which of the following? (Objec- small, ranging in size from 1 to 5 μm. Unlike the tives 7-11) other protozoa, Microsporidia spores are char- A. Naegleria acteristically equipped with extruding polar B. Cryptosporidium filaments (tubules), which initiate infection by C. Leishmania injecting sporoplasm (infectious material) into a D. Balantidium host cell. Quick Quiz! 7-19 Laboratory Diagnosis The most important Cyclospora prevention step that Diagnosis of the different species of Microspo- can be taken is: (Objective 7-7C) ridia varies. Serologic tests are available for the A. Proper water treatment detection of some species. In addition, some B. Wearing shoes when walking in sandy soil species will grow in cell culture. A number of C. Insecticide treatment of mosquito breeding areas stains may be used to detect all or part of the D. Thoroughly cooking beef and pork. spore microscopically. Thin smears stained with trichrome or acid-fast stain may show the desired spores. Microsporidia stain gram- positive and show partial positive staining when Microsporidia treated with acid-fast stain or the histologic (mi’kro-spor-i’dee-uh) Common associated disease and condition TA B L E 7 - 8 Microsporidia Spore: Typical names: Microsporidia infection, microsporidial Characteristics at a Glance infection. Parameter Description Although it is classified as a protozoal disease by the World Health Organization, Microspo- Size 1-5 μm ridia’s phylogenetic placement has been resolved Other features Equipped with extruding polar filaments (or tubules) that within the Fungi as a result of DNA testing. initiate infection by injecting There are a number of genera and species of sporoplasm (infectious material) parasites that are members of the phylum Micro- into host cell sporidia. Three of the five genera known to cause  CHAPTER 7 Miscellaneous Protozoa 177 stain periodic acid-Schiff (PAS). Giemsa-stained Notes of Interest and New Trends biopsy material and fecal concentrate specimens readily show the spores. It is important to Persons infected with C. cayetanensis in addition note that speciation of the Microsporidia to Microsporidia have been reported and are requires the use of transmission electron micro- considered somewhat common. scopy. Molecular diagnostic methods are being In recent years, the United States Environmen- developed. tal Protection Agency (EPA) has listed Microspo- ridia in the EPA Candidate Contaminate List, deeming it an emerging water-borne pathogen Life Cycle Notes needing monitorial attention. Transmission of Microsporidia may be direct or Although Microsporidia infection in humans may involve an intermediate host. On entering mostly occurs in immunocompromised patients, the host, human infection is initiated when the the further spread of AIDS worldwide increases infective spores inject sporoplasm into a host our need to understand and manage Microspo- cell. A complex reproductive process occurs, new ridia for the near future. spores emerge, and additional cells typically become infected. Spores are dispersed into the Quick Quiz! 7-20 outside environment in the direct transmission cycle in the feces or urine, or by the death of the How do Microsporidia spores differ from other pro- host. In addition, the spores may be ingested by tozoan spores? (Objective 7-11) a carnivorous animal. A. Double outer wall B. Extruding polar filaments C. Cilia Epidemiology D. Pseudopods Cases of E. bieneusi infection have been reported Quick Quiz! 7-21 in AIDS patients from Haiti, Zambia, Uganda, the United Kingdom, the United States, and Of the following, which laboratory technique is the Netherlands. Although most documented required for species identification of Microsporidia? infections of Microsporidia parasites have (Objective 7-8) occurred in AIDS patients, cases in persons A. Giemsa-stained biopsy material with normal immune systems have also been B. Electron microscopy described. C. Fecal concentration D. PAS stain Clinical Symptoms Quick Quiz! 7-22 Microsporidial Infection. Patients suffering from infections with Microsporidia have been The life cycle of Microsporidia is a complex process in known to develop enteritis, keratoconjunctivitis, which both the infective and diagnostic stages are and myositis. Infections involving peritonitis and spores. (Objective 7-5) hepatitis have rarely occurred. A. True B. False Treatment Toxoplasma gondii Albendazole is recommended for the treatment (tock”so-plaz’muh/gon’dee-eye) of E. bieneusi; oral fumagillin is recommended as an alternative treatment. Albendazole plus Common associated disease and condition fumagillin eye drops are recommended for the names: Toxoplasmosis, congenital toxoplasmo- treatment of Nosema infection. sis, cerebral toxoplasmosis. 178 CHAPTER 7 Miscellaneous Protozoa Bradyzoites. Although there is evidence to Morphology support an antigenic difference, the typical There are only two morphologic forms of tro- bradyzoite basically has the same physical phozoites seen in humans, tachyzoites and brady- appearance as the tachyzoite, only smaller (see zoites. The infective form for humans is the Fig. 7-13; Table 7-10). These slow-growing oocyst. This form may be encountered on occa- viable forms gather in clusters inside a host cell, sion, especially where veterinary parasitologic develop a surrounding membrane, and form a techniques are performed. Thus, all three of these cyst in a variety of host tissues and muscles morphologic forms are discussed in this section. outside the intestinal tract. Such cysts may Oocyst. The typical infective form of Toxo- contain as few as 50 and up to as many as several plasma gondii, the oocyst, is similar in appear- thousand bradyzoites. A typical cyst measures ance to that of Isospora belli. The most notable from 12 to 100 μm in diameter. difference between the two organisms is that T. gondii is smaller. The round to slightly oval form Laboratory Diagnosis measures 10 to 15 μm long by 8 to 12 μm wide. The transparent oocyst contains two sporocysts, The primary means of diagnosing T. gondii infec- each with four sporozoites. The organism is bor- tions is analyzing blood samples using serologic dered by a clear, colorless, two-layered cell wall. Tachyzoites. The actively multiplying, cres- cent-shaped tachyzoites range in size from 3 to TA B L E 7 - 9 Toxoplasma gondii Tachyzoites: Typical 7 μm by 2 to 4 μm (Fig. 7-13; Table 7-9). One Characteristics at a Glance end of the organism often appears more rounded than the other end. Each tachyzoite is equipped Parameter Description with a single centrally located nucleus, sur- General comment Actively multiplying morphologic rounded by a cell membrane. A variety of other form organelles are present, including a mitochon- Size 3-7 × 2-4 μm drion and Golgi apparatus; however, these struc- Shape Crescent-shaped, often more tures are not readily visible. rounded on one end Number of nuclei One Tachyzoites Other features Contains a variety of organelles that are not readily visible and Bradyzoites Cyst containing TA B L E 7 - 1 0 Toxoplasma gondii Bradyzoites Bradyzoites: Typical Characteristies at a Glance Parameter Description Tachyzoite size range: 3-7 !m by 2-4 !m General comment Slow-growing morphologic form Size Smaller than tachyzoites Physical appearance Similar to that of the tachyzoites Other features Hundreds to thousands of bradyzoites enclose Cyst size range: themselves to form a cyst 12-100 !m that may measure FIGURE 7-13 Toxoplasma gondii tachyzoites and 12-100 μm in diameter bradyzoites.  CHAPTER 7 Miscellaneous Protozoa 179 test methods. The recommended test for the present via hand-to-mouth transmission. Cat determination of immunoglobulin M (IgM) anti- litter boxes, as well as children’s sandboxes, are bodies present in congenital infections is the the primary sources of such infected fecal matter. double-sandwich ELISA method. Both IgM and The second route involves human ingestion of IgG levels may be determined using the indirect contaminated undercooked meat from cattle, fluorescent antibody (IFA) test. Additional sero- pigs, or sheep. These animals, as well as a wide logic tests for the IgG antibody include the indi- variety of other animals, may contract T. gondii rect hemagglutination (IHA) test and ELISA. The during feeding by ingesting infective oocysts actual demonstration of T. gondii trophozoites present in cat feces. The infective sporozoites are (tachyzoites) and cysts (filled with bradyzoites) released following ingestion and follow the same involves tedious microscopic examination of cycle in these animals as they do in the natural infected human tissue samples or the inoculation intermediate hosts. The resulting cysts form in of laboratory animals. The time and effort to the animal muscle and the parasites within them perform such testing is, in most cases, not may remain viable for years. practical. The third means of human T. gondii transfer is transplacental infection. This occurs when an asymptomatic infection in a mother is unknow- Life Cycle Notes ingly transmitted to her unborn fetus. In response Although the natural life cycle of T. gondii is to the parasite, the mother produces IgG, which relatively simple, the accidental cycle may involve also crosses the placenta and may appear for a number of animals and humans. The definitive several months in the circulation of the fetus/ host in the T. gondii life cycle is the cat (or other newborn. In addition, the mother produces IgM, felines). On ingestion of T. gondii cysts present which does not cross the placenta. However, the in the brain or muscle tissue of contaminated infant may demonstrate anti–T. gondii IgM from mice or rats, the enclosed bradyzoites are released birth to several months old. in the cat and quickly transform into tachyzoites. Although extremely rare, the fourth route of Both sexual and asexual reproduction occur in human infection occurs when contaminated the gut of the cat. The sexual cycle results in the blood is transfused into an uninfected person. production of immature oocysts, which are ulti- Once inside the human, T. gondii tachyzoites mately shed in the stool. The oocysts complete emerge from the ingested cyst and begin to grow their maturation in the outside environment, a and divide rapidly. The tachyzoite form is respon- process that typically takes from 1 to 5 days. sible for the tissue damage and initial infection. Rodents, particularly mice and rats, serve as the The tachyzoites migrate to a number of tissues intermediate hosts, ingesting the infected mature and organs, including the brain, where cysts T. gondii oocysts while foraging for food. The filled with bradyzoites then form. sporozoites emerge from the mature oocyst and rapidly convert into actively growing tachyzoites in the intestinal epithelium of the rodent. These Epidemiology tachyzoites migrate into the brain or muscle of the intermediate host, where they form cysts T. gondii is found worldwide, primarily because filled with bradyzoites. The cat becomes infected such a large variety of animals may harbor the on ingestion of a contaminated rodent and the organism. It appears from information collected cycle repeats itself. to date that no population is exempt from the Human infection of T. gondii is accidental and possibility of contracting T. gondii. One of the may be initiated in four ways. One route occurs most important populations at risk for con- when humans are in contact with infected cat tracting this parasite is individuals suffering feces and subsequently ingest the mature oocysts from AIDS. 180 CHAPTER 7 Miscellaneous Protozoa There are several epidemiologic consider- gondii are mild and mimic those seen in cases of ations worth noting. First, it has been docu- infectious mononucleosis. This acute form of the mented that T. gondii infections occur in 15% disease is characterized by fatigue, lymphadeni- to 20% of the population in the United States. tis, chills, fever, headache, and myalgia. In addi- Second, infection caused by the consumption of tion to the symptoms mentioned, chronic disease undercooked meat and its juices by women and sufferers may develop a maculopapular rash as their children in Paris was reported in 93% (the well as show evidence of encephalomyelitis, highest recorded rate) and 50%, respectively, of myocarditis, and/or hepatitis. Retinochoroiditis the local population. Third, there have been an with subsequent blindness has been known to estimated 4000 infants born with transplacen- occur on rare occasions. tally acquired T. gondii infections in the United Congenital Toxoplasmosis. This severe and States each year. Fourth, the T. gondii mature often fatal condition occurs in approximately oocysts are incredibly hardy and can survive for one to five of every 1000 pregnancies. Transmis- long periods under less than optimal conditions. sion of the disease occurs when the fetus is In the state of Kansas, it was documented infected (via transplacental means) unknowingly that these oocysts survived up to 18 months in by its asymptomatic infected mother. The degree the outside environment, withstanding two of severity of the resulting disease varies and is winter seasons. Finally, human infections in the dependent on two factors: (1) antibody protec- United States are usually acquired by hand-to- tion from the mother; and (2) the age of the fetus mouth contamination of infected oocysts in cat at the time of infection. Mild infections occur feces, ingesting contaminated meat, or transpla- occasionally and result in what appears to be a centally during pregnancy. As noted, transfusion- complete recovery. Unfortunately, these patients acquired T. gondii may also occur; however, it is may develop a subsequent retinochoroiditis years extremely rare. after the initial infection. Typical symptoms in an There are numerous other reports of T. gondii infected child include hydrocephaly, microceph- infections that have occurred worldwide. aly, intracerebral calcification, chorioretinitis, However, an in-depth discussion of these epide- convulsions, and psychomotor disturbances. miologic findings is beyond the scope of this Most of these infections ultimately result in chapter. mental retardation, severe visual impairment, or blindness. There are a number of important documented Clinical Symptoms statistics regarding the symptoms that infants Asymptomatic. Many patients infected with born with T. gondii infection are likely to T. gondii remain asymptomatic, especially chil- experience. dren who have passed the neonatal stage of their It is estimated that 5% to 15% of infected lives. Although well adapted to its surroundings, infants will die as a result of toxoplasmosis T. gondii appears to only cause disease in humans infection. when one or more of the following conditions Another 10% to 13% of infected infants have been met: (1) a virulent strain of the organ- will most likely develop moderate to severe ism has entered the body; (2) the host is in a handicaps. particularly susceptible state (e.g., those suffering Severe eye and brain damage will occur in from AIDS); and (3) the specific site of the para- approximately 8% to 10% of infected infants. site in the human body is such that tissue destruc- The remaining 58% to 72% of infected infants tion is likely to occur. will most likely be asymptomatic at birth. Toxoplasmosis: General Symptoms. Although Although the mechanism of this infection severe symptoms may be noted, the typical symp- reactivation is unknown, a small percentage of toms experienced by individuals infected with T. these infants will develop mental retardation or t ahir99-VRG & vip.p ersianss.ir  CHAPTER 7 Miscellaneous Protozoa 181 retinochoroiditis later in life, usually as children spiramycin. Spiramycin is used in Europe, Canada, or young adults. and Mexico but is still considered an experimen- Toxoplasmosis in Immunocompromised Patients. tal drug in the United States. However, it can be Patients immunosuppressed because of organ obtained by special permission from the FDA for transplantation or the presence of neoplastic toxoplasmosis in the first trimester of pregnancy. disease, such as Hodgkin’s lymphoma, have long Corticosteroids used as an anti-inflammatory been known to contract toxoplasmosis as an agent may also be of value. Folinic acid (leucovo- opportunistic infection. It is important to note, rin) may be administered to infected AIDS patients particularly in patients needing blood transfu- to counteract the bone marrow suppression sions, the importance of screening potential donor caused by pyrimethamine. An effective drug, par- units for toxoplasmosis prior to transfusion. ticularly for the treatment of toxoplasmic enceph- Cerebral Toxoplasmosis in AIDS Patients. A alitis in patients with AIDS, is atovaquone. focus of attention has been the association of T. gondii and AIDS patients. Since the 1980s, toxo- Prevention and Control plasmic encephalitis has been considered a signifi- cant complication in these individuals. In fact, one There are a number of measures that must be of the first apparent clinical symptoms of patients implemented and enforced to prevent the spread with AIDS may be that of central nervous system of T. gondii infections. One is the avoidance of (CNS) involvement by T. gondii. AIDS patients contact with cat feces. This may be accomplished suffering from infection with T. gondii may expe- by wearing protective gloves when cleaning out rience early symptoms of headache, fever, altered a cat litter box, disinfecting the litter box with mental status (including confusion), and lethargy. boiling water, and thorough hand washing after- Subsequent focal neurologic deficits, brain lesions, ward. In addition, placing a protective cover over and convulsions usually develop. children’s sandboxes when not in use will keep The T. gondii organisms do not spread into cats from using them as litter boxes. other organs of the body but rather stay confined T. gondii infections may also be prevented by within the CNS. A rise in spinal fluid IgG anti- the avoidance of ingesting contaminated meat. body levels is diagnostic, as is the demonstration This may be accomplished by thorough hand of tachyzoites in the cerebrospinal fluid (CSF) on washing after handling contaminated meat, as microscopic examination. The serum IgG level in well as the avoidance of tasting raw meat. In these patients does not respond, nor does that addition, all meat should be thoroughly cooked of the CSF. Most infected patients do not have prior to human consumption. Additional T. serum levels of IgM antibodies. The lack of gondii prevention and control measures include serum IgM coupled with the lack of change keeping cats away from potentially infective in serum IgG levels in these patients suggests that rodents, feeding cats only dry or cooked canned their infections occurred because of a reactiva- cat food, and/or not having cats at all. tion of a chronic latent infection and not because All humans should practice these preventive of an acquired primary infection. measures. However, pregnant women should be especially cautious around cat feces and contami- nated meat because of the possibility of contract- Treatment ing toxoplasmosis and transferring the disease to The treatment of choice for symptomatic their unborn children. cases of T. gondii infection consists of a combina- tion of trisulfapyrimidines and pyrimethamine Notes of Interest and New Trends (Daraprim). It is important to note that infected pregnant women should not be given pyrime- In 1908, the African rodent Ctenodactylus gondii thamine. An acceptable alternative drug is was the first animal discovered with T. t ahir99-VRG & vip.p ersianss.ir 182 CHAPTER 7 Miscellaneous Protozoa gondii—hence, the name. It was not until 1939 Quick Quiz! 7-25 that T. gondii was recognized as a cause of trans- placental infections. In which geographic area would you be likely to find Techniques using the polymerase chain reac- Toxoplasma gondii? (Objective 7-2) tion (PCR) assay have been developed. Successful A. Tropics results were achieved when analyzing samples of B. Africa venous blood from AIDS patients and amniotic C. United States fluid from pregnant women. D. All of the above Research has been conducted designed to detect specific IgE in patients suffering from toxoplasmosis. Known as an immunocapture Pneumocystis jiroveci assay, samples of CSF, fetal blood, umbilical cord (Pneumocystis carinii) blood, sera, and amniotic fluid were used. This (new-moe”sis-tis/kah-reye”nee-eye) technique is easy to perform and may prove to be helpful in diagnosing toxoplasmosis, particu- Common associated disease and condition larly in pregnant women. names: Pneumocystosis, atypical interstitial T. gondii tachyzoites, both invasive and plasma cell pneumonia. intracellular, have been successfully demon- strated in AIDS patients suffering from pulmo-