VII. Medical Parasitology CHAPTER 61. Thomas R. Fritsche, M.D., Ph.D., Rangaraj Selvarangan, B.V.Sc., Ph.D.

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1 CHAPTER Thomas R. Fritsche, M.D., Ph.D., Rangaraj Selvarangan, B.V.Sc., Ph.D. Laboratory Methods 1120 Examination of Blood 1121 Examination of Fecal Specimens 1122 Examination of Urogenital and Other Specimens 1124 (Sputa, Aspirates, Biopsies) Parasite Culture Techniques 1125 Immunodiagnostic Methods 1125 Molecular Diagnostic Methods 1127 Quality Assurance, Quality Improvement, and 1127 Safety Blood and Tissue Protozoa 1127 Malaria 1127 Babesiosis 1134 Hemoflagellates 1134 Toxoplasma gondii 1136 Opportunistic Free-Living Amebae 1137 Intestinal and Urogenital Protozoa 1138 Amebae and Blastocystis hominis 1138 Flagellates 1142 Ciliates 1144 Coccidia 1144 Microsporidia 1146 Intestinal Helminths 1146 Nematodes 1147 Cestodes 1150 Trematodes 1152 Tissue Helminths 1155 Nematodes 1155 Cestodes 1157 Trematodes 1159 Medically Important Arthropods 1159 Biological Characteristics 1159 Mechanisms of Injury 1159 Laboratory Approaches to Arthropod 1160 Identification Insects 1160 Arachnids 1162 Classes of Lesser Medical Importance 1163 Millipedes 1163 Parasitic Infections and the 1165 Immunocompromised Host References 1165 KEY POINTS Accurate diagnosis of parasitic infections usually depends on macroscopic or microscopic examination of specimens that have been appropriately collected and preserved. Thick and thin blood smears are useful to detect and characterize organisms found in the blood. Fecal specimens may either be fresh or placed into fixatives such as formalin and polyvinyl alcohol. Immunodiagnostic methods for parasitology include detection of both antibodies and antigens. Established enzyme immunoassays include antigen tests for Plasmodium spp. in blood or serum; Giardia lamblia, Cryptosporidium spp., and Entamoeba histolytica in feces; Trichomonas vaginalis in vaginal swabs. Direct fluorescent assays are also useful for identifying organisms in primary specimens. Molecular diagnostic methods are now emerging based on amplification techniques that offer high levels of sensitivity and specificity for both diagnosis and monitoring of parasitic diseases. The diagnosis of malaria should be considered in the differential diagnosis of unexplained fever with the history of travel in endemic geographic regions. Thick and thin blood smears are complementary for detecting and identifying the infecting Plasmodium spp. based on the varieties of developmental stages, presence of malarial pigment, and stippling seen in infected erythrocytes. Other protozoal infections include babesiosis and trypanosomiasis found in blood, leishmaniasis (causing cutaneous, mucocutaneous, and visceral forms of disease), and toxoplasmosis, often affecting the central nervous system following congenital infection and in patients with the acquired immunodeficiency syndrome (AIDS). Amebae are ingested as cysts, resulting in infection of the colon and passage of both cysts and trophozoites in the feces. The resulting diseases are amebic dysentery, amebic colitis, and liver abscesses. Diagnosis is made by microscopic examination of stool and by serological testing for antibodies in serum. Flagellates include Giardia lamblia, which causes diarrhea from ingestion of contaminated water and is diagnosed by finding trophozoites or cysts in feces. Trichomonas vaginalis can be detected in vaginal wet mounts by its characteristic motion. Coccidia (Isospora, Cryptosporidium, and Cyclospora) and microsporidia can cause protracted diarrhea in immunosuppressed individuals such as those with AIDS. Intestinal helminths include the nematodes (roundworms), cestodes (tapeworms), and trematodes (flukes). These organisms reside as adults in the gastrointestinal tract or in other locations (liver, lung, blood). Knowledge of their life cycles and zoogeography with intermediate hosts are critical for establishing the parasitic stage in presumed infection. Eggs, larvae, or adult forms (from 1 mm to >10 m in length, depending upon species) can be recovered from stool, urine, or sputum. Tissue helminths include filaria (larvae found in blood and also on skin biopsy), Trichinella (in muscle), Strongyloides (disseminated infection), and hydatidosis (large cystic lesions in the liver or lungs), among others. Arthropods cause disease through direct tissue invasion, envenomation, vesication, blood loss, transmission of infectious agents, hypersensitivity reactions, and psychological manifestations. Characteristics necessary for identification can be maintained by preserving the organisms in alcohol (ticks, mites, fleas, lice), by drying them (winged forms) after killing them with fumes of organic solvents, or killing with hot water (maggots) and subsequent storage in alcohol. VII

2 1128 Growing trophozoite Mature trophozoite EXOERYTHROCYTIC SCHIZOGONY Immature Mature Merozoites Penetrate red blood cells ERYTHROCYTIC SCHIZOGONY Immature Immature Ring-form trophozoite MAN Merozoites Mature Penetrate parenchymal cells of liver Microgametocyte Sporozoites injected by mosquito Macrogametocyte Sporozoites in salivary glands (infective stage) Oocyst ruptures sporozoites liberated Oocyst containing sporocytes MOSQUITO Oocyst on stomach wall Macrogametocyte Macrogamete Fertilization Microgamete Microgametocyte Exflagellating microgametocyte Ookinete Figure 61 1 Life cycle of malarial parasites. (Courtesy of the Centers for Disease Control, Parasitology Training Branch, Atlanta, GA.) generally not appropriate or available for smaller laboratories. The recent development of immunocapture assays for the detection of Plasmodiumspecific lactate dehydrogenase or HRP-II appears to provide a high degree of sensitivity and specificity in the diagnosis of malaria. Several versions of these tests, configured as dipstick methods, are especially promising in situations where ease of performance is critical and usual laboratory facilities are lacking (Palmer, 1998; Piper, 1999; Marx, 2005). Life Cycle Malarial parasites undergo a sexual phase (sporogony) in Anopheles mosquitoes that results in the production of infectious sporozoites and an asexual stage (schizogony) in humans that results in the production of s and merozoites (Fig. 61 1). In the blood stream, some merozoites eventually differentiate into gametocytes (gametogony), which, when ingested by female anopheline mosquitoes, mature into the male microgametes and the female macrogametes. Fusion of a microgamete and a macrogamete results in the formation of the motile ookinete, which migrates to the outside of the stomach wall and forms an oocyst. Within the oocyst, numerous spindle-shaped sporozoites are formed. The mature oocyst ruptures into the body cavity, releasing the sporozoites, which then migrate through the tissues to the salivary glands, from which they are injected into the vertebrate host as the mosquito feeds. The time required for development in the mosquito ranges from 8 21 days. The sporozoites injected into the vertebrate host reach the hepatic parenchymal cells within minutes and initiate the proliferative phase known as exoerythrocytic schizogony. Release of merozoites from ruptured hepatic s initiates the blood stream infection or erythrocytic schizogony and, eventually, the clinical symptoms of malaria. P. vivax and P. ovale differ from P. falciparum and P. malariae in that true disease relapses of the former species may occur weeks to months following subsidence of previous attacks. This occurs as a result of renewed exoerythrocytic and, eventually, erythrocytic schizogony from latent hepatic sporozoites, which are known as hypnozoites (Krotoski, 1982). Recurrences of disease due to P. falciparum or P. malariae, called recrudescences, arise from an increase in numbers of persisting blood stage forms to clinically

3 1132 Figure 61 5 Plasmodium ovale. 1, Young ring-shaped trophozoite. 2 5, Older ring-shaped trophozoites. 6 8, Older ameboid trophozoites. 9, 11, and 12, Doubly infected cells, trophozoites. 10, Doubly infected cell, young gametocytes. 13, First stage of the , Schizonts, progressive stages. 20, Mature gametocyte. 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, (Courtesy of Aimee Wilcox, National Institutes of Health Bulletin No. 180, USPHS.) months or more with the other Plasmodium species. The common presenting symptoms of malaria include chills and fever, which are often associated with splenomegaly. In the early stages of the disease, the febrile episodes occur irregularly but eventually become more synchronous, assuming the usual tertian (P. vivax, P. falciparum, and P. ovale) or quartan (P. malariae) periodicity. Patients with malaria may develop anemia and may have other manifestations, including diarrhea, abdominal pain, headache, and muscle aches and pains. P. falciparum malaria can result in high (50%) parasitemias, which can lead to severe hemolysis with hemoglobinuria and profound anemia. Erythrocytes infected with growing trophozoites and s of P. falciparum become sequestered in small vessels of the body and may lead to occlusion of these vessels, causing symptoms related to capillary obstruction and tissue anoxia. Involvement of the brain is known as cerebral malaria, in which the patient becomes disoriented, progressing to delirium, coma, and often death. Exchange transfusion may be lifesaving in severe P. falciparum infections (Nielson, 1979; Powell, 2002). The course of untreated malaria depends on the species. Most fatal cases of malaria are due to P. falciparum. In nonfatal cases, the febrile paroxysms become less severe with time and the disease gradually subsides. Patients with P. vivax or P. ovale infection may have relapses after many months or, occasionally, years. Persons with P. falciparum and P. malariae infection may have symptom-free periods but suffer from sporadic recrudescences owing to persisting low-grade parasitemia. Relapses and recrudescences may be associated with changes in the host s defense mechanisms or possibly with antigenic changes in the infecting organisms. Peripheral smears may show leukocytes that contain malaria pigment. Increased reticulocyte counts occur commonly and are associated with the rapid erythrocyte turnover. The presence of greatly enlarged platelets also may be noted on peripheral blood films and occur as a result of their rapid

4 1134 A B C D E F Figure 61 6 A, Human infection with Babesia microti; note high parasitemia and multiple-infected red cell (oil immersion). B, Trypanosoma sp. in stained blood film; note nucleus, kinetoplast, and undulating membrane (oil immersion). C, Leishmania mexicana amastigotes in impression smear of thigh lesion; Giemsa stain (oil immersion). D, Pseudocyst of Toxoplasma gondii in brain tissue (hematoxylin and eosin [H&E]; oil immersion). E, Cutaneous rosette of T. gondii tachyzoites in an immunocompromised patient (H&E; oil immersion). F, Tachyzoites of T. gondii recovered from a bronchoalveolar lavage specimen from an individual infected with the human immunodeficiency virus (Giemsa stain; oil immersion). recognized around growing trophozoites as a pink halo rather than the distinct granules seen in thin films. The ameboid character of P. vivax trophozoites is not as evident in thick films, but the number of merozoites in mature s is helpful. Macro- and microgametocytes cannot usually be differentiated. The distinctive sausage shape of P. falciparum gametocytes is still evident, although they may be more stubby than in thin films. Gametocytes of the other species can be detected and are easily differentiated from host cell nuclei by the presence of refractile hemozoin pigment. Mixed infections occur occasionally (about 5% of the time) but caution should be used in making such diagnoses unless there is definite evidence of two separate populations of parasites. The most common mixed infections are P. falciparum and P. vivax. Finding gametocytes of P. falciparum in a person obviously infected with P. vivax is diagnostic. There are multiple artifacts that may be confused with malarial parasites in thick and thin films. The most common artifact in thin films are blood platelets superimposed on red blood cells. These platelets should be readily identified because they do not have a true ring form, do not show differentiation of the chromatin and cytoplasm, and do not contain pigment. Clumps of bacteria or platelets may be confused with s. At times, masses of fused platelets may resemble gametocytes of P. falciparum but do not show the differential staining or the pigment. Precipitated stain and contaminating bacteria, fungi, or spores may also be confused with these parasites. Species-specific serologic tests for malaria are particularly useful for epidemiologic surveys and for detection of infected blood donors. Such tests do not reliably differentiate current from past infection, however. Sensitive and specific IFA tests using antigens from the four human species are available from the CDC (Wilson, 1995). Assays for the direct detection of malarial antigens in blood are especially promising (see Laboratory Methods). Babesiosis Like malarial parasites, the etiologic agents of babesiosis or piroplasmosis are apicomplexan protozoa found worldwide that infect erythrocytes, often producing a febrile illness of variable severity. Unlike malaria, babesiosis is transmitted by ticks and is found in a variety of animal species that serve as reservoirs (Krause, 2002). Human infections in the United States occur predominantly in the northeastern and midwestern states, where the rodent parasite Babesia microti is responsible for infection (Homer, 2000). Ixodes scapularis is the usual tick vector. Recent studies have implicated another, as yet unnamed, Babesia species (tentatively known as WA1) as being responsible for disease in the western United States. This parasite, associated with disease in Washington state and California, is thought to be transmitted by the western black-legged tick, Ixodes pacificus (Quick, 1993; Persing, 1995). In Europe, the canine parasite Babesia divergens, transmitted by Ixodes ricinus, infects humans and a recent report of a B. divergens-like infection in Washington State expands the range of known human cases (Herwaldt, 2004). The spectrum of babesiosis varies from latent, subclinical infection to fulminant, hemolytic disease. Fatalities have been reported, especially in splenectomized or immunocompromised individuals. Immunocompetent persons may experience symptoms similar to those of malaria, including fever, chills, malaise, and anemia, although without recognizable periodicity. Investigation of an outbreak caused by B. microti on Nantucket Island in New England showed that some symptomatic patients harbored the parasite for months and others showed serologic evidence of infection without a history of clinical disease (Ruebush, 1980). Other evidence is accumulating indicating that chronic subclinical infections may not be uncommon (Persing, 1995). Babesia parasites multiply in erythrocytes by schizogony but do not produce gametocytes. Although trophozoites of many species appear pearshaped at some point in their development, those of B. microti usually appear as delicate ring forms that may be easily confused with those of malarial parasites, especially P. falciparum (Fig. 61 6A) (Healy, 1980; Homer, 2000). Babesia trophozoites can be differentiated from those of malarial parasites by the presence of multiple rings in one cell that may form a tetrad (Maltese cross) and the absence of large, growing trophozoites and gametocytes. Also, Babesia-infected cells lack hemozoin pigment, which is present in Plasmodium-infected cells. History of residence in or travel to endemic areas, or of a recent tick bite, might suggest Babesia infection. Serologic tests (IFA) for both B. microti and WA1 are available from the CDC on referral from state health departments. Serology tests for malaria are negative in babesiosis, although patients with malaria may cross-react in the Babesia serologies (Wilson, 1995). Hemoflagellates The hemoflagellates of humans and animals are members of the order Kinetoplastida and are characterized by the presence of a large mitochondrion known as a kinetoplast, which contains enough DNA to be seen by light microscopy when treated with Giemsa stain. Two genera important in human disease are Trypanosoma and Leishmania. Members of both genera are transmitted by arthropod vectors and have animal hosts that serve as reservoirs. The kinetoplastida assume different morphologic forms depending on their presence in vertebrate hosts, including humans, or their insect vectors (Fig. 61 7). The amastigote stage is spherical, 2 5 μm in diameter, and displays a nucleus and kinetoplast. By definition an external flagellum is lacking, although an axoneme (the intracellular portion of the flagellum) is apparent at the ultrastructural level. Amastigotes may be found in human or animal hosts infected with either T. cruzi or Leishmania spp.,

5 Table 61 9 Morphology of Trophozoites of Intestinal Amebae 1141 Species Size (in Motility Nucleus Peripheral Karyosomal Cytoplasm Inclusions Diameter Numbers Chromatin Chromatin Appearance or Length) Entamoeba μm; usual Progressive, 1 Fine granules; Small, discrete; usually Finely granular Erythrocytes histolytica/e. range, μm with hyaline, Not visible in usually evenly central but occasionally occasionally in invasive dispar commensal form*; finger-like unstained distributed and eccentric forms. Noninvasive over 20 μm for pseudopods preparations uniform in size contain bacteria invasive form Entamoeba 5 12 μm; usual Usually 1 hartmanni range 8 10 μm nonprogressive Not visible in Similar to E. Small, discrete, often Finely granular Bacteria but may be unstained histolytica eccentric progressive preparations occasionally Entamoeba μm; usual Sluggish, 1 coli range μm nonprogressive Often visible in Coarse granules, Large, discrete, usually Coarse, often Bacteria, yeasts, other with blunt unstained irregular in size eccentric vacuolated materials pseudopods preparation and distribution Endolimax nana 6 12 μm; usual Sluggish, usually 1 None Large, irregularly shaped Granular, Bacteria range 8 10 μm nonprogressive Visible occasionally vacuolated with blunt in unstained pseudopods preparations Iodamoeba 8 20 μm; usual Sluggish, usually 1 None Large, usually central. Coarsely Bacteria, yeasts, or other bütschlii range μm nonprogressive Not usually Surrounded by refractile, granular, material visible in unstained achromatic granules. vacuolated preparations These granules are often not distinct even in stained slides Dientamoeba 5 15 μm; usual Pseudopods are 2 None Large cluster of 4 8 Finely granular, Bacteria fragilis range 9 12 μm angular, serrated, (In approximately granules vacuolated or broad lobed 20% of organisms and hyaline, only 1 nucleus is almost present.) Nuclei transparent invisible in unstained preparations * Usually found in asymptomatic or chronic cases; may contain bacteria. Usually found in acute cases; often contain red blood cells. Visibility is for unfixed material. Nuclei may sometimes be visible in fixed material. A flagellate (see text). Source: adapted from Brooke MM, Melvin DM: Morphology of Diagnostic Stages of Intestinal Parasites of Man. USDHEW PHS Publication No. 1966, VII Figure Nuclei of amebae. This drawing shows some of the various appearances of amebic nuclei in stained preparations. (Dientamoeba fragilis is a flagellate; see text.) The nuclei of I. bütschlii trophozoites and cysts have a large, centrally located karyosome frequently surrounded by achromatic granules that may not be distinct but appear only as a muddy karyolymph space or halo. In some nuclei, the halo is clear without evident achromatic granules, making the organism indistinguishable from E. nana. Cysts of I. bütschlii contain a single nucleus, in which the karyosome is often eccentric with a nearby crescent of achromatic granules (see Figs. 61 9, 61 10, and 61 12). The cyst is characterized by a prominent vacuole of glycogen that stains reddish brown in iodine-stained wet mounts, thus the name of the organism. Glycogen is dissolved by aqueous fixatives and may not be demonstrable in material that has been stored. Blastocystis hominis B. hominis is an ameba-like protozoan that inhabits the large bowel and is frequently found in stool specimens of asymptomatic individuals. Some studies have linked heavy infections to symptomatic intestinal disease, although this remains controversial (Markell, 1986; Sheehan, 1986; Miller, 1988; Zierdt, 1991; Stenzel, 1996). Blastocystis may assume one of

6 1164 Table Parasitic Infections in Compromised Hosts Infection Predisposing Host Abnormalities Comments References Intestinal Protozoa Cryptosporidiosis AIDS (especially CD4 + <200/μL), Severe protracted diarrhea (up to 17 L/day.) May have extraintestinal Wittner, 1993; transplantation, antineoplastic involvement including pancreas, biliary tract, and lungs. Limited Thielman, 1998 chemotherapy therapy available, is not curative Isosporiasis AIDS, transplantation, antineoplastic Severe protracted diarrhea. Extraintestinal involvement of regional lymph Wittner, 1993; chemotherapy nodes may be seen. Effective therapy is available Thielman, 1998 Cyclosporiasis AIDS, probably other immunosuppression Severe protracted diarrhea clinically similar to cryptosporidiosis and Wittner, 1993; isosporiasis. Responds to trimethoprim sulfamethoxazole Thielman, 1998 Microsporidiosis AIDS, other severe immunosuppression Various syndromes: Wittner, 1993; 1. Multisystem disease Thielman, Enteritis with severe diarrhea (most frequent) 3. Ocular infection 4. Hepatobiliary with granulomas, hepatic necrosis or cholangitis 5. Skeletal muscle disease Examination of urinary sediment with appropriate stain may allow diagnosis on nonenteric syndromes and some cases of enteric disease Giardiasis Common variable immunodeficiency, Prolonged diarrhea with malabsorption. AIDS is not a predisposing condition Thielman, 1998 X-linked agammaglobulinemia Blood and Tissue Protozoa Granulomatous amebic AIDS and other immunocompromised states Usually caused by amebae of the genera Acanthamoeba or Balamuthia. Martinez, 1997 encephalitis Produces subacute or chronic central nervous system infection but may be acute in severely immunosuppressed hosts with dissemination Toxoplasmosis AIDS with CD4 + usually <100/μL and Usually result of reactivation of cysts from previous infections. Often McCabe, 1993 other immunocompromised states. disseminated disease with multiorgan involvement or multifocal CNS Heart transplant, donor seropositive and lesions. Can cause pneumonitis resembling that caused by Pneumocystis recipient seronegative jiroveci. May cause chorioretinitis. Can occur after transplantation of bone marrow but usually mild. Heart transplant with donor serologically positive and recipient serologically negative may lead to severe, often fatal toxoplasmosis Leishmaniasis AIDS, other immunosuppression Limited influence on cutaneous leishmaniasis. Susceptibility to visceral Herwaldt, 1999 leishmaniasis increased but not disease severity. More likely to relapse after treatment American AIDS, lymphoblastic lymphoma, cardiac In AIDS central nervous system often involved, myocarditis, skin lesions Mileno, 1998; trypanosomiasis transplantation, other immunosuppression Markell, 1999 (Chagas disease) Babesiosis Splenectomy Usually subclinical infection in those with intact host defenses. Mileno, 1998; Splenectomized patients usually develop clinically evident disease which Markell, 1999 can be fatal Helminth Strongyloidiasis Immunosuppression for transplantation Because of endogenous autoinfection; hyperinfection or disseminated Mileno, 1998; or by cancer chemotherapy or adrenal infection can develop and present as pneumonia or severe intestinal Markell, 1999 corticosteroids or lymphoma. AIDS is not disease. Gram-negative sepsis or Gram-negative meningitis can result a major predisposing factor from translocation of intestinal bacteria via invading larvae. Should check for strongyloidiasis before immunosuppressing patient from highly endemic areas Arthropod Scabies Malignancy, transplant immunosuppression, AIDS Leads to Norwegian or crusted scabies with widespread Mileno, 1998; antineoplastic therapy, involvement by thick, crusted lesions. Sometimes has severe itching. Markell, 1999 Patient has numerous mites and is therefore highly contagious. Often less responsive to therapy AIDS = acquired immunodeficiency syndrome; CNS = central nervous system. Although systemic reactions may occur in individuals who have been previously sensitized, fatalities are rare (Goddard, 2002). Crustaceans Crustaceans of medical importance are primarily those species that serve as hosts for larval stages of several different helminths. Several genera of crabs and crayfish are intermediate hosts for the metacercariae of various species of lung fluke (Paragonimus spp.) found around the world. Copepods are common microscopic zooplankton, certain species of which serve as first intermediate hosts for the nematodes D. medinensis and Gnathostoma spinigerum and for cestodes of the genera Diphyllobothrium and Spirometra. Pentastomids Pentastomes, or tongue worms, are arthropods of uncertain affinities owing to a lack of morphologic characteristics. Adult stages are wormlike organisms that live in the nasal passages of certain predatory reptiles, birds, and mammals. Larval stages resemble mites and reside in rodents, herbivores, and freshwater fish. Human liver and lung infections with larval stages have been reported from Asia and Africa. Adult stages have been recovered from the nasopharynx of individuals from the Middle East and Africa, where they are responsible for an obstructive condition known as halzoun (Beaver, 1984; Strickland, 2000).