TABLE 3.

Diagnostic techniques available for various blood and tissue protozoan infections

Organism(s)	Techniques	Description
Microsporidia	Light microscopy, TEM, PCR, real-time PCR, IFAT, DAT	Light microscopy is cheap and applicable to any clinical specimen; however, differentiation of species may be difficult by light microscopy; transmission electron microscopy may be useful for differentiation of species, although it can be expensive; several PCR and real-time PCR assays have been developed for detection of various species, although PCR requires a special apparatus; real-time PCR can be more rapid than conventional PCR, as it does not require agarose gel electrophoresis; generally, real-time PCR is more sensitive than conventional PCR; sequencing of PCR products may be necessary for species differentiation; indirect fluorescent antibody tests and DAT are available for some species only
Leishmania	Conventional and real-time PCR, light microscopy, culture systems, serological techniques, DAT, rK39 dipstick test, latex agglutination test, QBC tube technique	Staining of blood films followed by light microscopy is definitive but is less sensitive than molecular techniques; real-time PCR can also be used to monitor patients' responses to therapy but requires a special apparatus; sequencing of PCR products can also allow differentiation of species; culture systems are useful but may be impractical; in Ethiopia DAT is preferred for diagnosis of L. donovani infection, as it is cheap, rapid, and sensitive; the rK39 dipstick test is cheap, rapid, and sensitive for diagnosis in ICT patients; however, the rK39 test demonstrated low sensitivities of ∼20% in European VL patients infected with HIV; the latex agglutination test is sensitive and noninvasive (applied to urine specimens); enzyme-linked immunosorbent assay and other serological techniques may be of limited use in IC patients, as circulating antibodies may not be detected during an active infection; the QBC tube technique is useful for the diagnosis of VL
Trypanosoma cruzi	Light microscopy, real-time PCR, PCR, ELISA, IFAT, QBC tube technique	Light microscopy is definitive; trypanosomes are more readily observed in peripheral blood specimens in the acute stage and in IC patients; the blood stains are useful for blood stages, and hematoxylin and eosin stains are used for diagnosis of the intracellular stages, as seen in solid tissues; live, motile trypanosomes may be observed microscopically in the buffy coat or CSF (if CNS involvement is apparent); PCR and real-time PCR are sensitive but require a special apparatus; PCR can also be useful for monitoring treatment efficacy; the QBC tube technique is useful for diagnosis during the acute phase of Chagas' disease but is less useful during the chronic phase of the disease
Trypanosoma brucei rhodesiense, Trypanosoma brucei gambiense	Light microscopy, PCR, real-time PCR, ELISA, CATT, QBC tube technique	The blood stains are useful for light microscopic diagnosis when applied to blood or CSF; the microhematocrit centrifugation technique also enables the visualization of live motile trypanosomes microscopically; several PCRs and real-time PCRs are also available; PCR is sensitive and can allow differentiation of T. brucei subtypes; enzyme-linked immunosorbent assay is also useful; the CATT is available for T. b. gambiense sleeping sickness and is the most widely used technique in areas of endemicity, as it is cheap, rapid, and sensitive; a CSF white blood cell count is useful to predict the stage of sleeping sickness; the QBC tube technique has demonstrated greater sensitivity for detection of African trypanosomes than the microhematocrit centrifugation technique
Non-human-infecting Trypanosoma	Light microscopy, various molecular techniques	No specific diagnostics are available, as human infections are rare; infections are typically diagnosed by a combination of light microscopy and various molecular techniques
Lower trypanosomatids	TEM, light microscopy, various molecular techniques	As with the non-human-infecting Trypanosoma spp., no specific diagnostics are available, as human infections are rare; infections were typically diagnosed by a combination of light microscopy and various molecular techniques; transmission electron microscopy was used in 1 case
Toxoplasma gondii	Light microscopy, PCR, real-time PCR, ELISA, IgG avidity test for pregnancy	Observation of live free tachyzoites in stained smears of blood or CSF is diagnostic of an active infection; enzyme-linked immunosorbent assay can be useful but fails to differentiate between active and benign infections; PCR performed on peripheral blood is also diagnostic for an active Toxoplasma infection; real-time PCR assays are available, which are extremely sensitive and specific. The IgG avidity test is useful in pregnancy to predict the risk that a Toxoplasma infection poses to the fetus; however, the results of serological tests can be complex and difficult to interpret for pregnant woman and neonates
Neospora caninum	Serology, PCR	Tachyzoites of Neospora are virtually indistinguishable from those of Toxoplasma; as such, light microscopy cannot differentiate the two; few diagnostics are available for human infections, as human neosporosis has never been reported; however, various serological techniques and PCR assays have been developed, which show good sensitivity and specificity
Babesia	Light microscopy, PCR, laboratory rodent inoculation, ELISA, IFAT, QBC tube technique	Microscopic analysis of stained blood smears is useful for diagnosis in the early stages of infection or in IC patients; live-rodent inoculation is sensitive and was once used for diagnosis but has been abandoned due to practicality issues; PCR can be applied to blood specimens and is sensitive and extremely useful for the characterization of new isolates; enzyme-linked immunosorbent assay and IFAT may be useful for diagnosis of B. microti infections but are less applicable to B. divergens infections; for definitive confirmation of Babesia infection at the species level, amplification of the SSU rDNA genes by Babesia-specific PCR followed by sequencing of the PCR product can be performed; the QBC tube technique can also be used for the microscopic diagnosis of Babesia infection
Acanthamoeba (GAE)	Light microscopy, cultivation, IFAT, PCR	Acanthamoeba spp. can be identified in Gram or Giemsa stains of CSF; calcofluor white can also be used for microscopic detection of Acanthamoeba; cultivation of trophozoites from CSF or tissue lesions by inoculation of the specimen onto neomycin-nalidixic acid agar plates seeded with a layer of Escherichia coli or Enterobacter aerogenes cells is a useful diagnostic tool; cultivation of Acanthamoeba can also provide the material for downstream molecular characterization; indirect fluorescent antibody techniques and PCR are also available; PCR is probably the most sensitive technique available for diagnosis of Acanthamoeba infection
Balamuthia (BAE)	Light microscopy, PCR, IFAT, electron microscopy	Several stains are useful for light microscopic diagnosis, although the hematoxylin and eosin stain is usually applied to fixed tissue sections; however, Balamuthia trophozoites may be difficult to see; the use of an immunofluorescent antibody technique is a sensitive and specific alternative to light microscopy and is the gold standard for diagnosis of Balamuthia infection; PCR is also useful for diagnosis, as it is sensitive; some PCR assays can distinguish between Balamuthia and other free-living amoebae; electron microscopy is useful, although it is expensive
Naegleria fowleri (PAM)	Light microscopy, PCR, real-time PCR, cultivation	As the onset of PAM is rapid, live, motile trophozoites can be readily observed in wet mounts of patient CSF; light microscopy of fixed, stained tissue smears is also useful. PCR and real-time PCR are available; cultivation of Naegleria from CSF enables downstream molecular analysis of isolates
Sappinia pedata	Light microscopy, PCR	Only 1 reported case, which was diagnosed by light microscopy; recently, 3 real-time PCR assays were also developed for Sappinia spp.