Informational Tables

Table 1.2  Body site, specimen and procedures, recommended methods, relevant parasites, and comments

Body site

Procedures and specimens

Recommended methods and relevant parasitesa

Comments

Amniotic fluid

PCR (and/or culture): fresh material

Animal inoculation -(toxoplasmosis)

PCR based on the detection of highly repetitive gene sequences is the method of choice

Only applicable to confirm suspected prenatal Toxoplasma infections.

Blood

Microscopyb: thin and thick blood films; fresh blood or EDTA blood (fill EDTA tube completely with blood, then mix)

Giemsa or other blood stains (all blood parasites); hematoxylin-based stain (sheathed microfilariae); for malaria, thick and thin blood films are mandatory and should be prepared within 30–60 min of blood collection via venipuncture (other tests may be used as well); Wright-Giemsa stain or Diff-Quik (rapid stains) can also be used

Most drawings and descriptions of blood parasites are based on Giemsa-stained blood films. Although Wright’s stain (or Wright-Giemsa combination stain) will work, stippling in malaria may not be visible and the organisms’ colors will not match the descriptions. However, with other stains (those listed above, in addition to some of the “rapid” blood stains), the organisms are detectable on the blood films. The use of blood collected with anticoagulant (rather than fresh) has direct relevance to the morphology of malaria organisms seen in peripheral blood films. If the blood smears are prepared after more than 1 h, stippling may not be visible, even if the correct pH buffers are used. Also, if blood is kept at room temperature (with the stopper removed), the male microgametocyte may exflagellate and fertilize the female macrogametocyte, and development continues within the tube of blood (as it would in the mosquito host). The ookinete may actually resemble a Plasmodium falciparum gametocyte. The microgamete may resemble spirochetes; white blood cells serve as the internal quality control for every blood film, if the leukocytes exhibit acceptable morphology, parasites present will also be acceptable per staining and morphology characteristics 

Concentration methods: EDTA blood

Buffy coat, fresh blood films for detection of moving microfilariae or trypanosomes; QBC, a screening method for blood parasites (hematocrit tube contains acridine orange), has been used for malaria, Babesia, trypanosomes, and microfilariae; it is usually impossible to identify malaria organisms to the species level; this requires high levels of training; confirmation to species often requires examination of thin blood films

Antigen detection: EDTA-blood for malaria, serum or plasma for circulating antigens (hemolyzed blood can interact in some tests)

Commercial test kits for malaria and some microfilariae

PCR: EDTA-blood, ethanol- fixed or unfixed thin and thick blood films, coagulated blood, possible with hemolyzed or frozen blood samples

Sensitivity not higher than thick films for Plasmodium spp., much more sensitive for Leishmania (peripheral blood is used from immunodeficient patients only). Sequencing of PCR product is often used for species or genotype identification

No commercial tests available at this time; some reference laboratories offer PCR or other molecular procedures for all five species of human malaria, Babesia, Leishmania, and trypanosomes.; high laboratory standards required (may work with frozen, coagulated, or hemolyzed blood samples).

Specific antibody detection: serum or plasma, anticoagulated or coagulated blood (hemolyzed blood can cause problems in some tests)

Most commonly used are EIA (many test kits commercially available), EITB (commercially available for some parasites), and IFA

Many laboratories are using in-house tests; only a few fully defined antigens are available; sensitivities and specificities of the tests should be validated by the laboratory

Bone marrow

Biopsy specimens or aspirates Microscopy: thin and thick films with aspirate collected in EDTA

Giemsa or other blood stains (including rapid stains) (all blood parasites)

Leishmania amastigotes are recovered in cells of the reticuloendothelial system; if films are not prepared directly after sample collection, infected cells may disintegrate. Sensitivity of microscopy low, so it should be used only in combination with other methods.

Cultures: sterile material in EDTA or culture medium

Culture for Leishmania (or Trypanosoma cruzi)

Positive control cultures should be performed with patient cultures

PCR: aspirate in EDTA

PCR for blood parasites including Leishmania and Toxoplasma and rare other parasites

May be available in specialized testing laboratories

Central nervous system

Microscopy: spinal fluid and CSF (wet examination, stained smears), brain biopsy specimen (touch or squash preparations, stained)

Stains: Giemsa or other blood stains, Warthin-Starry stain (trypanosomes, Toxoplasma); Giemsa, Warthin-Starry stain, trichrome, or calcofluor (amebae [Naegleria—PAM, Acanthamoeba or Balamuthia—GAE]); Giemsa, acid-fast, PAS, modified trichrome, silver methenamine (microsporidia) (tissue Gram stains also recommended for microsporidia in routine histologic preparations); H&E, routine histology (larval cestodes, Taenia solium cysticerci, Echinococcus spp.)

If CSF is received (with no suspect organism suggested), Giemsa would be the best choice; however, modified trichrome or calcofluor is also recommended as a second stain (amebic cysts, microsporidia). If brain biopsy material is received (particularly from an immunocompromised patient), cultivation is recommended for microsporidial isolation and PCR for identification to the species or genotype level. A small amount of the sample should always be stored frozen for PCR analyses in case the results of the other methods are inconclusive.

Culture: sterile aspirate or biopsy material (in physiologic NaCl)

Free-living amebae (exception: Balamuthia does not grow in the routine agar/bacterial overlay method), microsporidia, and Toxoplasma (can be cultured in tissue culture media

PCR: aspirate or biopsy material fresh, frozen, or fixed in ethanol

Protozoa and helminths, species and genotype characterization

Cutaneous ulcers

Microscopy: aspirate, biopsy (smears, touch or squash preparations, histologic sections)
Cultures (less common)PCR: aspirate, biopsy material fresh, frozen, or fixed in ethanol

Giemsa or other blood stains (Leishmania); H&E, routine histology (Acanthamoeba spp., Entamoeba histolytica)
Leishmania (often bacterial contamination, culture problems), free-living amebae, Leishmania (species identification)

The most likely causative parasites would be Leishmania, which would stain with Giemsa. PAS could be used to differentiate Histoplasma capsulatum from Leishmania in tissue. In immunocompromised patients, skin ulcers have been documented with Acanthamoeba or Entamoeba histolytica amebae as causative agents; cultures of material from cutaneous ulcers may be contaminated with bacteria; PCR would be the method of choice.

Eyes

Microscopy: smears, touch or squash preparations; biopsy specimens, scrapings, contact lens, sediment of lens solution

Calcofluor for cyst only (amebae [Acanthamoeba]); Giemsa for trophozoites and cysts (amebae); modified trichrome (preferred) or silver methenamine stain, Warthin-Starry stain, PAS, acid-fast, (microsporidial spores), H&E, routine histology, Warthin-Starry stain (cysticerci, Loa loa, Toxoplasma)

Some free-living amebae (most commonly Acanthamoeba) have been implicated as a cause of keratitis. Although calcofluor stains the cyst walls, it does not stain the trophozoites. Therefore, in suspect cases of amebic keratitis, both stains should be used. H&E (routine histology) can be used to detect and confirm cysticercosis. The adult worm of Loa loa, when removed from the eye, can be stained with a hematoxylin-based stain (Delafield’s) or can be stained and examined by routine histology.

Culture: fresh material (see above) in PBS supplemented with antibiotics if possible to avoid bacterial growth

Cultures: free-living amebae, Toxoplasma, microsporidia

PCR: fresh material in physiologic NaCl or PBS, ethanol or frozen

Free-living amebae, Toxoplasma, microsporidian species and genotype identification

Microsporidia: confirmation to the species or genotype levels can be done by PCR and sequence analyses or EM; however, the spores could be found by routine light microscopy with modified trichrome, calcofluor, and/or tissue Gram stains.

Intestine

Stool and other intestinal material

Concentration methods: ethyl acetate sedimentation of SAF-fixed stool samples (most protozoa); flotation or combined sedimentation flotation methods (helminth ova); agar or Baermann concentration (larvae of Strongyloides spp., fresh stool required)

Stool fixation with formalin or formalin-containing fixatives preserves parasite morphology (trophozoites marginal), allows prolonged storage (room temperature) and long transportation, and prevents hatching of Schistosoma eggs, but makes Strongyloides larval concentration difficult and impedes further PCR analyses. Single vial Universal fixative now available (no mercury, no formalin, no PVA); can be used for O&P, most fecal immunoassays, special stains, some PCR

Microscopy: stool, sigmoidoscopy material, duodenal contents (all fresh or preserved), direct wet smear, concentration methods

Direct wet smear (direct examination of unpreserved fresh material is also used (motile protozoan trophozoites; helminth eggs, protozoan cysts may also be detected)

Agar plate for Strongyloides more sensitive than routine O&P

Taeniid eggs cannot be identified to the species level

Stains: trichrome or iron hematoxylin (intestinal protozoa); modified trichrome (microsporidia); modified acid-fast (Cryptosporidium, Cyclospora, Cystoisospora)

Microsporidia: confirmation to the species or genotype levels requires PCR or EM; however, modified trichrome and/or calcofluor stains can be used to confirm the presence of spores.

Anal impression preparation

Adhesive cellulose tape, no stain (Enterobius vermicularis)

Four to six consecutive negative tapes are required to rule out infection with pinworm (Enterobius vermicularis).

Adult worms or tapeworm segments (proglottids)

Carmine stains (rarely used for adult worms or cestode segments). Proglottids can usually be identified to the genus level (Taenia, Diphyllobothrium, Hymenolepis) without using tissue stains

Worm segments can be stained with special stains. However, after dehydration through alcohols and xylenes (or xylene substitutes) without prior staining, the sexual organs and the branched uterine structure will be visible, allowing identification of the proglottid to the species level, providing the proglottids are gravid

Antigen detection (fresh or frozen material, suitability of fixation is test dependent)

Commercial immunoassays, e.g., EIA, FA, cartridge formats (Entamoeba histolytica, the Entamoeba histolytica/E. dispar group, Giardia lamblia, Cryptosporidium spp.); in-house tests for T. solium and T. saginata and other parasites

Coproantigens can be detected in the prepatent period and independently from egg excretion.

PCR: fresh, frozen, or ethanol- fixed material

Few commercial tests available; primers for genus or species identification of most helminths and protozoa are published; commercial instrumentation available, Entamoeba histolytica, Giardia lamblia, Cryptosporidium, Cyclospora cayetanensis

Due to potential inhibition after DNA extraction from stool samples, concentration or isolation methods may be required prior to DNA extraction. However, new DNA isolation kits facilitate isolation of high-quality DNA from stool. Sequence analyses may be required for species or genotype identification.

Biopsy specimens Microscopy: fixed for histology or touch or squash preparations for staining

H&E, routine histology, Warthin-Starry stain, (Entamoeba histolytica, Cryptosporidium, Cyclospora, Cystoisospora belli, Giardia, microsporidia); less common findings would include Schistosoma spp., hookworm, or Trichuris

Special stains may be helpful for the identification of microsporidia: tissue Gram stains, silver stains, Warthin-Starry stain, PAS, and Giemsa or modified acid-fast stains for the coccidia

Liver and spleen

Biopsy specimens or aspirates

Culture: Microscopy: unfixed material in physiologic NaCl; fixed for histology

Examination of wet smears for Entamoeba histolytica (trophozoites), protoscolices of Echinococcus spp. or eggs of Capillaria hepatica; Giemsa (Leishmania, other protozoa and microsporidia); H&E (routine histology)

There are definite risks associated with punctures (aspirates and/or biopsy) of spleen or liver lesions (Echinococcus). Always keep a small amount of material frozen for PCR.

Culture: sterile preparation of fresh material

For Leishmania (not common)

Animal inoculation: sterile preparation of fresh material

Intraperitoneal inoculation of E. multilocularis cyst material for viability test after long-term chemotherapy

PCR: fresh, frozen, or ethanol fixed

Species or genotype identification (e.g., Echinococcus spp.)

Respiratory tract

Sputum, induced sputum, nasal and sinus discharge, bronchoalveolar lavage fluid, transbronchial aspirate, tracheobronchial aspirate, brush biopsy specimen, open-lung biopsy specimen

Helminth larvae (Ascaris, Strongyloides), eggs (Paragonimus, Capillaria), or hooklets (Echinococcus) can be recovered in unstained respiratory specimens

Immunoassay reagents (FA) are available for the diagnosis of pulmonary cryptosporidiosis (may require validation). Routine histologic procedures allow the identification of any of the helminths or helminth eggs present in the lungs. Disseminated toxoplasmosis and microsporidiosis are well documented, with organisms being found in many different respiratory specimens.

Microscopy: unfixed material, treated for smear preparation

Stains: Stains include Giemsa for many protozoa including Toxoplasma tachyzoites, modified acid-fast stains (Cryptosporidium), and modified trichrome, Warthin-Starry stain, tissue Gram stains (microsporidia); routine histology with H&E; silver methenamine stain, PAS, acid-fast, and tissue Gram stains for helminths, protozoa, and microsporidia

PCR: fresh, frozen, or fixed in ethanol

Microsporidian identification to the species level requires subsequent sequencing

Muscles

Biopsy material Microscopy: unfixed, touch and squash preparations or fixed for histology and EM

Larvae of Trichinella spp. can be identified unstained (species identification with single larvae by PCR); H&E, routine histology (Trichinella spp., cysticerci); silver methenamine stain, PAS, acid-fast, tissue Gram stains, EM (several genera of microsporidia)

If Trypanosoma cruzi is present in the striated muscle, the organisms could be identified in routine histology preparations. Modified trichrome, tissue Gram stains, and/or calcofluor stains can be used to confirm the presence of microsporidial spores.

PCR: fresh, frozen, or ethanol fixed

Microsporidian identification to the species level requires subsequent sequencing

Skin

Aspirates, skin snips, scrapings, biopsy specimens

See cutaneous ulcer (above)

Any of the potential parasites present can be identified by routine histology procedures.

Microscopy: wet examination, stained smear (or fixed for histology or EM)

Wet preparations (microfilariae), Giemsa-stained smears or H&E, routine histology (Onchocerca volvulus, Dipetalonema streptocerca, Dirofilaria repens, other larvae causing cutaneous larva migrans [zoonotic Strongyloides spp.], hookworms), Leishmania, Acanthamoeba spp., Entamoeba histolytica, microsporidia, and arthropods (Sarcoptes and other mites)

The larger the amount of material received, the better the chance of finding parasites. Visual identification may require several tissue samples and additional cut sections from multiple tissue blocks to find the organisms (parasites tend to be isolated in tissue, not disseminated throughout the specimen like bacteria or fungi).

PCR: fresh, frozen, or fixed in ethanol

Primers for most relevant parasite species available

Urogenital system

Vaginal discharge, saline swab, transport swab (no charcoal), air-dried smear for FA, urethral discharge, prostatic secretions, urine (single unpreserved, 24-h unpreserved, or early-morning specimens)

Giemsa, immunoassay reagents (FA) (Trichomonas vaginalis); Delafield’s hematoxylin (microfilariae); modified trichrome (microsporidia); H&E, routine histology PAS, acid-fast, tissue Gram stains (microsporidia); direct examination of urine sediment for Schistosoma haematobium eggs or microfilariae

Although T. vaginalis is probably the most common parasite identified, there are others to consider, the most recently implicated organisms being in the microsporidian group. Microfilariae could also be recovered and stained. Fixation of urine with formalin prevents hatching of Schistosoma eggs. Perform concentration using saline to prevent premature hatching of schistosome eggs. Material must be put into culture medium immediately after collection; do not cool or freeze (T. vaginalis).

Microscopy: wet smears, smears of urine sediment, stained smears
Cultivation: vaginal or urethral discharge or swab preparations

Identification and propagation of T. vaginalis (commercialized plastic-envelope culture systems available); moving trophozoites can be detected using microscopy (or in Giemsa-stained smears) Commercial immunoassay (rapid formal), T. vaginalis

Antigen detection (vaginal swab) Probe
PCR: fresh, frozen, or fixed in ethanol

Commercial instrumentation, T. vaginalis

a CSF, cerebrospinal fluid; EIA, enzyme immunoassay; EITB, enzyme-linked immunoelectrotransfer blot (Western blot); EM, electron microscopy; FA, fluorescent antibody; GAE, granulomatous amebic encephalitis; GI, gastrointestinal; H&E, hematoxylin and eosin; IFA, indirect fluorescent-antibody assay; PAM, primary amebic encephalitis; PAS, periodic acid-Schiff stain; PBS, phosphate-buffered saline; QBC, quantitative buffy coat; SAF, sodium acetate-acetic acid-formalin.

b Many parasites or parasite stages may be detected in standard histologic sections of tissue material. However, species identification is difficult and additional examinations may be required. Usually, these techniques are not considered first-line methods. Additional methods like EM (electron microscopy) are carried out only by specialized laboratories and are not available for standard diagnostic purposes. EM examination for species identification has largely been replaced by PCR assays, and other molecular methods.

c The differences between the two approaches stem from the following: the U.S. FDA approach assesses the device’s effectiveness as well as its risk of harm; the CE mark, on the other hand, affirms simply that the product “meets high safety, health and environmental protection requirements” (European Commission 2015). The U.S. approval would ensure not only that the product poses no harm to consumers, but also that it does what it claims to do.

Critics of the FDA system suggest that this goal adds time and unpredictability to the approval process without in fact establishing the effectiveness of the device. Thus, while the CE mark is less difficult to obtain, it is a less powerful certification. FDA approval means that the device is approved for use in all parts of the world, while the CE mark has restrictions, sometimes even within the EU.

NOTE: Molecular testing has become more widely used in diagnostic parasitology. Often these procedures provide better sensitivity and specificity than are found in routine non-molecular methods. A number of molecular diagnostic panels are available (primarily for GI parasites), and newer, more comprehensive panels are pending FDA clearance (Entamoeba histolytica, Giardia lamblia, Dientamoeba fragilis, Blastocystis spp, Cryptosporidium spp., Cyclospora cayetanensis, microsporidia (at least two species), and Strongyloides stercoralis). Other panel options may include Trichomonas vaginalis.