Parasitology: Specimens, Blood and Tissue Protozoa

Travel, residence, immune status, symptoms, exposure, treatment, and the suspected parasite should accompany the specimen. These details determine which concentration method, stain, antigen assay, molecular target, or reference laboratory is appropriate.

The thick and thin films serve different purposes. A thick film concentrates blood after erythrocytes lyse during staining, which makes it the more sensitive screening preparation. A thin film preserves erythrocyte shape, parasite architecture, and the relationship between the organism and its host cell. Species identification and percentage parasitemia depend mainly on the thin film.

1. Collect capillary blood or venous blood into EDTA. For a fingerstick, wipe away the first drop and use freely flowing blood. Do not collect blood from an exposed venipuncture needle. EDTA is acceptable and practical, but storage progressively distorts parasites and erythrocytes.
2. Prepare at least 2 thick and 2 thin films. A standardized thick film commonly uses about 6 µL spread into a circle 10 to 12 mm in diameter. Validate the volume, diameter, and drying time that give complete dehemoglobinization without washing the film from the slide.
3. Air-dry films flat and protect them from insects, dust, heat, and formalin fumes. Fix only the thin film in absolute methanol. Methanol or heat fixation prevents the erythrocyte lysis needed in the thick film.
4. Stain with a validated Romanowsky method. Giemsa in buffered water at approximately pH 7.2 remains the reference method for malaria because it preserves parasite chromatin, cytoplasm, pigment, and stippling. Wright or Wright-Giemsa can demonstrate parasites on a thin film when the procedure is validated. A methanol-containing stain must not fix the thick film before dehemoglobinization.
5. Scan both preparations at 10× or 20× for distribution, stain quality, and large blood parasites. Examine the thick film under 100× oil in an evenly stained area containing roughly 10 to 20 leukocytes per field. Examine the thin film in the monolayer where erythrocytes touch with little overlap. Cells at the extreme feathered edge are distorted.
6. For suspected malaria, examine at least 100 thick-film oil-immersion fields before reporting a negative conventional examination. A laboratory may require 200 to 300 fields or the whole usable film when parasite density is expected to be low. Follow the validated procedure and document the extent of review.^{33 Centers for Disease Control and Prevention. DPDx: blood specimens, specimen processing, staining, and…}

Stain precipitate is irregular and usually lies above or between cells. A platelet over an erythrocyte may resemble a chromatin dot but lacks a surrounding parasite cytoplasm. Howell-Jolly bodies are round, dense nuclear remnants without blue parasite cytoplasm. A true malaria or Babesia call requires a reproducible population of organisms with internally consistent structure.

A calibrated ocular micrometer is useful whenever size helps narrow an identification, especially for stool protozoa, oocysts, eggs, larvae, and tissue forms. Calibrate each objective used for measurement. Size supports identification together with nuclear structure, cytoplasmic features, host-cell changes, specimen source, and clinical context.

Peripheral blood concentration methods have narrower roles. A buffy-coat preparation can increase recovery of trypanosomes and microfilariae. The Knott method and membrane filtration concentrate selected microfilariae.

Female Anopheles mosquitoes transmit human malaria naturally. Malaria can also be acquired congenitally or through transfusion, transplantation, shared needles, or laboratory exposure. Six taxa are established causes of human malaria: Plasmodium falciparum, P. vivax, P. malariae, P. knowlesi, P. ovale curtisi, and P. ovale wallikeri. The two P. ovale species are reported together as the P. ovale complex when they have not been distinguished molecularly.^{4,54 Centers for Disease Control and Prevention. Evaluation and diagnosis of malaria.5 Centers for Disease Control and Prevention. DPDx: malaria.}

The mosquito injects sporozoites, the stage infective to humans. Hepatic schizogony (asexual multiplication inside liver cells) releases merozoites, which invade erythrocytes and develop through ring, growing trophozoite, and schizont stages. Some become gametocytes, the stage infective to the mosquito. The mosquito is the definitive host because sexual reproduction occurs there. Humans are intermediate hosts. P. vivax and the P. ovale complex form dormant hepatic hypnozoites that can cause relapse. Every malaria species can persist or recur from blood-stage infection, termed recrudescence.

The approximate erythrocytic cycles are 24 hours for P. knowlesi, 48 hours for P. falciparum, P. vivax, and the P. ovale complex, and 72 hours for P. malariae. Clinical fever patterns are often irregular, especially early in disease, in mixed infection, or after antimalarial exposure. Never make the laboratory species call from periodicity.

Emergency testing sequence

A patient with fever or compatible illness and possible exposure in a malaria-endemic area requires an immediate thick and thin film. Report the presence of parasites, the best-supported species identification, and parasitemia as soon as each is available. If the first films are negative and malaria remains possible, repeat films every 12 to 24 hours for a total of 3 sets. A rapid, laboratory-validated molecular test may run concurrently and can shorten that sequence when its negative result has been validated to exclude malaria in the tested population. Reference PCR is especially useful for low-density infection, mixed infection, and species overlap, but a send-out result must not delay initial microscopy.^{4,64 Centers for Disease Control and Prevention. Evaluation and diagnosis of malaria.6 Centers for Disease Control and Prevention. Malaria diagnostic tests.}

Species morphology on thin films

Babesia species are intraerythrocytic apicomplexans transmitted by ticks. Transfusion is a well-established route, and congenital transmission occurs rarely. In the northeastern and upper midwestern United States, B. microti cycles mainly between Ixodes scapularis and the white-footed mouse, Peromyscus leucopus. Rare western US infections are caused by B. duncani. Current evidence supports Dermacentor albipictus as its vector and mule deer as an important reservoir. Do not assign Ixodes pacificus as the vector for B. duncani. European human babesiosis includes B. divergens and B. venatorum, commonly associated with Ixodes ricinus.^{8,98 Centers for Disease Control and Prevention. DPDx: babesiosis.9 Swei A, et al. Evidence for transmission of the zoonotic apicomplexan parasite… 2019.}

The tick injects sporozoites, which enter erythrocytes directly without a hepatic stage. Asexual trophozoites and merozoites are the diagnostic forms. Some parasites become gametes that can continue the sexual cycle after ingestion by the tick, the definitive host, but those gametes cannot be distinguished by routine light microscopy. Humans usually do not maintain the natural enzootic cycle.

Blood-film findings

Babesia trophozoites range from tiny delicate rings to pleomorphic, vacuolated, ameboid, or pyriform forms. Infected erythrocytes remain normal in size. Multiple infection, paired pear-shaped forms, and occasional extracellular parasites support babesiosis. Pigment is absent. A four-member tetrad, the Maltese cross, is diagnostic when present but is uncommon. Its absence does not exclude babesiosis. Species cannot be assigned by morphology alone.^{88 Centers for Disease Control and Prevention. DPDx: babesiosis.}

Examine both thick and thin films, recognizing that tiny Babesia forms may be difficult to see in a dehemoglobinized thick film. Review a broad area of the thin film, often 200 to 300 oil-immersion fields in a low-density infection under a validated procedure. If films are negative and suspicion persists, use PCR. A species-specific B. microti PCR can miss another Babesia species, so exposure in the western United States, Europe, or another region may call for a pan-Babesia assay or additional species targets. If travel and morphology leave malaria in the differential, pursue molecular testing for both groups without waiting to find a Maltese cross.

Calculate percent infected erythrocytes with the same thin-film equation used for malaria. Quantitation establishes baseline burden and helps follow response in selected patients. A single positive antibody result does not prove active babesiosis because antibody can persist. A 4-fold rise in B. microti IgG in paired sera supports recent infection. Smear or PCR establishes current parasitemia. Blood-donor antibody and NAT screening protects the blood supply. Those assays are not interchangeable with clinical diagnostic tests.^{1010 Krause PJ, et al. Clinical practice guidelines by the Infectious Diseases Society… 2021.}

Trypanosoma and Leishmania are kinetoplastids. Their kinetoplast is a compact mass of mitochondrial DNA that stains as a dark red-purple dot or short rod near the nucleus on a Romanowsky-stained preparation.

African trypanosomiasis

A tsetse fly in the genus Glossina injects metacyclic trypomastigotes. Bloodstream trypomastigotes multiply by longitudinal binary fission in blood, lymph, and later CSF. Trypanosoma brucei gambiense occurs in West and Central Africa, usually causes a slowly progressive illness, and has humans as its principal reservoir, although animals can be infected. T. b. rhodesiense occurs in eastern and southern Africa, usually progresses more rapidly, and is a zoonosis maintained in cattle and wildlife.^{11,1211 Centers for Disease Control and Prevention. DPDx: African trypanosomiasis.12 World Health Organization. Trypanosomiasis, human African (sleeping sickness).}

The two subspecies are morphologically indistinguishable. Trypomastigotes are approximately 14 to 33 µm long, with a small posterior kinetoplast, central nucleus, undulating membrane, and free flagellum. Slender and stumpy forms may occur, and dividing forms can be seen. T. b. rhodesiense often produces a higher parasitemia, so fresh blood, thick and thin films, or a buffy-coat concentration may show organisms readily. Gambiense disease has a lower parasitemia and often requires repeated examination and concentration.

Confirmation requires demonstration of the parasite in chancre fluid, lymph-node aspirate, blood, bone marrow in selected cases, or CSF. Antibody rapid tests and the card agglutination test for trypanosomiasis screen for T. b. gambiense in appropriate populations. They do not confirm infection and do not diagnose T. b. rhodesiense. Where treatment selection still requires CSF staging, trypanosomes in CSF or more than 5 leukocytes/µL supports second-stage disease. A patient with no CSF trypanosomes and 5 or fewer leukocytes/µL meets the conventional first-stage laboratory definition. Molecular assays are specialized adjuncts and are not used alone for routine confirmation.

Chagas disease

Triatomine insects transmit Trypanosoma cruzi. Infective metacyclic trypomastigotes are passed in bug feces and enter through the bite site, conjunctiva, or another mucosal surface. Oral, congenital, transfusion, transplantation, and laboratory transmission also occur. In mammalian cells the parasite becomes an amastigote, divides, and differentiates into a bloodstream trypomastigote. Bloodstream forms circulate without dividing.

Acute infection, congenital infection, reactivation during immunosuppression, and some orally acquired outbreaks can produce detectable parasitemia. A T. cruzi trypomastigote is about 12 to 30 µm long, often curves into a C or U shape, and has a large terminal or subterminal kinetoplast, central nucleus, undulating membrane, and free flagellum. Shape alone is not specific. Tissue amastigotes occur in clusters and cannot be reliably separated from Leishmania amastigotes without epidemiologic and ancillary evidence.

Use fresh blood concentration, stained blood films, and a validated PCR for acute, congenital, or reactivated disease. Chronic infection usually has parasitemia below the sensitivity of microscopy and intermittently below the sensitivity of PCR. Diagnosis therefore requires positive results from 2 serologic assays that use different antigens or formats. Use a third assay to resolve discordant results. A negative blood PCR does not exclude chronic Chagas disease. Chronic cardiomyopathy and gastrointestinal disease reflect persistent infection with inflammatory injury to myocardium, smooth muscle, and the autonomic nervous system. Current evidence favors this mechanism over a purely autoimmune loss of parasympathetic cells.^{1313 Centers for Disease Control and Prevention. Clinical testing and diagnosis for Chagas disease.}

Leishmaniasis

Female phlebotomine sand flies transmit Leishmania: Phlebotomus in the Old World and several New World genera historically grouped as Lutzomyia. Reservoirs vary by species and include humans, dogs, and wild mammals. The metacyclic promastigote infects the mammal. Macrophages ingest it, and intracellular amastigotes multiply in the phagolysosomal compartment. The sand fly ingests amastigotes during a blood meal, and promastigotes develop in the fly.

Only amastigotes occur in human tissue. They are usually 1 to 5 µm long by 1 to 2 µm wide and contain a nucleus and a smaller rod-shaped kinetoplast. Organisms may be extracellular when cells rupture. Species are morphologically indistinguishable. T. cruzi amastigotes have the same basic structure. Histoplasma yeasts lack a kinetoplast and may show narrow-based budding. Gomori methenamine silver (GMS) stain highlights fungal cell walls. Fungal stains do not reliably stain Leishmania.

Cutaneous and mucosal disease require sampling from active abnormal tissue. Prepare direct smears or touch preparations, histopathology, culture, and molecular testing as the specimen allows. Visceral disease is associated chiefly with the L. donovani complex, including L. donovani and L. infantum. The latter was historically reported in the Americas as L. chagasi. Bone marrow is commonly sampled first in North America. Splenic aspirate has the highest sensitivity but also a serious hemorrhage risk. Blood or buffy coat may be useful, especially in immunocompromised patients.

Microscopy or histopathology can establish infection when characteristic amastigotes are demonstrated. Culture in Novy-MacNeal-Nicolle medium or a suitable tissue-culture medium yields promastigotes and may require up to 4 weeks of incubation. PCR improves sensitivity and enables species identification, which can affect prognosis and management. Serology supports visceral leishmaniasis in an immunocompetent patient but performs poorly for cutaneous disease and may be falsely negative in patients with HIV. A positive antibody result does not prove active visceral infection in an endemic population.^{1414 Centers for Disease Control and Prevention. DPDx: leishmaniasis.}

Felids are the definitive hosts of Toxoplasma gondii. They shed unsporulated oocysts, which usually become infective after 1 to 5 days in the environment. A mature oocyst is about 10 to 12 µm and contains 2 sporocysts, each with 4 sporozoites. Humans acquire infection by ingesting sporulated oocysts or tissue cysts in undercooked meat. Congenital, transplantation-associated, transfusion-associated, and laboratory transmission also occur.

Tachyzoites are crescent shaped, approximately 4 to 8 µm by 2 to 3 µm, and multiply during active dissemination. Tissue cysts are commonly 5 to 50 µm or larger and contain numerous slender bradyzoites. A bradyzoite is the slow-growing organism inside the tissue cyst. Each cyst contains many bradyzoites. Tissue cysts persist in neural and muscle tissue and can reactivate during immunosuppression.^{1515 Centers for Disease Control and Prevention. DPDx: toxoplasmosis.}

Diagnostic patterns

• Immunocompetent patient: Begin with serum IgG and IgM when recent systemic infection is suspected. IgG documents infection at some time. IgM may persist for 18 months or longer and can be falsely positive, so a result used to date infection requires confirmation by a toxoplasmosis reference laboratory. High IgG avidity early in pregnancy can exclude infection acquired within the assay's stated recent interval. Low avidity does not prove recent infection.
• Pregnancy and fetus: Interpret maternal IgG, IgM, confirmatory serology, avidity, and serial specimens together. PCR of amniotic fluid collected at the appropriate gestational interval detects many fetal infections. Maternal blood PCR is not the routine test for fetal infection.
• Newborn: Maternal IgG crosses the placenta, so neonatal IgG alone cannot establish congenital infection. Reference-laboratory IgM and IgA, comparison of maternal and infant antibody profiles, PCR or direct detection when available, and serial infant IgG are used together. Persistence or rise of infant IgG beyond the expected loss of maternal antibody supports congenital infection.
• Immunocompromised patient: Toxoplasmic encephalitis usually represents reactivation. Serum IgG is commonly positive and IgM commonly absent. CSF PCR is highly specific but has limited sensitivity, especially after treatment begins. A negative result does not exclude encephalitis. Demonstration of tachyzoites or tissue cysts by histology, immunohistochemistry, or PCR in CSF, blood, bronchoalveolar lavage, ocular fluid, or biopsy can establish disease in the appropriate syndrome.

Earlier maternal infection is less likely to transmit across the placenta but more likely to cause severe fetal injury when transmission occurs. Transmission becomes more frequent later in gestation. Many congenitally infected infants appear well at birth. Report wording should not imply that an asymptomatic newborn is uninfected or that a single antibody class shows whether or when infection occurred.

Run positive, negative, and internal controls with special stains, antigen assays, and NAATs at the frequency required by each method. New reagent lots need performance checks. Reference slides and image atlases support morphology review. Unknowns and competency specimens assess whether the examiner can recognize low-density and mixed populations. Ocular micrometers require objective-specific calibration.

Name the organism only to the level the evidence supports. When the evidence does not support a species, report a provisional Plasmodium or kinetoplastid identification and state the differential, urgency, and confirmation plan. Preserve representative positive films, document the fields or cells counted, and arrange prompt expert or public health review when the morphology is unusual, the parasite is rare, or the result conflicts with exposure history. At the bench, treat every malaria request as a stat test, from the first film to the parasitemia.