Parasitology: Specimens, Blood and Tissue Protozoa

A laboratory may limit its specialized parasitology menu. It must still detect malaria and primary amebic meningoencephalitis the same day, often STAT, because both are life-threatening. Specimen type follows the suspected organism’s life cycle: blood and stool account for most requests; urogenital secretions, sputum, aspirates, and biopsy make up the remainder. Size is diagnostic for many organisms, so a calibrated ocular micrometer is mandatory bench equipment. It is the only way to separate Entamoeba histolytica from E. hartmanni, or Cryptosporidium from Cyclospora oocysts.

Blood films

Thin films are spread in a single cell layer, exactly as for a hematologic differential. Intact, nonoverlapping cells let the examiner judge whether infected erythrocytes are enlarged and whether intracellular inclusions are present. Thick films concentrate a much larger volume of blood. During staining, erythrocytes lyse, leaving leukocyte nuclei, platelets, and any parasites. The thick film is more sensitive and is used to screen for low-density infection.

Blood is best obtained by fingerstick, earlobe puncture, or the first anticoagulant-free drop from a venipuncture needle. Anticoagulant distorts parasite morphology and impairs staining, though EDTA blood is the specimen most laboratories receive and is acceptable if smears are made promptly. Spread 1-2 small drops to a dime-sized (~1.5 cm) circle for a thick film, air-dry flat, and do not heat-fix: heat prevents lysis. A properly thin thick film lets newsprint be faintly read through it.

Giemsa at pH 7.0-7.2 is the reference stain; it best displays erythrocyte inclusions (Schüffner stippling, Maurer clefts) that help species-identify Plasmodium. Wright and Wright-Giemsa stain thin films adequately but poorly demonstrate these inclusions and are unsuitable for thick films. Dilute Giemsa stock freshly each day in phosphate-buffered water, and verify staining time and dilution for each new lot.

Scan the entire thick and thin film at low power (10×) first for microfilariae, which concentrate at the feathered edge of the thin film. Screen at 50× oil, then confirm or rule out the smallest parasites (Plasmodium, Babesia) at 100× oil in the feathered-edge region where erythrocyte central pallor is preserved.

The buffy coat concentrates Leishmania donovani amastigotes, trypanosomes, and microfilariae. The Knott technique (2% formalin lysis plus centrifugation) and 5-μm membrane filtration concentrate microfilariae. Acridine-orange microhematocrit centrifugation is more sensitive than routine films for Plasmodium and other blood parasites but cannot replace confirmatory thick/thin-film species identification and parasitemia quantitation once a case is positive.¹

Fecal specimens

Trophozoites predominate in liquid stool but degrade within 30-60 minutes unless fixed; cysts, more resistant, predominate in formed stool. Formalin does not preserve trophozoite morphology. A complete ova-and-parasite (O&P) exam therefore includes a concentrated wet preparation and a permanently stained smear.

Do not collect specimens within 1 week of nonabsorbable antidiarrheals, antacids, bismuth, barium, or antimalarials; crystalline residues obscure the field. Antibiotics and contrast media can suppress protozoan shedding for weeks. Ideal fresh-specimen windows are liquid stool within 30 minutes, semiformed within 1 hour, and formed within 24 hours; when those windows cannot be met, fix immediately. Refrigerate unpreserved specimens awaiting pickup; preserved specimens are stable at room temperature. Collect into a clean, dry container or a toilet-mounted collection sheet; urine, toilet water, or soil either destroy trophozoites or introduce confusable free-living organisms.

The minimum work-up is three specimens collected over a maximum 10-day span, to catch organisms that shed intermittently. Strongyloides stercoralis may need up to seven specimens, or antigen/NAAT or a concentration/culture technique, because larval shedding is erratic. Formalin-ethyl acetate sedimentation recovers essentially all parasite forms, including operculate trematode and cestode eggs for which flotation is unreliable. Zinc-sulfate flotation is faster but under-recovers heavy, operculate, or unembryonated eggs. Permanent stains (trichrome, iron hematoxylin) resolve internal nuclear and cytoplasmic detail that wet mounts cannot; differential identification of intestinal amebae and flagellates depends on them.

Immunodiagnostics, molecular methods, quality, and safety

Commercial immunoassays exist for amebiasis, cryptosporidiosis, giardiasis, malaria, and trichomoniasis. They offer speed and often outperform routine O&P sensitivity, but none substitutes for microscopy when helminths or other non-targeted organisms are clinically possible. DFA methods visualize Giardia cysts and Cryptosporidium oocysts in fixed stool; sensitivity improves with centrifugation (500 × g for 10 minutes).

Malaria rapid diagnostic tests detect histidine-rich protein II (HRP-II, P. falciparum), parasite lactate dehydrogenase (pLDH), or parasite aldolase (both pan-Plasmodium). In the United States the FDA-approved RDT is BinaxNOW Malaria (HRP-II and aldolase). RDTs perform adequately for moderate-to-heavy P. falciparum infection but lose sensitivity at low density and for non-falciparum species. HRP-II can remain detectable for about 2 weeks after parasite clearance and can be falsely negative with hrp2/hrp3 deletions. Every RDT result, positive or negative, still requires confirmatory quantitative blood-film microscopy.¹

Serology is an adjunct when the organism cannot be recovered directly: deep-tissue infection (toxoplasmosis, toxocariasis), organ-confined disease where biopsy is undesirable (cysticercosis, echinococcosis), and light or subclinical infection (filariasis, schistosomiasis, strongyloidiasis). IgG documents exposure but rarely distinguishes active from past infection. Toxoplasmosis and babesiosis are exceptions in which IgM/IgA testing helps stage acute infection, although those antibodies can persist for up to 2 years. Toxoplasma IgG avidity helps date infection, particularly in pregnancy. Most parasitic serologies are sent to public-health or reference laboratories.

FDA-cleared NAATs exist for Trichomonas vaginalis, Giardia duodenalis, Cryptosporidium spp., Entamoeba histolytica, and Cyclospora cayetanensis, largely on multiplex gastrointestinal panels. Laboratory-developed NAATs extend coverage to Plasmodium, Babesia, Leishmania, Toxoplasma gondii, and Trypanosoma through reference laboratories. Open formats that manipulate amplified product require strict workflow separation.

Unpreserved parasitology specimens are potentially infectious. Malarial parasites and hemoflagellates remain infective in blood. Fresh stool can carry infective protozoan cysts, Taenia solium, Enterobius vermicularis, and Hymenolepis nana eggs, and Strongyloides filariform larvae. Trichuris, Ascaris, and hookworm eggs remain infective even in older specimens.

Plasmodium (malaria)

Malaria is an apicomplexan infection of erythrocytes transmitted exclusively by female Anopheles mosquitoes. Four species classically infect humans (P. falciparum, P. vivax, P. ovale, and P. malariae) plus the zoonotic P. knowlesi (Southeast Asia, morphologically confused with P. malariae and requiring PCR to distinguish). P. falciparum causes most malaria deaths. Any unexplained fever with travel to an endemic area requires STAT thick and thin films; if the first set is negative and suspicion remains, repeat every 12-24 hours for a total of three sets before ruling malaria out.¹

An infected mosquito injects sporozoites, which undergo exoerythrocytic schizogony in hepatocytes. Hepatic schizont rupture releases merozoites that invade erythrocytes: ring-form trophozoites → growing trophozoites → either erythrocytic schizonts that release more merozoites, or gametocytes taken up by the mosquito. The erythrocytic cycle takes about 48 hours (tertian: P. falciparum, P. ovale, P. vivax) or about 72 hours (quartan: P. malariae). P. vivax and P. ovale uniquely form dormant hepatic hypnozoites that cause true relapse. P. falciparum and P. malariae recrudesc from persisting blood-stage parasites. Transfusion- or congenitally acquired P. vivax/P. ovale infection never relapses because only mosquito-delivered sporozoites establish liver infection.

Young P. falciparum rings are about one-sixth the erythrocyte diameter versus one-third for the others. Appliqué/accolé forms and finding only rings (without mature asexual stages) strongly suggest P. falciparum. Finding schizonts or mature trophozoites in the periphery signals very severe disease. Require both red chromatin and blue cytoplasm before calling a structure a parasite; platelets superimposed on red cells, bacterial clumps, and precipitated stain can mimic parasites.

Percent parasitemia is calculated from the thin film: 96 parasitized cells among 2,400 erythrocytes = 96 ÷ 2,400 × 100 = 4.0%. Quantitative parasitemia is reported at diagnosis and trended during treatment of P. falciparum. Species-specific IFA cannot diagnose acute infection. Mixed infections occur most often with P. falciparum and P. vivax and should be called only when two genuinely distinct parasite populations are seen.

Babesia

Babesia is a tick-borne apicomplexan erythrocyte parasite with a broad animal reservoir. It can also be transmitted by transfusion or transplacentally. B. microti dominates in the northeastern and midwestern United States (Ixodes scapularis); B. duncani in the Pacific Northwest and northern California (I. pacificus); B. divergens and B. venatorum in Europe (I. ricinus). Fatality concentrates in asplenic or immunocompromised patients.

Babesia trophozoites are delicate ring forms easily confused with P. falciparum. Distinguishing features are a tetrad (“Maltese cross”) merozoite arrangement unique to Babesia; absence of ameboid trophozoites and true gametocytes; heterogeneous ring shapes; and extracellular forms in heavy infection. NAAT is available through CDC and some reference laboratories. IFA does not detect acute disease reliably but is used to screen blood donors. An FDA-licensed nucleic acid assay for B. microti in whole blood and an FDA-licensed antibody assay for plasma are available for blood-supply screening.

Hemoflagellates

Both Trypanosoma and Leishmania belong to the Kinetoplastea. Their kinetoplast is a dense mass of circular DNA within a single large mitochondrion, visible on Giemsa stain.

African trypanosomiasis is transmitted by tsetse flies (Glossina). T. brucei rhodesiense causes acute zoonotic disease in East Africa and may kill before CNS involvement. T. brucei gambiense causes chronic sleeping sickness in West Africa and can produce posterior cervical lymphadenopathy (Winterbottom sign). Bloodstream trypomastigotes are up to 30 μm, with a small kinetoplast; dividing forms may be seen (unlike T. cruzi). Diagnosis uses thick/thin films, buffy coat, lymph-node or bone-marrow aspirate, or spun CSF. Total IgM is elevated in blood and CSF, and CSF shows 50-500 mononuclear cells/μL.

American trypanosomiasis (Chagas disease, T. cruzi) is transmitted by reduviid kissing bugs, which defecate infective trypomastigotes at the bite site. T. cruzi trypomastigotes are shorter (~20 μm) and have a larger kinetoplast. They replicate only as intracellular amastigotes and never divide as bloodstream trypomastigotes. Acute disease in children under 5 years features malaise, fever, hepatosplenomegaly, myocarditis, periorbital swelling at the inoculation site (Romaña sign), or swelling elsewhere (chagoma). Chronic disease with megaesophagus, megacolon, and cardiac conduction disease reflects autoimmune-mediated destruction of parasympathetic effector cells. Trypomastigotes are visible on blood film only during acute disease or reactivation. Molecular testing is preferred when morphologic diagnosis is not feasible.

Leishmaniasis, transmitted by sand flies (Phlebotomus, Old World; Lutzomyia, New World), takes amastigote form in the mammalian host and promastigote form in the vector. Species cannot be distinguished morphologically. Cutaneous disease includes Old World L. tropica/L. major/L. aethiopica and New World L. mexicana (Chiclero ulcer of the earlobe) and L. braziliensis/Viannia. Mucocutaneous disease (espundia) is chiefly L. braziliensis. Visceral disease (kala-azar) is L. donovani or L. infantum (syn. L. chagasi). Diagnosis rests on demonstrating 2-4 μm amastigotes in lesion-edge imprints or in buffy coat, lymph node, bone marrow, spleen, or liver. Unlike Histoplasma yeasts, they do not stain with GMS or PAS. Promastigotes can be cultured in Novy-MacNeal-Nicolle or supplemented Schneider’s Drosophila medium (2-5 days to positivity, held 4 weeks).

Toxoplasma gondii

Cats and other felids are the only definitive hosts. Oocysts shed in feces become infective after 1-5 days of environmental sporulation, typically 48-72 hours. Humans acquire infection by eating meat (especially lamb or pork) harboring tissue cysts or by ingesting oocyst-contaminated material. Transfusion, transplantation, and transplacental transmission also occur.

Most immunocompetent infection is asymptomatic or mimics mononucleosis. Congenital infection in the first half of pregnancy risks intrauterine death, microcephaly, or hydrocephaly with intracranial calcification; second-half infection is often asymptomatic at birth but may later show fever, hepatosplenomegaly, jaundice, or chorioretinitis. In immunocompromised hosts, especially AIDS, disease is usually CNS reactivation.

Actively dividing tachyzoites are crescent/oval, about 3 × 7 μm; latent tissue cysts (bradyzoites) are spherical, up to 30 μm. Serology remains the primary diagnostic route in immunocompetent hosts. Antibodies appear at 1-2 weeks and peak at 6-8 weeks. IgM assays help date congenital or acute infection but false positives and prolonged IgM persistence limit their specificity. PCR is now central to testing pregnant women, neonates, and immunocompromised hosts, and to diagnosing encephalitis and disseminated disease.

Opportunistic free-living amebae

Examine fresh CSF immediately, at room temperature, for motile N. fowleri trophozoites. Do not refrigerate or freeze before the wet mount. A wet mount is supportive, not confirmatory; confirm with PCR and/or immunofluorescence. Avoid Gram stain because heat fixation can destroy the amebae. A negative wet mount does not exclude PAM. Acanthamoeba keratitis presents as a painful paracentral corneal ring infiltrate; diagnosis is by demonstrating trophozoites or cysts in corneal scrapings (Giemsa, PAS, trichrome, or calcofluor white for cysts) or by culture on non-nutrient agar overlaid with bacteria.²