Henry's Clinical Diagnosis and Management by Laboratory Methods PDF

PART 7 C Figure 65.5—cont’d C, Plasmodium falciparum. 1, Very young ring form (early trophozoite). 2, Double infection of single cell with young trophozoites—one a “marginal” or “applique” form, the other a “signet ring” form. 3 and 4, Young trophozoites showing double ch...

PART 7 C Figure 65.5—cont’d C, Plasmodium falciparum. 1, Very young ring form (early trophozoite). 2, Double infection of single cell with young trophozoites—one a “marginal” or “applique” form, the other a “signet ring” form. 3 and 4, Young trophozoites showing double chromatin dots (“headphone” forms). 5 to 7, Developing trophozoite forms. 8, Three late-­stage trophozoites in one cell. Although these forms are not generally seen in freshly obtained peripheral blood with P. falciparum infection due to sequestration in the peripheral capillaries, they may be seen when there is a delay between collection and examination. 9, Trophozoite showing pigment in a cell containing Maurer dots. These intracytoplasmic inclusions can be differentiated from the stippling seen with P. vivax and P. ovale infection in that the Maurer dots are larger and fewer in number. 10 and 11, Two late-­stage trophozoites in each of two cells, showing variation in forms that parasites may assume. 12, Late-­stage trophozoite showing haze of pigment through- out cytoplasm. Maurer dots are also present. 13, Unusual slender forms of P. vivax. 14, Mature trophozoite, showing clumped pigment. 15, Parasite in the process of initial chromatin division to form a schizont (schizonts are not usually found in the peripheral circulation in P. falciparum infection). 16 to 19, Various phases of development of the schizont (early segmenting schizonts). 20, Mature schizont. 21 to 24, Successive forms in development of the gametocyte—usually not found in the peripheral circulation. 25, Immature macrogametocyte. 26, Mature macrogametocyte. 27, Immature microgametocyte. 28, Mature microgametocyte. (Courtesy of United States Public Health Service, National Institutes of Health.) Continued 1307 CHAPTER 65 MEDICAL PARASITOLOGY D Figure 65.5—cont’d D, Plasmodium ovale. 1, Young ring form (early trophozoite). 2 to 5, Older ring-­shaped trophozoites. 6 to 8, Older trophozoites, some with ameboid features reminiscent of P. vivax infection. Generally, P. ovale late-­stage trophozoites have a more compact appearance than those of P. vivax. 9, 11, and 12, Doubly infected cells, trophozoites. 10, Doubly infected cell, young gametocytes. 13, Early segmenting schizont with only 2 merozoites. 14 to 19, Schizonts, progressive stages. 20, Mature gameto- cyte. (Free translation of legend accompanying original plate in Guide pratique d’examen microscopique du sang appliqué au diagnostic du paludisme by Georges Villain. Reproduced with permission from Biologie Medicale Supplement, 1935. Courtesy of Aimee Wilcox, National Institutes of Health Bulletin No. 180, United States Public Health Service.) detecting B. microti antibodies in human plasma samples as well as the Imu- periodicity. Investigation of an outbreak caused by B. microti on Nantucket gen Babesia microti nucleic acid test for detection of DNA in whole blood Island in New England showed that some symptomatic patients harbored (https://www.fda.gov/news-­events/press-­announcements/fda-­approves-­ the parasite for months and others showed serologic evidence of infection first-­tests-­screen-­tickborne-­parasite-­whole-­blood-­and-­plasma-­protect-­us-­ without a history of clinical disease. Other evidence indicates that chronic blood-­supply). subclinical infections may not be uncommon (Westblade et al., 2017). The spectrum of babesiosis varies from latent, subclinical infection to While asymptomatic infections in immunocompetent patients generally fulminant, hemolytic disease. Fatalities have been reported, especially in do not require treatment, symptomatic patients, including those at risk for splenectomized or immunocompromised individuals. Immunocompetent severe disease (e.g., asplenic individuals), are generally treated using atova- persons may experience symptoms similar to those of malaria, includ- quone plus azithromycin or clindamycin plus quinine (Drugs for Parasitic ing fever, chills, malaise, and anemia, although without recognizable Infections, 2013). 1308  PART 7 A B C D E F Figure 65.6 A, Plasmodium falciparum in Giemsa-­stained thick blood film showing thin, delicate rings and a “headphone” form (arrow) (1000×). B, Plasmodium falciparum in Giemsa-­stained thin blood film; note high parasitemia, multiply infected red blood cells, and delicate ring forms with appliqué and headphone forms (1000×). C, Babesia microti in Giemsa-­stained thin blood film; note high parasitemia, multiply infected red blood cells with irregular forms, and Maltese cross configuration (arrow) (1000×). D, Trypanosoma brucei in thin blood film; note nucleus, small kinetoplast (arrow), anterior flagellum, and undulating membrane (Giemsa stain; 1000×). E, Trypanosoma cruzi in thin blood film; note nucleus, large kinetoplast (arrow), anterior flagellum, and undulating membrane (Giemsa stain; 1000×). F, Trypanosoma cruzi amastigotes in a cardiac biopsy; individual amastigotes with a small nucleus and rod-­shaped kinetoplast (arrow) are visible (hematoxylin and eosin [H&E], 1000×). Babesia parasites multiply in erythrocytes by binary fission, producing suggest Babesia infection. NAATs for Babesia species are available from morphologically indistinguishable trophozoites and gametes. Although the CDC on referral from state health departments and from some com- trophozoites of many species may be highly variable in size and shape, mercial laboratories. Serologic tests (e.g., IFA) may also be available but those of B. microti usually appear as delicate ring forms that may be eas- are generally not useful for detection of acute disease. However, they may ily confused with those of malarial parasites, especially P. falciparum (see be useful for screening blood donors in endemic settings. Serology tests Fig. 65.6C) (Westblade et al., 2017). Babesia can be differentiated from for malaria are negative in babesiosis, although patients with malaria may those of malarial parasites by the presence of a tetrad (Maltese cross) for- cross-­react in the Babesia serologies (Westblade et al., 2017; Pritt, 2019). mation of the merozoites and the absence of large ameboid trophozoites and morphologically distinguishable gametocytes; extracellular forms may be seen in heavy infections. Also, Babesia species usually have a heteroge- HEMOFLAGELLATES neous appearance with round, oval, spindled, and “racket” forms coexist- The hemoflagellates of humans and animals are members of the group ing on the same peripheral blood smear. Finally, Babesia-­infected cells lack Kinetoplastea and are characterized by the presence of a kinetoplast, a com- hemozoin pigment, which is present in Plasmodium-­infected cells. History plex of abundant circular DNA within a large mitochondrion, which can of residence in or travel to endemic areas, or of a recent tick bite, might be seen by light microscopy when treated with Giemsa stain. Two genera 1309 CHAPTER 65 MEDICAL PARASITOLOGY G H I Figure 65.6—cont’d G, Leishmania sp. amastigotes in an impression smear of lymph node (Giemsa stain; 1000×). H, Pseudocyst (left) and free tachyzoites (arrows) of Toxoplasma gondii in brain tissue (H&E; 1000×). I, Intracellular and free T. gondii tachyzoites in an impression smear of brain tissue from an immunocompromised patient with reactivated disease (Giemsa; 1000×). important in human disease are Trypanosoma and Leishmania. Members of a small kinetoplast. Those of T. cruzi are somewhat shorter (20 μm) and both genera are transmitted by arthropod vectors and have animal hosts display a larger kinetoplast. Also, dividing forms may be seen in blood films that serve as reservoirs. with T. brucei but not T. cruzi, as the latter replicates only in the amastigote Kinetoplastids assume different morphologic forms depending on their stage in the human host. presence in vertebrate hosts, including humans, or in their insect vectors (Fig. 65.7). The amastigote stage is spherical, 2 to 5 μm in diameter, and African Trypanosomiasis displays a nucleus and kinetoplast. By definition, an external flagellum is In equatorial Africa, parasites of the T. brucei group infect both animals and lacking, although an axoneme (the intracellular portion of the flagellum) is humans and are transmitted by the bite of tsetse flies in the genus Glossina. apparent at the ultrastructural level. Amastigotes may be found in human Multiplication of organisms at the bite site often produces a transient chan- or animal hosts infected with T. cruzi or Leishmania spp., where they mul- cre. East African trypanosomiasis is caused by T. brucei rhodesiense, which tiply exclusively within cells, but not with T. brucei. The promastigote is has a number of animal reservoir hosts. The disease is characterized by an elongated and slender organism with a central nucleus, an anteriorly a rapidly progressive acute febrile illness with lymphadenopathy. Patients located kinetoplast and axoneme, and a free flagellum extending from the may die before central nervous system (CNS) involvement is prominent. anterior end. This stage occurs in the insect vectors of Leishmania and is The infection in western Africa is caused by T. brucei gambiense, which the stage detected in culture but is not detected in clinical specimens unless is responsible for classic African sleeping sickness. The disease has a more there is a substantial delay in processing. The epimastigote is similar to the chronic course that begins with intermittent fevers, night sweats, and mal- promastigote, but the kinetoplast is found closer to the nucleus and has a aise. Lymphadenopathy, especially of the posterior cervical lymph nodes small undulating membrane that becomes a free flagellum. All species of (Winterbottom sign), may be pronounced. Involvement of the CNS Trypanosoma that infect humans assume an epimastigote stage in the insect becomes prominent with time. Somnolence, confusion, and fatigue prog- vector or in culture. Neither the promastigote nor epimastigote are seen ress, leading to stupor, coma, and eventual death. Humans are the primary in humans. In the trypomastigote, the kinetoplast is found at the poste- reservoir for this disease (Bruckner & Labarca, 2019). rior end and the flagellum forms an undulating membrane that extends the Treatment varies by stage of infection (hemolymphatic vs. late disease length of the cell, emerging as a free flagellum at the anterior end. Trypo- with CNS involvement) and is potentially toxic. The organoarsenic com- mastigote forms occur predominantly in the bloodstream of mammalian pound melarsoprol is the only drug available for treating T. b. rhodesiense hosts infected with various Trypanosoma spp. Infectious stages found in late-­stage disease and carries an associated 1% to 5% mortality rate (Drugs appropriate insect vectors following transformation from the epimastigote for Parasitic Infections, 2013; WHO, 2019). Consultation with the CDC form are known as metacyclic trypomastigotes. or other public health experts is indicated in nonendemic settings. The diagnosis is suspected on the basis of geographic history and clini- Trypanosoma spp. (African and American cal findings. Patients show high total IgM levels in blood and cerebrospi- Trypanosomiasis) nal fluid (CSF). Pleocytosis occurs with 50 to 500 mononuclear cells per Infections with trypanosomes include those caused by Trypanosoma bru- microliter in CSF. The diagnosis is established by demonstrating the para- cei (African trypanosomiasis) and T. cruzi (American trypanosomiasis, or sites on thick and thin films of peripheral blood, buffy coat preparations, or Chagas disease). Both are of great importance in endemic areas. A third aspirates of lymph nodes or bone marrow, or in spun CSF that is stained species, Trypanosoma rangeli, has been described in humans in the Americas with Giemsa (Bruckner & Labarca, 2019). Culture or animal inoculation but does not cause clinical illness. Bloodstream trypomastigotes of the T. may be helpful if it is available; a number of molecular methods have also brucei group (see Fig. 65.6D) are up to 30 μm long with graceful curves and been described. 1310  Epimastigote Trypomastigote Promastigote Flagellum PART 7 Flagellum Amastigote Undulating Axoneme membrane Basal body Axoneme Kinetoplast Nucleus Basal body Kinetoplast Nucleus Nucleus Granules Undulating membrane Axoneme Basal body Figure 65.7 Morphology of hemoflagellates commonly Kinetoplast infecting humans. American Trypanosomiasis Diagnosis of Chagas disease can be challenging. The trypomastigotes American trypanosomiasis, or Chagas disease, is caused by T. cruzi. In its (Fig. 65.6E) can be observed in peripheral blood or CSF only during the sylvatic form, the parasite occurs in the United States, Mexico, Central acute stage of the disease or during reactivation. Amastigotes can also America, and most of South America. Human infections are common in be identified in heart biopsies (Fig. 65.6 F), although this method is not parts of Mexico and Central and South America, where they are transmit- commonly employed. Molecular diagnosis may be employed when mor- ted by kissing bugs in the family Reduviidae. In contrast, only rare cases phologic diagnosis is inappropriate; examples of such clinical scenarios of locally acquired Chagas have been documented in the United States. include (1) a person with a bug bite who has emigrated from or returned However, Chagas is now recognized as an important parasite in the United from an endemic area within 2 months, (2) monitoring organ transplant States due to the large number of immigrants that the country receives from recipients after initial serologic testing, (3) laboratory accidents (e.g., endemic areas. As such, it has been designated as an NPI targeted for public accidental inoculation), and (4) suspected congenital cases. Often, more health action (CDC, 2018b). The CDC and WHO estimate that more than than one molecular assay is performed as different assays have differ- 8 million individuals are infected worldwide and that greater than 300,000 ent molecular targets (Qvarnstrom et al., 2012). Diagnosis of chronic infected individuals currently live in the United States (CDC, 2018b). Chagas disease is best achieved by antibody detection. Other clinical Genera and species of reduviid bugs involved in transmission vary from scenarios that may warrant antibody detection include (1) screening one country to another and among different ecologic niches. Some redu- of blood and organ donors, (2) symptomatic patients with appropriate viids are responsible for maintaining the sylvatic cycle in animal reservoirs, travel or exposure history, (3) initial transplant recipients with appro- whereas others are adapted to an anthropophilic life in which they infest priate epidemiologic history or who received donated organs from an poorly constructed houses, usually in rural areas. At the time of feeding, individual with appropriate epidemiologic history, and (4) possible con- the reduviid bug defecates. The bug feces contain infective trypomasti- genital cases. No single serologic assay is sensitive and specific enough to gotes that, as a result of scratching or rubbing, enter the body at the bite be relied on alone. Therefore, per current recommended guidelines and site or through intact mucosa of the mouth or conjunctiva. Infective forms the CDC, serologic confirmation of chronic T. cruzi infection requires actively enter nearby tissue cells, where they transform into dividing amas- reactivity on two tests using two different methodologies and/or two tigotes. When the infected cell is filled with amastigotes, transformation different T. cruzi antigen preparations. When results are discordant, a to trypomastigotes occurs, followed by cell rupture. Trypomastigotes are testing by a third assay is recommended to resolve the initial results or released into the peripheral blood and reach distant tissues, where they repeat testing on a new sample may be required. The CDC performs transform into amastigotes and start the reproductive cycle de novo. Other an ELISA (Chagatest, Wiener Laboratories, Rosario, Argentina) and important means of infection are vertical transmission, blood transfusion, a Trypomastigote-­ Excreted Secreted Antigen (TESA) immunoblot organ transplantation from an infected donor, and, rarely, laboratory acci- (LDT, CDC) for primary testing and an LDT immunofluorescence dent and through ingestion of contaminated food or drink (CDC, 2018b). assay (IFA) for discordant results. A TESA blot can also be obtained Given the risk of transmission via blood transfusion, blood donor units are commercially (BioMérieux, Rio de Janeiro, Brazil) (Bern et al., 2007; routinely screened in the United States for T. cruzi using FDA-­approved Afonso et al., 2012). In endemic areas, xenodiagnosis (examination of serologic tests. the gut contents of laboratory-­raised reduviids that have been allowed to Chagas disease may cause acute or chronic infection. Acute disease is feed on a patient) may be used. In the chronic stage, serodiagnosis is the most common in children younger than 5 years of age and is characterized method of choice. by malaise, chills, fever, hepatosplenomegaly, and myocarditis. Swelling Management and treatment of Chagas disease is complex and varies of the tissues around the eye (Romaña sign) may be present if inocula- with the age of the patient and stage of infection (CDC, 2018b; Drugs tion of the organisms occurs on the face. Swelling of tissues at other loca- for Parasitic Infections, 2013). Antiparasitic therapy using nifurtimox or tions following the bite of an infected reduviid is called a chagoma. In older benznidazole is indicated for all cases of acute and reactivated disease and individuals, the acute course is milder and often asymptomatic, and the is also recommended for cases of chronic infection in children ≤18 years of patient remains infected for life. Chronic manifestations of the infection, age. Antiparasitic treatment is also strongly recommended for adults up to including megaesophagus, megacolon, and alterations in the conduction 50 years old with chronic disease in which advanced-­stage cardiac involve- system of the heart, are related to destruction of the effector cells of the ment is not present. The potential benefits and risks of treatment for older parasympathetic system by autoantibodies. Infection can be transmit- individuals and those with advanced cardiomyopathy must be individually ted by blood transfusion, and quiescent infections may be exacerbated by weighed prior to offering therapy (CDC, 2018b; Drugs for Parasitic Infec- immunosuppression. tions, 2013). 1311 CHAPTER 65 MEDICAL PARASITOLOGY Leishmania spp. (leishmaniasis) Operation Desert Storm (Magill et al., 1993). In some areas, humans may Leishmaniasis is a disease of the reticuloendothelial system caused by serve as the disease reservoir, although a variety of animals, including dogs kinetoplastid protozoa of the genus Leishmania. All species that infect and cats, usually assume this role. humans have animal reservoirs and are transmitted by sand flies belong- The infection is usually benign and often subclinical, although some ing to the genera Phlebotomus in the Old World and Lutzomyia in the New individuals, especially young children and malnourished individuals, have World. The parasites assume the amastigote form in mammalian hosts and marked involvement of the viscera, especially liver, spleen, bone marrow, the promastigote form in insect vectors. Species of Leishmania cannot be and lymph nodes. In some cases, death occurs after months to years unless differentiated by examination of amastigotes or promastigotes. Leishmani- it is treated appropriately. The infection is called kala-­azar in India in ref- asis may assume many different clinical forms; cutaneous, mucocutaneous, erence to the darkening of the skin. Treatment is liposomal amphotericin and visceral diseases are best known. The form and severity of disease vary B, sodium stibogluconate, meglumine antimoniate, miltefosine, ampho- with the infecting species, the particular host’s immune status, and prior tericin, or paromomycin (Drugs for Parasitic Infections, 2013). Visceral exposure (Bruckner & Labarca, 2019). leishmaniasis is an opportunistic infection in individuals with concurrent human immunodeficiency virus (HIV); the condition responds poorly to Cutaneous Leishmaniasis therapy in such circumstances (Bruckner & Labarca, 2019). Old World cutaneous leishmaniasis occurs in southern Europe, northern and eastern Africa, the Middle East, Iran, Afghanistan, India, and south- Diagnosis of Leishmaniasis ern Russia. Infections are caused by Leishmania tropica, Leishmania major, The diagnosis usually is established by visualization of amastigotes in and Leishmania aethiopica, although L. donovani and Leishmania infantum smears, imprints, or biopsies, or by growth of promastigotes in culture. In may also produce cutaneous lesions. The lesions of cutaneous leishmaniasis integumentary leishmaniasis, the border of the most active lesion should be may be variable in appearance, with “wet,” “dry,” and warty appearances. biopsied and the fresh biopsy should be used to make imprints. The CDC Locally, they may be referred to by a variety of names such as oriental sore, provides instructions for specimen collection at https://www.cdc.gov/para Aleppo boil, desert boil, and Delhi boil. Leishmania tropica produces the sites/leishmaniasis/resources/pdf/cdc_diagnosis_guide_leishmaniasis_201 “urban” dry ulcer, which is more long-­lived than the “rural” wet ulcer of L. 6.pdf. Both the imprints and smears should be stained with Giemsa. Biop- major. Ulcers caused by these species usually develop on an exposed area of sies should be examined by histopathology and/or submitted for culture. the body and heal spontaneously. Infection produces long-­lasting immu- Specimens that may be submitted when visceral leishmaniasis is suspected nity. L. tropica may become viscerotropic, as was demonstrated in mili- include buffy coat preparations, lymph node and bone marrow aspirates, tary personnel who participated in Operation Desert Storm (Magill et al., spleen and liver biopsies, and antibody detection (Garcia, 2016). 1993). Leishmania aethiopica causes a more aggressive cutaneous infection, A culture is desirable because it is more sensitive than microscopic which in some individuals metastasizes to produce mucosal lesions or dif- examination and allows determination of the species or subspecies, a prac- fuse cutaneous leishmaniasis, the latter of which is characterized by mul- tice that may help in clinical management of the patient. Biopsy or aspirate tiple skin nodules resembling lepromatous leprosy. specimens collected aseptically are cultured in Novy-­MacNeal-­Nicolle Cutaneous leishmaniasis of the New World is caused by many spe- medium or in Schneider’s Drosophila medium supplemented with fetal calf cies, including Leishmania mexicana, Leishmania braziliensis, Leishmania serum. Cultures usually begin to show promastigotes in 2 to 5 days but amazonensis, Leishmania venezuelensis, Leishmania peruviana, Leishmania should be held for 4 weeks. The CDC provides culture collection kits after panamensis, and Leishmania guyanensis, among others (Bruckner & Labarca, consultation and will perform culture and species identification using PCR 2019). Lesions produced by L. mexicana often involve the earlobe (Chiclero and sequencing analysis (de Almeida et al., 2011). ulcer), are self-­limiting, and are not known to metastasize to the mucosa. Amastigotes found in imprints, smears, and tissue sections are rec- However, L. mexicana and L. amazonensis may produce diffuse cutaneous ognized by their size (2–4 μm) and the presence of delicate cytoplasm, a lesions similar to those produced by L. aethiopica. A focus of cutaneous nucleus, and a kinetoplast (see Fig. 65.6G). In tissue sections, they may leishmaniasis exists in the southern part of Texas, where infections are appear smaller because of shrinkage during fixation. Amastigotes must be caused by one or more species (McIlwee et al., 2018). Leishmania peruviana, differentiated from other intracellular organisms, including yeast cells of which has been found on the western slopes of the Peruvian Andes, causes Histoplasma capsulatum and trophozoites of T. gondii. Leishmania spp. have an infection called uta, a benign cutaneous lesion that occurs predomi- a kinetoplast and do not stain with Gomori methenamine silver (GMS) or nantly in children. L. peruviana is acquired in the home, where the main periodic acid–Schiff (PAS). In contrast, Histoplasma lack the kinetoplast, reservoirs are domestic dogs. This epidemiologic situation contrasts with and the cell wall stains with PAS and GMS. According to one study (Weigle other cutaneous leishmaniases, which usually are acquired in forests and et al., 1987), the sensitivity of histologic sections stained with hematoxylin have wild animals as reservoir hosts. Treatment of cutaneous leishmani- and eosin (H&E) is 14%; imprints 19%, cultures 58%, and all methods asis is based on the causative species and location and extent of disease. In combined, 67%. It should be noted that the amastigotes of Leishmania spp. endemic settings outside of the Americas, where there is no risk of mucosal and T. cruzi are morphologically indistinguishable. dissemination, cosmetically unimportant lesions may be treated topically (e.g., cryotherapy) or allowed to self-­heal. For individuals at risk of aggres- sive, disfiguring, or disseminated disease, systemic therapies with sodium TOXOPLASMA GONDII (TOXOPLASMOSIS) stibogluconate, meglumine antimoniate, miltefosine, or paromomycin may Toxoplasma gondii is a protozoan parasite of the Apicomplexa clade that be used (Drugs for Parasitic Infections, 2013). Topical paromomycin may has a worldwide distribution in humans and in domestic and wild animals, also be used. especially carnivores. Infection in immunocompetent persons is generally asymptomatic or mild, but immunocompromised patients may experience Mucocutaneous (Mucosal) Leishmaniasis serious complications. Infection in utero may result in serious congenital Mucocutaneous leishmaniasis (espundia) is caused primarily by L. brazil- infection with sequelae or stillbirth (McAuley & Singh, 2019). The CDC iensis and species in the Viannia subgenus, which produce typical cutaneous estimates that more than 40 million individuals in the United States alone lesions that generally are more aggressive, last longer, and often dissemi- are infected with this parasite and have identified it as one of five NPIs nate to mucous membranes, especially in the nasal, oral, or pharyngeal targeted for public health action (CDC, 2018b). areas. In these locations, they may produce disfiguring lesions secondary to The sexual stage in the life cycle of this coccidian parasite is completed erosion of soft tissues and cartilage. L braziliensis is distributed in Mexico in the intestinal epithelium of cats and other felines, which serve exclusively and Central and South America. Treatment is with sodium stibogluconate, as definitive hosts. During this enteroepithelial cycle, asexual schizogony meglumine antimoniate, amphotericin, or miltefosine (Drugs for Parasitic and sexual gametogony occur, leading to the development of immature Infections, 2013). oocysts that are passed in the feces. Oocysts mature to the infective stage (which contain two sporocysts with four sporozoites each) in the environ- Visceral Leishmaniasis ment in 2 to 21 days. Ingestion of infective oocysts may lead to infection Visceral leishmaniasis of the Old World occurs sporadically over a wide of a wide variety of susceptible vertebrate hosts in which actively growing geographic area and is caused by L. donovani or L. infantum. L. donovani trophozoites (tachyzoites) may infect any nucleated cells. Proliferation of predominates in Africa, India, and Asia, and L. infantum (syn. L. chagasi) tachyzoites results in cell death and injury to the host during acute infec- occurs in the Mediterranean region, Middle East, Central Asia, and Cen- tion. Once immunity has developed, the organisms form tissue cysts that tral and South America. New World visceral leishmaniasis is caused by L. may eventually contain hundreds or thousands of slowly growing bradyzo- chagasi and occurs sporadically throughout Central and South America. On ites. The presence of tissue cysts is characteristic of chronic infection. All occasion, some species that cause cutaneous disease have been responsible stages of the life cycle occur in felines, but only trophozoite and cyst stages for visceral disease, as demonstrated in some troops who participated in occur in humans and other intermediate hosts. 1312  Humans acquire infection with T. gondii by ingestion of inadequately are crescent-­shaped or oval, measuring approximately 3 × 7 μm; cysts mea- cooked meat, especially lamb or pork, that contains tissue cysts or by inges- sure up to 30 μm in diameter and are usually spherical, except in muscle tion of infective oocysts from material contaminated by cat feces. Out- fibers, where they appear elongate (McAuley & Singh, 2019) (see Figs. breaks have occurred from inhaling contaminated dust in an indoor riding 65.6H and 65.6I). stable (Teutch et al., 1979) and from drinking contaminated water or In recent years, PCR has been increasingly used to detect toxoplasmic unpasteurized goat’s milk (Benenson et al., 1982; Sacks et al., 1982; Bowie encephalitis, disseminated disease, and intrauterine infection. Testing is PART 7 et al., 1997). Transmission via blood transfusion, organ transplantation, available from most reference laboratories, the CDC, and select research and transplacentally to the developing fetus also can occur. laboratories. PCR is now an important component of testing for pregnant Most acute infections are asymptomatic or mimic other infectious dis- women, neonates, and immunocompromised hosts (McAuley & Singh, eases in which fever and lymphadenopathy are prominent, such as infec- 2019). tious mononucleosis and acute HIV infection. Congenital infection may occur when the mother develops acute infection during gestation. The risk for infection to the neonate is unrelated to the presence or absence OPPORTUNISTIC FREE-­LIVING AMEBAE of symptoms in the mother, but severity of infection depends on the stage Amebae of the genera Naegleria, Acanthamoeba, and Balamuthia are inhab- of gestation at which it is acquired. Intrauterine death, microcephaly, or itants of soil, water, and other environmental substrates, where they feed hydrocephaly with intracranial calcifications may develop if infection is on other microscopic organisms, especially bacteria and yeasts. All three acquired in the first half of pregnancy. Infections in the second half of genera have been associated with opportunistic infection of the CNS, and pregnancy are usually asymptomatic at birth, although fever, hepatospleno- Acanthamoeba spp. can also cause keratitis (Visvesvara et al., 2007; Cope megaly, and jaundice may appear. Chorioretinitis, psychomotor retarda- et al., 2019). There has also been a single report of Paravahlkampfia franci- tion, and convulsive disorders may appear months or years later (Montoya nae being detected in the CSF of a patient with primary amebic meningo- & Remington, 2008). Given the risks of congenital toxoplasmosis, expect- encephalitis (PAM) (Visvesvara, et al., 2009), and Sappinia pedata in a left ant mothers are advised against handling cat feces (e.g., cleaning the cat temporal lobe brain biopsy in a patient with amebic encephalitis (Cope litter box) if the cat spends times outdoors and eating undercooked meat. et al., 2019; Gelman et al., 2001; Qvarnstrom et al., 2009). In immunosuppressed individuals, especially those with acquired PAM, caused by the ameboflagellate Naegleria fowleri, typically affects immunodeficiency syndrome (AIDS), infection with T. gondii usually children and young adults who have been swimming, jumping, or diving in manifests with CNS involvement. Other possible clinical and pathologic warm freshwater lakes or pools, or people of any age by the improper use manifestations include pneumonitis, myocarditis, retinitis, pancreatitis, of nasal irrigation systems. The ameboflagellate enters the brain via the or orchitis (McAuley & Singh, 2019). Toxoplasmosis may be difficult to cribriform plate and olfactory bulbs and reaches the frontal lobes, where diagnose clinically and is often discovered at autopsy. These infections usu- it produces an acute hemorrhagic meningoencephalitis that is usually fatal ally result from reactivation of a latent infection acquired months or years within 1 week of onset of symptoms. The disease has an extremely poor before but occasionally result from a primary infection. Treatment is based prognosis, despite vigorous therapeutic intervention, including induc- on numerous factors, including the immune status of the host and form tion of therapeutic coma. Drugs that have been used with limited suc- of infection. Treatment is indicated for congenitally infected neonates cess include amphotericin B, rifampin, fluconazole, azithromycin, and and immunocompromised patients but not typically for acute infection in miltefosine (Drugs for Parasitic Infections, 2013). Antemortem diagnosis immunocompetent adults. Pyrimethamine is the standard therapy along is made occasionally by identifying typical trophozoites in CSF on direct with leucovorin to protect against bone marrow suppression (CDC, 2018b; wet mounts, in stained preparations (Fig. 65.8A), or in culture. However, Drugs for Parasitic Infections, 2013). Management during pregnancy varies diagnosis is usually established at autopsy examination by the finding of with treatment center. Spiramycin may be administered to prevent spread trophozoites (only) in tissue sections (Fig. 65.8B). Trophozoites measure to the fetus or when infection is diagnosed prior to 18 weeks’ gestation; 10 to 35 μm; have a large, round, central karyosome; and if exposed to however, pyrimethamine, sulfadiazine, and leucovorin are recommended warm distilled water, convert to flagellated forms in 1 to 2 hours. Cysts are when infection of the fetus is suspected or when diagnosed at or after 18 not seen in clinical specimens. Culture usually is performed on nonnutri- weeks’ gestation (CDC, 2018b; Drugs for Parasitic Infections, 2013). ent agar plates (1.5% agar, 0.5% sodium chloride, pH 6.6–7.0) seeded with Serology remains the primary approach in establishing a diagnosis of a lawn of heat-­killed or living Escherichia coli (Garcia, 2010, 2016). Amebae toxoplasmosis in immunocompetent hosts (Robert-­Gangneux & Dardé, ingest the bacteria, leaving tracks in the bacterial lawn, which may be seen 2012; Wilson & Nutman, 2015; McAuley & Singh, 2019). The Sabin-­ under low-­power magnification using reduced light. Feldman dye test and the IFA test are standards against which other meth- Granulomatous amebic meningoencephalitis (GAE) may be caused by ods are compared, although the former is performed in only a few centers. several species of Acanthamoeba, including A. castellani, A. culbertsoni, A. EIA tests are commercially available and generally provide results similar polyphaga, and A. astronyxis, among others (Marciano-­Cabral & Cabral, to those of the IFA. Antibodies appear in 1 to 2 weeks, and titers peak at 6 2003). It is usually a subacute or chronic opportunistic infection of chroni- to 8 weeks. Tests for IgM-­specific antibodies are especially useful for diag- cally ill, debilitated, and immunosuppressed individuals, leading to death nosis of congenital and acute infection, but knowledge of test limitations, weeks to months following onset of symptoms. Infection is thought to specifically the occurrence of false-­positive reactions, is extremely impor- spread hematogenously from primary foci in skin, the pharynx, or the tant. The persistence of IgM-­specific antibodies, sometimes for 1 year or respiratory tract. Systemic infections occur in individuals with AIDS and longer, also is problematic and must be interpreted in conjunction with may present as ulcerative skin lesions, subcutaneous abscesses, or erythem- IgG antibody results. Because many persons have had asymptomatic infec- atous nodules (Cope et al., 2019). Exposure to fresh water is not necessary tion, low IgG titers have little significance. Titers in patients with chronic because cysts of Acanthamoeba readily become airborne and may be recov- ocular infection may also be low. Immunocompromised patients, such as ered from the throat and nasal passages (Cope et al., 2019). The pathologic those with AIDS who have active Toxoplasma infection, almost always have reaction in tissues is necrosis with acute inflammation. Despite the name preexisting specific IgG antibodies, although titers may be low and IgM of the disease, granulomas are not commonly seen but may be present in antibodies are infrequently detected. As discussed in the Serologic Diag- immunocompetent hosts. Trophozoites, and less commonly cysts, are seen nosis section earlier in the chapter, IgG avidity testing may be useful for within brain parenchyma, skin, and, rarely, lung sections. Organisms are differentiating between acute and remote infection. Interpretation of IgG commonly clustered around blood vessels, reflecting their hematogenous and IgM antibody titers varies by test method and by manufacturer. The origin. Diagnosis usually is established at autopsy, but organisms may be laboratory performing the test should provide the necessary interpretive recognized in brain biopsies or recovered using the culture technique criteria (McAuley & Singh, 2019). described for Naegleria. Therapeutic drugs that have been used with some Diagnosis of toxoplasmosis may also be established by examination of success are pentamidine, sulfadiazine, flucytosine, fluconazole, and milt- tissues, blood, or body fluids (McAuley & Singh, 2019). Demonstration efosine (Drugs for Parasitic Infections, 2013). of tachyzoites or tissue cysts is definitive but may prove difficult to dem- Acanthamoeba trophozoites are somewhat larger than Naegleria, onstrate in H&E-­stained sections; fluorescence or immunohistochemical measuring 15 to 45 μm, and display needlelike filamentous projections stains, if available, are useful. Giemsa is good for staining smears of body from the cell known as acanthopodia. Cysts measure 10 to 25 μm and are fluids and tissue imprints. Organisms may be demonstrated by inoculating double-­walled, displaying a wrinkled outer wall (ectocyst) and a polygo- appropriate material into tissue culture or uninfected mice, although this nal, stellate, or round inner wall (endocyst). Identification to the species method is not widely available. Recovery in routine viral cultures also has level is problematic and reflects uncertainty as to the validity of the 18 been described but requires extended incubation. Isolation of organisms or more described species, although it is usually not required for clini- from blood or body fluid serves as evidence of acute infection, whereas cal management. Currently, genotyping is the preferred approach used in recovery from tissues may reflect chronic infection. In smears, tachyzoites differentiating types of Acanthamoeba (Marciano-­Cabral & Cabral, 2003). 1313 CHAPTER 65 MEDICAL PARASITOLOGY A B C D E F Figure 65.8 A, Naegleria fowleri trophozoites in the cerebrospinal fluid in a case of primary amebic meningoencephalitis (PAM). Note the pale nucleus with large karyosome (arrow) (Giemsa; 1000×). B, Naegleria fowleri trophozoites in an H&E-­stained brain section in a case of PAM; note the small nucleus with large central karyosome (arrow) (1000×). C, Acanthamoeba sp. trophozoites and cysts on an agar culture (1000×). D, Acanthamoeba sp. cysts (left) and trophozoite (right) in contact lens fluid in a case of acan- thamebic keratitis. There are abundant bacteria also present (H&E; 1000×). E, Double-­walled cysts of Acanthamoeba sp. within corneal stroma (H&E; 1000×). F, Trophozoites in brain tissue in a case of Balamuthia mandrillaris granulomatous amebic encephalitis. The small nucleus with central large karyosome is highlighted (arrow) (H&E; 400×). Immunofluorescence and immunoperoxidase techniques may prove useful cornea. Incomplete or infrequent disinfection and use of homemade saline in identifying and differentiating species and are available from the CDC and multipurpose solutions are known risk factors for acquiring the infec- (Cope et al., 2019). tion (Cope et al., 2019). The disease is characterized by development of a GAE may also be caused by the leptomyxid ameba, Balamuthia man- paracentral ring infiltrate of the corneal stroma, which progresses to ulcer- drillaris (Cope et al., 2019). Treatment and prognosis are similar to GAE ation and possible perforation, with loss of the eye. The infection may be caused by Acanthamoeba spp. Morphologically, Balamuthia cannot be eas- confused with fungal, bacterial, or herpetic keratitis but is characteristically ily differentiated from Acanthamoeba by routine histology, although dif- refractory to commonly used antimicrobials. Optimal treatment varies ferences may be detected at the ultrastructural level. These organisms based on the extent of disease, with topical biguanides and diamidines for are antigenically distinct and may be identified using specific monoclonal uncomplicated disease (Clark et al., 2012; Drugs for Parasitic Infections, or polyclonal antisera in DFA or immunoperoxidase assays (Cope et al., 2013). Keratoplasty is required in cases of extensive and refractory disease. 2019). Balamuthia does not grow on agar plates used for Naegleria and Diagnosis usually is established by demonstrating amebic trophozoites Acanthamoeba but can be recovered in tissue culture using mammalian cell or cysts in corneal scrapings or biopsies (Fig. 65.8E). A variety of perma- lines. nent stains can be used to highlight the organisms, including Giemsa, Acanthamoeba keratitis is an increasingly recognized painful infection PAS, and trichrome. Use of the fluorochrome Calcofluor white is espe- of the cornea that is most likely to occur in persons who use daily-­wear or cially helpful in recognizing amebic cysts (Garcia, 2016). While cultures extended-­wear soft contact lenses or who have experienced trauma to the (described earlier, Fig. 65.8C) provide increased sensitivity over staining 1314  Amebae Entamoeba Entamoeba Entamoeba Entamoeba Endolimax Iodamoeba Dientamoeba histolytica/dispar hartmanni coli polecki nana buetschlii fragilis PART 7 Trophozoite 10µm 10µm 10µm 10µm 10µm 10µm 10µm Cyst No cyst 10µm 10µm 10µm 10µm 10µm 10µm Figure 65.9 Amebae found in human stool specimens. (Dientamoeba fragilis is a flagellate but morphologically similar to amebae.) methods and are often available from clinical laboratories, the sensitivity degeneration. Formalin does not preserve trophozoites well; thus, parasites achieved by PCR may equal or exceed that of culture (Boggild et al., 2009). may be missed from formalin-­based preparations unless permanent stained smears are also prepared and examined. Therefore, as mentioned previ- ously, a complete stool examination should include examination of both a INTESTINAL AND UROGENITAL PROTOZOA concentrated wet prep and permanently stained slide. Protozoa inhabiting the intestinal tract of humans include amebae, flagel- lates, ciliates, and coccidia, many of which are considered nonpathogens. Microsporidia also inhabit the human intestinal tract. They were histori- INTESTINAL AMEBAE cally grouped with the intestinal protozoa but are now known to be highly Three genera of amebae inhabit the intestinal tract of humans: Entamoeba, specialized fungi and are discussed elsewhere. Infection rates vary widely Endolimax, and Iodamoeba. Cysts are ingested and excyst in the small intes- by population tested and the method employed. Most intestinal infections tine. Resulting trophozoites proliferate by binary fission in the lumen of with protozoa are thought to be acquired by fecal-­oral contamination the colon. Both cysts and trophozoites may be passed in feces, but only directly from food handlers or indirectly via contaminated water. mature cysts are infective. Entamoeba histolytica is the only ameba in which For most laboratorians, identification of intestinal protozoa can be one the trophozoites can invade tissues and cause clinical disease. of the more difficult aspects of parasitology. These organisms are small, The genus Entamoeba, characterized by the presence of chromatin on and pathogenic species must be differentiated from nonpathogenic spe- the nuclear membrane, includes E. histolytica, the etiologic agent of ame- cies and from inflammatory cells, epithelial cells, yeasts, pollen, and other biasis; E. dispar, a nonpathogenic species morphologically identical to E. confusing objects. Numerous characteristics assist in identifying intestinal histolytica; E. hartmanni and E. coli, two commensal species; and Entamoeba protozoa. Motility patterns are classically described for many amebae, flag- polecki, which is occasionally found in people who have contact with pigs or ellates, and the ciliate Balantioides coli, but these are appreciated only when primates (Fig. 65.10) (Ash & Orihel, 2007). More recently, E. moshkovskii wet mount examination of fresh material is available. More importantly, and E. bangladeshi have also been identified in human stool specimens and features such as size, shape, and nuclear and cytoplasmic characteristics are morphologically indistinguishable from E. histolytica. Their potential are used for identification in wet mounts of concentrated specimens and pathogenicity is unknown at this writing. Entamoeba gingivalis, which does permanently stained preparations. not have a known cyst stage, inhabits the oral cavity of people with poor Size is an important feature (Figs. 65.9 and 65.10); thus, a properly oral hygiene (Ash & Orihel, 2007). Entamoeba polecki (Fig. 65.10F), E. calibrated ocular micrometer must be available for routine diagnostic moshkovskii, E. bangladeshi, and E. gingivalis are seen infrequently and are use. Shape is also a helpful feature; flagellates generally are elongated and not described further. Endolimax nana and I. buetschlii are nonpathogenic tapered, with a nucleus or nuclei at one end, whereas amebic trophozo- species. Dientamoeba fragilis is recognized as a flagellate, although it lacks ites are rounded to oval with occasional pseudopod projections. Other external flagella, and is discussed with the flagellates in the text but may important identifying morphologic features are the number and size of be found with amebae in tables and figures because it is morphologically nuclei and the pattern of chromatin distribution, best seen in permanent similar to them (Garcia, 2016). stained preparations, and cytoplasmic structures such as fibrils in flagel- lates, ingested materials in amebic trophozoites, and glycogen masses and Entamoeba histolytica (Amebiasis) chromatoid bodies in amebic cysts. Most patients with Entamoeba histolytica infection are asymptomatic. During examination by any method, both nuclear and cytoplasmic However, Entamoeba histolytica may cause various clinical diseases, most characteristics should be assessed from a number of individual organisms commonly amebic dysentery, amebic colitis, and amebic liver abscesses to complete the identification. When reporting the presence of two or (Meyers et al., 2013). General host defense mechanisms, previous contact more species in a sample, the observer should be able to define distinct with the parasite, diet, and the strain of E. histolytica may influence the populations of organisms to prevent confusion with an occasional organ- severity of infection. ism with an atypical appearance. Amebic dysentery, which occurs infrequently in the United States, is As mentioned in the previous section on Laboratory Methods, tro- an acute disease characterized by bloody diarrhea with abdominal pain and phozoites typically predominate in liquid stool but degenerate within 30 cramping. Trophozoite invasion of the intestinal mucosa occurs, produc- minutes to 1 hour after passage unless the specimen is placed into a fixa- ing ulceration that may lead to perforation and peritonitis. The more com- tive. Cysts typically predominate in formed stool and are more resistant to mon form of disease seen in this country is amebic colitis, which may mimic 1315 CHAPTER 65 MEDICAL PARASITOLOGY A B C D E F Figure 65.10 Intestinal protozoa, trichrome stain, oil immersion at 1000×, except as noted. A, Trophozoites of Entamoeba histolytica/dispar. B, Cyst of E. histolytica/dispar showing 3 of the 4 nuclei and a chromatoid bar with rounded ends. C, E. histolytica trophozoites with ingested red blood cells from a colonic lesion (arrow) (H&E). D, Tropho- zoites of E. coli. E, Cyst of E. coli. F, Cysts of E. polecki with numerous chromatoid bars. Continued ulcerative colitis and other forms of inflammatory bowel disease. Symptoms in the stool in less than half of patients at the time liver abscess is manifest; generally are less severe than in amebic dysentery but may include nonbloody therefore, stool parasite examination is not a reliable means for diagno- diarrhea, constipation, abdominal cramping, and weight loss. Small, pinpoint sis of infection. Rarely, amebic abscesses appear in other organs, such as mucosal ulcerations may develop and expand within the submucosa to form the lung, brain, or skin, by hematogenous spread from the intestine or by flask-­shaped ulcers. All of the colon may be involved or only a portion, most contiguous spread from a liver abscess (e.g., across the diaphragm to the commonly the cecum, rectosigmoid, or ascending colon. Less commonly, lungs). When the brain is involved, the trophozoites must be differentiated masses of granulomatous tissue, known as amebomas, may form in the intes- from those of the free-­living amebae. This is usually accomplished by care- tine in response to the presence of amebae, producing a so-­called napkin ring ful examination of the trophozoite morphology, associated host response, lesion that could be mistaken for a carcinoma (Meyers et al., 2013). and clinical history. It is recommended that all patients with amebiasis be As E. histolytica trophozoites invade the intestinal wall, they may enter treated with suitable antiparasitic agents; asymptomatic individuals are the bloodstream and disseminate via the portal blood supply to the liver treated with iodoquinol, paromomycin, or diloxanide furoate to eliminate and other organs. Amebic liver abscess (ALA) is the most common form of the luminal stage of intestinal infection and prevent environmental shed- extraintestinal amebiasis, occurring in approximately 5% of patients with ding of infectious cysts (Drugs for Parasitic Infections, 2013). Patients a history of intestinal amebiasis. Symptoms include fever and right upper with invasive or disseminated disease are first treated with metronidazole quadrant pain. These liver abscesses are usually diagnosed by radiographic or tinidazole, both of which have good systemic penetration. This therapy scans, ultrasound, and detection of parasite-­specific host antibodies. It is should then be followed by either iodoquinol or paromomycin to eradicate important to note that E. histolytica cysts and/or trophozoites are present any intestinal parasites (Drugs for Parasitic Infections, 2013). 1316  PART 7 G H I Figure 65.10—cont’d G, Trophozoites and cyst (arrow) of Iodamoeba buetschlii. The latter has a characteristic glycogen vacuole. H, Trophozoites of Endolimax nana. I, Cysts of E. nana. Epidemiology Panel (Luminex, Austin, TX). It is important to note that cross-­reactivity Most infections with E. histolytica are acquired by ingestion of contami- with E. dispar may still occur with some molecular methods. The BioFire nated food or water, although one historic outbreak was caused by a con- assay specifically mentions in its package insert that large numbers of E. taminated colonic irrigation machine (Istre et al., 1982). Infection may also dispar may result in a positive result for E. histolytica. be transmitted by oral-­anal sexual activities (Pritt & Clark, 2008). Amebia- Unlike intestinal infection, ALA and other forms of disseminated dis- sis is relatively uncommon in the United States, seen primarily in travelers ease are best detected by serologic testing. Approximately 95% of patients and immigrants. In contrast, infection with the morphologically indistin- with ALA are seropositive; this decreases to 70% for patients with active guishable ameba, E. dispar, is thought to outnumber E. histolytica infection intestinal infection and to 10% in asymptomatic carriers. Detectable titers in the order of 9:1 (Pritt & Clark, 2008). may persist for months or years after successful treatment; as such, anti- body detection cannot distinguish between past and present infection Diagnosis (Wilkins & Nutman, 2015). Examination of a series of stool specimens should be sufficient for diagno- Trophozoites of E. histolytica vary from 10 to 60 μm in diameter, with sis of intestinal amebiasis in most cases, although differentiation of E. his- the nonpathogenic forms usually 15 to 20 μm and the invasive forms greater tolytica from E. dispar, E. moshkovskii, and E. bangladeshi requires additional than 20 μm in greatest dimension (Table 65.8; see Figs. 65.9, 65.10A, and molecular testing. The only exceptions to this are when ingested erythro- 65.10C) (Brooke, 1969). In direct wet mounts, trophozoites show progres- cytes are seen within trophozoites in stool or trophozoites are observed sive motility via rapidly formed hyaline pseudopodia that demonstrate a in biopsy specimens or in extraintestinal sites since this is considered to sharp demarcation between endoplasm and ectoplasm; unstained nuclei are be pathognomonic for invasive E. histolytica infection (Fig. 65.10C). If the not easily visible. In invasive disease, some trophozoites contain ingested patient has been given antibiotics or contrast media, the amebic infection erythrocytes (see Fig. 65.10C), a feature diagnostic of E. histolytica infection. may be masked for a period of time. Aspirated material from liver abscesses In stained preparations, the peripheral nuclear chromatin is often evenly dis- can be examined microscopically to detect trophozoites, although they tributed along the nuclear membrane as fine granules. The karyosome is are not commonly identifiable within the aspirated necrotic material. The small and is often centrally located, with fine fibrils that generally are not last material aspirated is most likely to contain trophozoites and may be visible, attaching it to the nuclear membrane. Variations in nuclear structure examined by direct microscopic examination or permanently stained slides. occur, with some karyosomes located eccentrically and peripheral chromatin If tissue is available, sections may show organisms that stain prominently irregularly distributed. As mentioned previously, the only characteristic that with PAS. is pathognomonic for E. histolytica in stool is phagocytosis of erythrocytes. Culture procedures are not widely used for diagnosis but are useful The cytoplasm is finely granular and, in cases of invasive disease, it is com- for research. EIA antigen detection tests that are specific, sensitive, and mon to see only ingested erythrocytes. In contrast, the cytoplasm of nonin- able to differentiate E. histolytica from E. dispar are commercially available vasive organisms may contain ingested bacteria or yeasts, which should not for stool specimens (see Table 65.4) (Garcia et al., 2018). Amplification be mistaken for erythrocytes. In degenerating organisms, the cytoplasm may techniques and DNA probes are also useful for differentiating E. histolytica become vacuolated and nuclei may show abnormal chromatin clumping. from E. dispar (Qvarnstrom et al., 2005). At the time of this writing, there Cysts of E. histolytica are spherical and measure 10 to 20 μm (usu- are three FDA-­approved commercial molecular assays that can detect E. ally 12–15 μm) in diameter (Table 65.9; see Figs. 65.9 and 65.10B). The histolytica: (1) BioFire FilmArray Gastrointestinal GI Panel (BioMérieux, rounded precyst stage has a single nucleus but does not have a refractile Marcy-­ I’Étoile, France), (2) BD MAX Enteric Parasite Panel (Becton cyst wall. As it matures, the cyst develops four nuclei, each approximately Dickinson, Franklin Lakes, NJ), and (3) xTag Gastrointestinal Pathogen one-­sixth the diameter of the cyst. Cyst nuclei appear similar to those of 1317  Ciliate Coccidia Blastocystis CHAPTER 65 MEDICAL PARASITOLOGY Balantioides Cystoisospora Sarcocystis Cryptosporidium Blastocystis coli belli spp. spp. spp. Trophozoite Immature Mature Mature oocyst oocyst oocyst Cyst Single sporocyst 0 20 40 Mature 0 10 20 30 0 10 20 A oocyst µm µm µm Flagellates Pentatrichomonas Chilomastix Giardia Enteromonas Retortamonas hominis mesnili duodenalis hominis intestinalis Trophozoite No cyst Figure 65.11 A, Ciliate, Coccidia, and Blastocystis Cyst spp. found in stool specimens of humans. B, Flagel- lates found in stool specimens of humans. (Modified from Brooke MM, Melvin DM: Morphology of diag- 0 5 10 nostic stages of intestinal parasites of man, Publica- tion No. [CDC] 848116, Washington, DC, 1984, U.S. B µm Department of Health and Human Services.) trophozoites, but their smaller size makes them less useful as differenti- as a distinct species. Differentiation requires careful measurement of a rep- ating features. The cyst cytoplasm may contain glycogen vacuoles and resentative sample of organisms with a properly calibrated ocular microm- chromatoid bodies with blunted or rounded ends. The number and size of eter. The shrinkage of E. histolytica/E. dispar cysts and trophozoites in nuclei and the appearance of chromatoid bodies are important diagnostic fixatives may result in an overreporting of E. hartmanni. criteria for identifying cysts. Entamoeba coli, a common lumen-­dwelling ameba, may also be diffi- Those laboratories that do not use one of the immunologic or molecu- cult to differentiate from E. histolytica given its morphologic similarities. lar methods to differentiate E. histolytica from E. dispar should report find- In general, the cysts and trophozoites of E. coli are larger than those of ings of E. histolytica/E. dispar or similar. A reporting comment may be E. histolytica, but significant size overlap exists and, therefore, size is not added to suggest additional species-­level testing if clinically indicated. a reliable feature. The most definitive morphologic feature is the pres- ence of 8 (or, rarely, 16) nuclei in mature cysts of E. coli compared with Nonpathogenic Amebae the usual 4 of E. histolytica, E. dispar, and E. hartmanni. Other helpful Laboratory personnel must be able to differentiate nonpathogenic or features are the characteristics of the cytoplasm. The cytoplasm of E. commensal intestinal amebae from E. histolytica/E. dispar and D. fragilis coli trophozoites stains somewhat more darkly than the cytoplasm of E. (a flagellate), which are potential pathogens. Identification characteris- histolytica and is more vacuolated, containing numerous ingested bacteria, tics, best visualized in permanent stained sections, are summarized in yeasts, and other materials. Chromatoid bodies, when present in E. coli Tables 65.8 and 65.9 and in Figures 65.9 and 65.10. Identification of cysts, are irregular in shape, with splintered or pointed ends rather than trophozoites is based on size and nuclear and cytoplasmic characteris- the rounded ends seen in E. histolytica. Although nuclear characteristics tics; identification of cysts is based on size, number and characteristics differ slightly from those of E. histolytica (see Figs. 65.10D and 65.10E), of nuclei, and presence and character of chromatoid bodies and glyco- significant overlap may occur, especially in specimens that have not been gen masses. promptly preserved. Therefore, distribution of peripheral chromatin and Entamoeba hartmanni has morphologic characteristics that are extremely karyosomes should not be given great emphasis in identification of Ent- similar to those of E. histolytica except that the trophozoites have a maxi- amoeba spp. mum diameter of 12 μm and cysts have a maximum diameter of 10 μm The nuclei of I. buetschlii trophozoites and cysts have a large, centrally but are generally consistently less than 10 μm in diameter. Historically, E. located karyosome frequently surrounded by achromatic granules that may hartmanni was called the small race of E. histolytica until it was recognized not be distinct but appear only as a muddy karyolymph space or halo. In 1318  TABLE 65.8 Morphology of Trophozoites of Intestinal Amebae Size (in Diameter Nucleus Peripheral Karyosomal Cytoplasm Species or Length) Motility Number* Chromatin Chromatin Appearance Inclusions Entamoeba 10–60 μm; usual Progressive, 1 Fine granules; Small, discrete; Finely granular Erythrocytes PART 7 histolytica/E. range, 15–20 μm with hyaline, Not visible usually evenly usually central occasionally in dispar commensal form†; fingerlike pseu- in unstained distributed and but occasionally invasive forms; over 20 μm for inva- dopods preparations uniform in size eccentric noninvasive, sive form‡ contain bacteria Entamoeba 5–12 μm; usual Usually non- 1 Similar to E. Small, discrete, Finely granular Bacteria hartmanni range, 8–10 μm progressive but Not visible histolytica often eccentric may be progres- in unstained sive occasionally preparations Entamoeba 15–50 μm; usual Sluggish, non- 1 Coarse granules, Large, discrete, Coarse, often Bacteria, yeasts, coli range, 20-­25 μm progressive with Often visible irregular in size usually eccentric vacuolated or other materi- blunt pseudo- in unstained and distribution als pods preparation Endolimax 6–12 μm; usual Sluggish, usu- 1 None Large, irregu- Granular, vacu- Bacteria nana range, 8–10 μm ally nonprogres- Visible oc- larly shaped olated sive with blunt casionally in pseudopods unstained preparations Iodamoeba 8–20 μm; usual Sluggish, usu- 1 None Large, usu- Coarsely granu- Bacteria, yeasts, buetschlii range, 12–15 μm ally nonprogres- Not usu- ally central; lar, vacuolated or other materi- sive ally visible surrounded by als in unstained refractile, achro- preparations matic granules; these granules often are not distinct even in stained slides Dientamoeba 2 (In approxi- None Large cluster of Finely granular, Bacteria fragilis mately 20% 4–8 granules vacuolated of organisms, only 1 nucleus is present) Nuclei invisible in unstained preparations Modified from Brooke MM, Melvin DM: Morphology of diagnostic stages of intestinal parasites of man, PHS Publication No. 1966, Bethesda, MD, 1969, U.S. Department of Health, Education, and Welfare. *Visibility is for unfixed material. Nuclei sometimes may be visible in fixed material. †Usually found in asymptomatic or chronic cases; may contain bacteria. ‡Usually found in acute cases; often contain red blood cells. some nuclei, the halo is clear without evident achromatic granules, making isolates appear to have animal reservoirs. Nine (possibly 10) primary sub- the organism indistinguishable from E. nana. Cysts of I. buetschlii contain types have been found in mammals and birds, with subtypes 1 and 3 making a single nucleus, in which the karyosome is often eccentric with a nearby up most of the cases of human blastocystosis. It is becoming more common crescent of achromatic granules (Fig. 65.10G). The cyst is characterized by for clinical laboratories to report Blastocystis only at the genus level (Stens- a prominent vacuole of glycogen (Fig. 65.10G) that stains reddish brown in vold et al., 2007). Some studies have linked heavy infection to symptomatic iodine-­stained wet mounts. Glycogen is dissolved by aqueous fixatives and intestinal disease, although this remains controversial (Coyle et al., 2012). may not be demonstrable in material that has been stored. Treatment options include metronidazole, trimethoprim/sulfamethoxa- Endolimax nana is the smallest ameba to infect humans. Trophozo- zole, iodoquinol, and nitazoxanide (Drugs for Parasitic Infections, 2013). ites often have atypical nuclei that contain a triangular chromatin mass, a Traditionally, the forms in human stool were referred to as cyst-like band of chromatin across the nucleus, or two discrete masses of chromatin forms; however, those are not believed to be one of the different vaculoar on opposite sides of the nuclear membrane (Fig. 65.10H). A clear halo forms seen in stool specimens (Fig. 65.12G). Occasionally, these forms may or karyolymph space surrounds the karyosome and extends to the nuclear be seen dividing on stained slides. Because the morphologic separation of the membrane. Atypical nuclear forms may be helpful in differentiating E. various stages is not as clear-­cut as with other protozoa, it is not common to nana from I. buetschlii, which is similar in appearance but larger. Cysts of identify and/or report the stages of Blastocystis. The forms in stool can vary Endolimax (Fig. 65.10I) usually contain four nuclei, although smaller num- greatly in size, from 5 to 40 μm in diameter. Characteristically, stool forms bers may be seen. Glycogen, when present, occurs diffusely in the cyto- contain a large central body (similar to a vacuole) surrounded by a thin rim of plasm rather than as a discrete mass. cytoplasm that contains nuclei and other organelles. In a properly preserved specimen stained with trichrome, the central body is usually green while the surrounding organelles stain red (Fig. 65.12G). Blastocystis should be reported BLASTOCYSTIS SPP. and quantified when present, especially when they are numerous (five or Blastocystis is an enigmatic protozoan that inhabits the large bowel and is more per 1000× field), since quantity may correlate with clinical presentation frequently found in stool specimens of asymptomatic individuals. Although (Garcia, 2016). This is the only protozoan parasite (and the only intestinal appearing in stains as an ameba-­like protozoan, it is now known to be a parasite in the United States) in which the quantity is routinely reported. Stramenopile (a group that includes diatoms and algae) and is placed in the Stramenopiles-­ Alveolata-­ Rhizaria (SAR) clade (Adl et al., 2012, 2019). FLAGELLATES Traditionally, Blastocystis spp. have been described based on host data (B. hominis from humans, B. ratti from rats, etc.) and historically all isolates of Dientamoeba fragilis humans were referred to as B. hominis. However, molecular analyses appear Dientamoeba fragilis is an ameboid-­appearing protozoan that infects the to demonstrate that there is no population unique to humans and all human colon and has been associated with diarrheal disease, especially in young 1319 CHAPTER 65 MEDICAL PARASITOLOGY TABLE 65.9 Morphology of Cysts of Intestinal Amebae Nucleus Peripheral Karyosomal Cytoplasm Chro- Species Size Shape Number Chromatin Chromatin matoid Bodies Glycogen Entamoeba 10–20 μm; Usually spherical 4 in mature cyst; Peripheral chro- Small, discrete, Present; elongate Usually diffuse; histolytica/E. usual range, immature cysts matin present; usually central bars with bluntly concentrated dispar 12–15 μm with 1 or 2 oc- fine, uniform rounded ends mass often casionally seen granules, evenly present in young distributed cysts; stains red- dish brown with iodine Entamoeba 5–10 μm; usual Usually spherical 4 in mature cyst; Similar to E. Similar to E. Present; elongate Similar to E. hartmanni range, 6–8 μm Immature cysts histolytica histolytica bars with bluntly histolytica with 1 or 2 often rounded ends seen Entamoeba coli 10–35 μm; Usually spherical; 8 in mature cyst; Peripheral chro- Large, discrete, Present; usually Usually diffuse, usual range, occasionally oval, occasionally matin present; usually ec- splinterlike with but occasion- 15–25 μm triangular, or of supernucleate coarse granules centric, but pointed ends ally well-­defined another shape cysts with 16 or irregular in size occasionally mass in imma- more are seen; and distribu- central ture cysts; stains immature cysts tion, but often reddish brown with 2 or more appear more with iodine occasionally seen uniform than in trophozoites Endolimax nana 5–10 μm; usual Spherical, ovoid, 4 in mature None Large, usu- Occasionally, Usually diffuse; range, 6–8 μm or ellipsoidal cysts; immature ally centrally granules or small concentrated cysts with fewer located oval masses seen, mass seen occa- than 4 rarely but bodies as seen sionally in young seen in Entamoeba spp. cysts; stains red- are not present dish brown with iodine Iodamoeba 5–20 μm; Ovoid, ellipsoi- 1 in mature cyst None Large, usually Granules occasion- Compact, buetschlii usual range, dal, triangular, or eccentric; re- ally present, but well-­defined