Informational Tables

3.3 Body site, specimen, and recommended stain(s)^a

Body site

Specimen(s)

Recommended stain for suspect organism

Comments

Blood

Whole or anticoagulated blood

Blood stains (Giemsa,Wright’s, Wright-Giemsa combination, Field’s, rapid blood stains)
All blood parasites
Hematoxylin-based stain
Microfilariae (sheathed)
Note: The QBC tube is also available as a screening tool for blood parasites (hematocrit tube contains acridine orange) and has been used for malaria, Babesia, trypanosomes, leishmania, and microfilariae; however, the sensitivity and specificity of the QBC tube do not equal those seen with thick and thin blood films.

Stat request (collection, processing, staining, examination, and reporting). Most drawings and organism descriptions of blood parasites were originally based on Giemsa-stained blood films.
Although Wright’s stain (or Wright- Giemsa combination stain) works, stippling in malaria parasites may not be visible and the organism colors do not match the descriptions. However, if other stains (those listed above, in addition to some of the “quick” blood stains) are used, the organisms should be visible on the blood film.
The use of anticoagulated blood (EDTA recommended) is acceptable.
For detection of stippling, the smears should be prepared within 1 h after the specimen is drawn. After that time, stippling may not be visible on stained films, and after 4 to 6 h, organisms begin to disappear. It is important to maintain the correct pH of the buffers when staining blood films.
Overall QC: If the WBCs look good, any parasites present will also exhibit good morphology (both thick and thin blood films).

However, if other stains (those listed above) are used in addition to some of the "quick" blood stains, organisms should be visible on blood films.

Body site

Specimen(s)

Recommended stain for suspect organism

Comments

Bone marrow

Aspirate

Blood stains (Giemsa, Wright’s, Wright-Giemsa combination, Field’s, rapid blood stains) All blood parasites

See comments above for blood.

Central nervous system

Spinal fluid, brain biopsy specimen

Giemsa or other blood stains for trypanosomes, Toxoplasma gondii

Stat request (collection, processing, staining, examination, and reporting). If cerebrospinal fluid is received (with no suspected organism suggested), any of the blood stains would be acceptable; however, calcofluor white is also recommended as a second stain. If brain biopsy material is received (particularly from an immunocompromised patient), electron microscopy studies may be required in order to identify microsporidia to the genus or species level. Immunofluorescent methods (along with routine histologic morphology) may be required to identify free-living amebae to the correct genus.

Giemsa, trichrome, or calcofluor for amebae (Naegleria, Acanthamoeba, and Balamuthia spp.) (cysts only)

NAAT, Giemsa, acid-fast PAS, modified trichrome, calcofluor, and silver methenamine for microsporidia

Hematoxylin and eosin (routine histology) for larval cestodes

Cutaneous ulcer

Aspirate, biopsy specimen

Giemsa or other blood stains for leishmaniae

Most likely causative agents would be leishmaniae, all of which would stain with Giemsa. Hematoxylin and eosin (routine histology) could also be used to identify these organisms.

Hematoxylin and eosin (routine histology) for Acanthamoeba spp., Entamoeba histolytica

Body site

Specimen(s)

Recommended stain for suspect organism

Comments

Eye

Biopsy specimen, scrapings, contact lens, lens solution if opened; unopened lens care solutions subject to FDA regulations are not tested by routine laboratories

Calcofluor for amebae (Acanthamoeba spp.) (cysts only)

Some free-living amebae (most commonly Acanthamoeba spp.) have been implicated as a cause of keratitis. Although calcofluor will stain cyst walls, it will not stain trophozoites. Therefore, in suspected cases of amebic keratitis, use both stains. Hematoxylin and eosin (routine histology) can be used to detect and confirm cysticercosis.

Blood stains for amebae (trophozoites, cysts)

Adult Loa loa worm, when removed from the eye, can be stained with hematoxylin-based stain (Delafield’s) for routine microscopy) or can be stained and examined via routine histology. Toxoplasma infection could be diagnosed by using routine histology and/or serology results. Confirmation of microsporidia to the genus and species levels may require EM or molecular NAAT studies.

Hematoxylin and eosin (routine histology) for cysticerci, Loa loa, Toxoplasma gondii
Silver methenamine, PAS, acid-fast stains, calcofluor, and EM studies for microsporidia

Intestinal tract

Stool, sigmoidoscopy material, duodenal contents

Trichrome or iron hematoxylin for intestinal protozoa

Although trichrome, iron hematoxylin, or EcoStain stains can be used on almost all specimens from the intestinal tract, actual worm segments (tapeworm proglottids) can be stained with special stains. However, after routine dehydration with alcohols and xylenes (or xylene substitutes), the branched uterine structure will be visible, allowing identification of proglottids to the species level.

NAAT, modified trichrome and calcofluor for microsporidia/p>

Immunoassay detection kits are also available for the identification of Giardia duodenalis (G. lamblia, G. intestinalis), Entamoeba histolytica/E. dispar, Entamoeba histolytica, and Cryptosporidium spp. Confirmation of microsporidia to the genus or species level may require EM or molecular NAAT studies.

Modified acid-fast stain for Cryptosporidium spp., Cyclospora cayetanensis, and Cystoisospora belli
Immunoassay reagents (EIA, FA, cartridge format) for Entamoeba histolytica/E. dispar group, Entamoeba histolytica, Giardia duodenalis (G. lamblia, G. intestinalis), Cryptosporidium spp., and microsporidia (experimental)

Anal impression preparation

No stain, clear cellulose tape, or egg collection device (paddle)

Three consecutive negative tapes are recommended to rule out infection or testing on three separate days.

Adult worm or worm segments

Carmine stains (rarely used)

Proglottids can usually be identified to the species level without using tissue stains.

Body site

Specimen(s)

Recommended stain for suspect organism

Comments

Biopsy specimen

Hematoxylin and eosin (routine histology) for Entamoeba histolytica (also PAS), Cryptosporidium spp., Cyclospora cayetanensis, Cystoisospora belli, Giardia duodenalis (G. lamblia, G. intestinalis), and microsporidia

Special stains may be helpful in the identification of microsporidia: tissue Gram stains, silver stains, PAS, and Giemsa stain.

Liver, spleen

Aspirates Biopsy specimen

Giemsa or other blood stains for leishmaniae

Aspirates and/or touch preparations from biopsy material can be routinely stained with Giemsa stain. This will allow identification of leishmaniae. Definite risks are associated with spleen aspirates and/or biopsy material. Other parasites, such as larval cestodes, trematodes, amebae, or microsporidia, can be seen and identified by routine histological staining.

Hematoxylin and eosin (routine histology); NAAT for microsporidia

Leishmaniae, microsporidia, trematodes (adults, eggs)

Lung

Sputum, induced sputum, bronchoalveolar lavage fluid, transbronchial aspirate, tracheobronchial aspirate, brush biopsy specimen, open lung biopsy specimen

Silver methenamine
stain, calcofluor white
(cysts only)
(cysts only)
Pneumocystis jirovecii
Giemsa (trophozoites only)
Pneumocystis jirovecii
Modified acid-fast stains
Cryptosporidium spp.
Hematoxylin and eosin (routine histology)
Strongyloides stercoralis
Paragonimus spp.
Amebae (Entamoeba histolytica, Acanthamoeba spp., Balamuthia mandrillaris)
Silver methenamine stain
PAS, acid-fast stains, tissue
Gram stains, modified trichrome, electron microscopy or NAAT for microsporidia

Pneumocystis jirovecii is also a diagnostic possibility (now classified with the fungi) recovered and identified from the lungs by using silver or Giemsa stains monoclonal reagents (FA).
Monoclonal reagents (FA) are also available for the diagnosis of pulmonary cryptosporidiosis.
Routine histology procedures would allow the identification of any helminths or helminth eggs present in the lungs.

Muscle

Biopsy specimen

Hematoxylin and eosin (routine histology) for Trichinella spiralis, Taenia solium (cysticerci), Sarcocystis spp.
Silver methenamine, PAS, acid-fast, and tissue Gram stains and EM studies for microsporidia; NAAT

If Trypanosoma cruzi is present in the striated muscle, it can be identified from routine histological preparations. Confirmation of microsporidia to the genus or species level may require EM or NAAT studies

Nasopharynx Sinus cavities

Modified trichrome,acid-fast stain, Giemsa, optical brightening agent (calcofluor white), methenamine silver, electron microscopy or NAAT for Microsporidia Giemsa, trichrome Acanthamoeba sp. Naegleria sp.

Microsporidium identification to the genus or species level may require electron microscopy studies. Some free-living amebae (most commonly Acanthamoeba or Naegleria) have been found to colonize the nasopharynx and/or sinus cavities. Although calcofluor white stains the cyst walls, it does not stain the trophozoites. Therefore, in suspected cases, both calcofluor white and one of the routine stains should be used.

Skin

Aspirates Skin snip, scrapings, biopsy specimens

See Cutaneous Ulcer above. Hematoxylin and eosin (routine histology) for Onchocerca volvulus, Mansonella streptocerca Acanthamoeba spp. Balamuthia mandrillaris Entamoeba histolytica

Any of these parasites can be identified by using routine histological procedures and stains.

Urogenital system

Vaginal discharge

Giemsa

Although Trichomonas vaginalis is probably the most common parasite identified, there are others to consider, the most recently implicated organisms being in the microsporidia. Microfilariae can also be recovered and stained.

Urethral discharge, prostatic secretions

Immunoassay reagents (FA) for Trichomonas vaginalis

Urine
Biopsy specimen

Delafield’s hematoxylin for microfilariae
Hematoxylin and eosin (routine histology) for Schistosoma haematobium, microfilariae
NAAT, silver methenamine, PAS, acid-fast, modified trichrome, and tissue Gram stains and EM studies for microsporidia