Забір і обробка зразків для мікроскопічної діагностики паразитарних інфекцій

Blood

Babesia species

Thick and thin smears as for Plasmodium species

Collect as for Plasmodium species.

Use Wright or Giemsa stain.

Morphology is similar to Plasmodium species ring forms but without pigment and gametocytes. Tetrads are diagnostic of Babesia species but are infrequent.

Thick blood smears can be difficult to interpret and should be performed only by an experienced microscopist.

Filarial worms

Thick and thin smears from 1 mL of anticoagulated blood; if first specimen is negative, 5–10 mL, concentrated by centrifugation or filtration

Microfilariae of Wuchereria bancrofti and Brugia malayi: Draw blood between 10 PM and 2 AM.

Loa loa, Dipetalonema perstans, and Mansonella ozzardi: Draw blood between 10 AM and 6 PM.

Use Giemsa or hematoxylin-eosin stain directly or, for greater sensitivity, after concentration in 2% formalin (Knott technique) or after filtration through a Nucleopore membrane.

Plasmodium species

Thick and thin smears of capillary blood (ie, finger or earlobe, using a disposable lancet) or 5–10 mL of fresh anticoagulated blood (preferably in collection tubes that contain EDTA)

Collect multiple samples during acute illness.

Prepare smears from capillary or anticoagulated blood within 3 hours after collection.

Use Wright or Giemsa stain.

Ensure that glass slides are clean.

Trypanosoma species

Thin smears of capillary blood or 5–6 mL of anticoagulated blood

Collect capillary or anticoagulated blood. Smear on glass slides.

Various concentration techniques are used to enhance sensitivity.

Motile trypanosomes are seen in wet preparations; Giemsa (or Field) stain is used to identify them in fixed preparations.

Bone marrow, other reticuloendothelial tissue, or cerebrospinal fluid

Leishmania species (visceral leishmaniasis)

Aspirates of bone marrow, spleen, liver, or lymph nodes or smears from the buffy coat

Smear on glass slides.

Use Giemsa, Wright-Giemsa, or hematoxylin-eosin stain.

Naegleria

Acanthamoeba

Balamuthia

Fresh cerebrospinal fluid

Use aseptic collection technique.

Examine specimen as soon as possible.

Examine using light or phase-contrast microscopy.

Parasites may be detected by their movements; they can be fixed and stained with Giemsa or cultured.

Rhodesiense

Trypanosoma brucei gambiense

Aspirates of lymph nodes or chancre

Fresh cerebrospinal fluid

Use aseptic collection technique.

Use wet mount to identify motile parasites, or fix and stain with Giemsa or Field stain before or after concentration by centrifugation.

Duodenal aspirate or jejunal biopsy

Giardia species

Cryptosporidium species

Cystoisospora species

Cyclospora species

Microsporidia

Strongyloides species

Duodenal aspirate or jejunal biopsy specimen

Examine aspirates immediately, or fix and stain them. Do histopathologic examination of biopsy specimens.

Use a wet mount of aspirate to identify ova or Strongyloides larvae. Multiple stains may be used for diagnosis (see Feces below for details).

Transmission electron microscopy has been the primary method for detection and species identification of microsporidia.

Rectal biopsy

Schistosoma mansoni

Schistosoma japonicum

Rectal biopsy specimen from level of dorsal fold (Houston valve), about 9 cm from anus

Fix for histopathologic examination, and crush a segment between slides for increased sensitivity.

Speciation is based on the morphology of ova.

Sigmoidoscopy (proctoscopy)

Entamoeba histolytica

Fresh scrapings with a curet or Volkmann spoon, a piece of mucosa snipped off with a surgical instrument, or aspirate from a lesion via a 1-mL pipette with a rubber bulb (cotton-tipped swabs are not satisfactory)

Examine specimen immediately or after fixation and staining.

Use wet mounts or fixed stained slides (eg, with Trichrome stain) to detect trophozoites and cysts. Stool should be assayed for E. histolytica antigen or DNA; these tests are more sensitive and can differentiate E. histolytica from E. dispar and other nonpathogenic amebas.

Feces

Cryptosporidium species

Multiple freshly passed stools (≥ 3) collected daily or every other day

Refrigerate and examine fresh samples, or preserve in formalin or another fixative.

Handle with care; fresh and dichromate-preserved stools are infectious.

Duodenal aspirate or biopsy can be diagnostic.

Examine wet mounts by conventional light, differential interference contrast, and immunofluorescence microscopy.

Stain specimens with modified acid-fast or modified safranin stain. Assays for fecal antigens or DNA are more sensitive.

Cyclospora species

Multiple freshly passed stools collected daily or every other day

Specimens should be refrigerated and examined fresh or frozen, or preserved in 10% formalin and 2.5% potassium dichromate. Different laboratory tests require different preservation techniques. Concentration techniques increase sensitivity.

Examine wet mounts by conventional light, bright-field differential interference contrast, and UV fluorescence microscopy. Oocysts are autofluorescent under UV light. Fixed specimens can be stained with modified acid-fast or modified safranin stain. A sporulation assay can differentiate Cyclospora from blue-green algae.

Assays for DNA in feces are specific and more sensitive.

Cystoisospora species

Multiple freshly passed stools collected daily or every other day

Examine fresh, or preserve in formalin or other fixative. Concentration techniques enhance sensitivity.

Oocysts can be visualized in wet mounts by bright-field differential interference contrast or epifluorescence microscopy. Stain fixed specimens with modified acid-fast stain. When stools are negative, examination of duodenal aspirate or a biopsy specimen can be diagnostic.

Entamoeba dispar

Entamoeba histolytica

Other amebas

Multiple freshly passed stools (≥ 3) collected in AM

Examine unformed or diarrheal specimens within 15 minutes.

Keep formed stools refrigerated until examination. Preserve in formalin or another fixative.

Use wet mounts and permanent stained slides (eg, Trichrome stain) and concentration techniques for cysts.

Stool should be assayed for specific E. histolytica antigen or DNA, which is more sensitive and can differentiate E. histolytica from E. dispar and other nonpathogenic amebas.

Enterobius species

Ova collected from area around the anus on cellophane tape and placed on glass slide

Collect from area around the anus in the AM before a bowel movement or bath.

Enterobius ova are occasionally seen in a stool specimen or in vaginal contents obtained during a Papanicolaou test. Adult worms may be observed on the perianal region or in the vagina.

Giardia species

Multiple freshly passed stools (≥ 3) collected in AM every other day

Examine fresh, or preserve in formalin or another fixative. Trophozoites can also be detected in duodenal aspirates.

Examine direct and concentrated specimens. Cysts are usually seen in wet mounts and trophozoites are seen in fixed, Trichrome-stained slides. Assays for fecal antigens or DNA are more sensitive.

Microsporidia

Multiple stools collected daily or every other day

Small-bowel biopsies may be necessary if stools are negative.

Specimens stained by chromotropic methods are most widely used. Chemofluorescent agents such as calcofluor white can also be used for quick identification.

Electron microscopy is the most sensitive method and used for speciation.

Assays for DNA in stool or tissue are available for some species.

Cestodes (tapeworms)

Nematodes (roundworms)

Ascaris

Hookworms

Strongyloides

Trichuris

Trematodes (flukes)

Others

Multiple stools collected daily (up to 7 needed for Strongyloides)

Refrigerate specimen, and examine fresh, or fix in 10% formalin and concentrate using formalin–ethyl acetate sedimentation.

Active larvae are seen with Strongyloides; ova are seen with other intestinal helminths.

If stool is held at ambient temperature, hookworm ova may hatch releasing larvae that must be differentiated from Strongyloides larvae.

When Strongyloides is suspected and direct examination is negative, one or more of the following specialized stool tests should be done; formalin-ethyl acetate concentration, recovery of larvae by the Baermann funnel technique, culture by the Harada-Mori filter plate method, or agar plate culture.

Sputum or aspirate from respiratory tract

Paragonimus species

Fresh sputum

Examine specimen as soon as possible, or preserve for later examination.

Concentration techniques may be necessary. Occasionally, ova are present in pleural fluid.

Strongyloides species (hyperinfection)

Sputum, any aspirated material, fluid obtained by BAL or drainage material

Examine specimen as soon as possible, or preserve it for later examination.

Active larvae may be seen in wet mounts or can be fixed and stained with Giemsa.

Lung biopsy

Paragonimus species

Open lung biopsy or percutaneous biopsy guided by fluoroscopy or CT

Collect and place in sterile container with sterile saline. Fix and stain with Giemsa or hematoxylin-eosin.

Ova and adult flukes can be identified.

Skin

Leishmania species (cutaneous leishmaniasis)

Biopsy of a nonulcerated area of the lesion and touch preparations or slip smear scrapings

Look for amastigotes in Giemsa-stained touch preparations or smears and in hematoxylin-eosin–stained biopsy specimens.

Leishmania amastigotes are morphologically indistinguishable from those of Trypanosoma cruzi. Leishmania can be cultured from skin biopsies, but growth in vitro may take weeks. Molecular assays for leishmania DNA are available.

Onchocerca volvulus

For patients infected in Africa, skin snips from the thigh, buttocks, or iliac crest

For patients infected in Latin America, skin snips from the head, scapula, or buttocks

For skin snips, disinfect the skin with alcohol, insert a 25-gauge needle just under the epidermis, raise it, and slice off small piece of tissue with a scalpel or razor blade, or use a sclerocorneal punch biopsy tool. Bleeding should not occur. Examine fresh, or fix in methanol and stain with Giemsa or hematoxylin-eosin.

Examine the specimen suspended in saline for motile microfilaria migrating from the skin snip. Microfilariae may be seen in tissue sections.

Urogenital secretions or biopsy

Schistosoma haematobium, occasionally S. japonicum

Fresh urine or biopsy of the urinary bladder, particularly the area around the trigone

Recommended time for urine collection is between noon and 3 PM. Centrifugation increases detection.

Ova can be seen in wet mounts of urine or in biopsy specimens from the bladder.

Trichomonas species

Sterile swabs of vaginal, urethral, or prostatic secretions placed in a tube with a small amount of sterile saline

Tell female patients not to douche for 3–4 days before collecting the specimen.

Send specimen to the laboratory as soon as possible.

Identification of motile organisms by wet mount is the most rapid. Direct fluorescent antibody for parasites is more sensitive; culture is most sensitive but takes 3–7 days. Nucleic acid amplification tests are available.