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Approach to stool microscopy

Approach to stool microscopy
Author:
Danny A Milner, Jr, MD, MSc(Epi)
Section Editor:
Edward T Ryan, MD, DTMH
Deputy Editor:
Elinor L Baron, MD, DTMH
Literature review current through: Dec 2022. | This topic last updated: Jun 02, 2021.

INTRODUCTION — Stool microscopy is a diagnostic tool for identification of parasitic organisms including protozoa and helminths; it is also useful for quantification of fecal leukocytes. Protozoa represent one group within the kingdom Protista; other Protista include protophyta and certain molds.

Issues related to stool molecular testing are discussed separately. (See "Approach to the adult with acute diarrhea in resource-rich settings", section on 'Multipathogen molecular panels'.)

CLINICAL APPROACH — Analysis of stool for parasites has its highest clinical utility when caring for patients with diarrhea or intestinal symptomatology lasting two weeks or longer or during outbreaks of intestinal infection. Most intestinal infections resolve within a few days of onset, and microscopic evaluation of stool for parasites in such short-term enteritis is usually unrevealing.

Analysis of stool for parasites has its highest epidemiologic utility when screening large at-risk populations (eg, school children in endemic areas or areas of poor fecal-oral contamination control) for guidance regarding public health campaigns. (See "Mass drug administration for control of parasitic infections".)

The approach to organism identification via stool microscopy should be guided by the clinical presentation, including geographic exposure, age, and nature of clinical symptoms. The differential diagnosis of intestinal parasitic infection also includes bacterial and viral pathogens; in such cases, useful diagnostic tests include culture, antigen detection, fecal leukocyte count, serology, and/or specialized testing for viral pathogens. (See "Approach to the adult with acute diarrhea in resource-rich settings", section on 'Evaluation'.)

Rapid antigen testing is more useful than microscopy for diagnosis of Giardia, Cryptosporidium, and Entamoeba infection. In general, the presence of nonbloody diarrhea that occurs in association with camping, animal contact, travel to endemic areas, an outbreak, and/or HIV infection should prompt consideration of these organisms [1].

Stool microscopy remains the primary tool for identification of a broad range of other stool pathogens. Its use is appropriate in the setting of bloody diarrhea, eosinophilia, HIV infection, bacteremia with an enteric organism, and/or exposure to a region where parasitic infections are endemic. Patients with eosinophilia should also undergo microscopy evaluation of other clinical specimens tailored to clinical symptoms, such as bronchoalveolar lavage fluid, cerebrospinal fluid, and/or urine.

Most helminths do not cause diarrhea; for cases in which helminth infection is suspected, both gross evaluation (for proglottids, larvae, or adult worms/worm fragments) and microscopic evaluation (for eggs and larvae) are appropriate.

The diagnosis of pinworm (Enterobius vermicularis) is established using alternative techniques such as the "scotch tape test" on anal mucosa, as pinworms are not routinely found on stool examination.

SPECIMEN COLLECTION AND PREPARATION — Ideally, fresh stool samples should be sent to the laboratory as soon as possible after collection; stool should not be refrigerated prior to receipt by the laboratory. A fresh stool sample, if still warm, has the highest yield for visualization of trophozoites (the active, infective form of protozoa). Loose stool samples must be received by the laboratory within 30 minutes to optimize evaluation for presence of trophozoites, although in many clinical settings this is not feasible. Sample collection into Cary-Blair media is a preferred method [2]. Because prompt receipt by the laboratory is not always possible, preservation solution should be utilized for any sample with delayed review (eg, home collection, outpatient clinics, off-site laboratories). Evaluation of stool following use of preservation solution is usually sufficient for the diagnosis of most parasitic pathogens.

Stool should be processed using a 3:1 ratio of fixative to sample [3]. Formalin (10 percent, neutral buffered) is commonly available in hospital settings and is used in processing for visualization (formalin-ethyl acetate concentration). Among symptomatic and chronic carriers, preservation of stools has higher yield for intestinal protozoa (60 percent) than fresh examination (36 percent) [4].

To optimize visualization of cysts and eggs, formed stool samples should be broken up thoroughly in preservative prior to slide preparation. Techniques to concentrate eggs and cysts include flotation (which refers to use of a solution with high specific gravity, allowing eggs to float to surface) or sedimentation (which refers to use of a solution with low specific gravity, allowing eggs to settle to bottom). Most laboratories use sedimentation with a solution of formalin and ethyl-acetate, which preserves egg morphology and is relatively easy to perform.

Clinical laboratory procedures can be variable regarding number and types of specimens accepted. In the setting of clinical suspicion for intestinal parasitic infection, evaluation of multiple stools (up to three over a 10-day period) and use of a preserved sample may increase yield (1.75-fold) over a single stool exam [5].

MICROSCOPY TECHNIQUES — Microscopy techniques for stool examination include wet mount preparation and permanently stained slides. An ocular micrometer on the microscope is useful for making assessments of relative size.

Wet mount — A wet mount is a slide preparation consisting of a drop of stool (in saline if formed), with or without iodine for contrast enhancement, examined under a cover slip. The slide is first examined at low power (10x or 20x objective) in a systematic field-by-field approach for identification of helminth eggs and/or larvae; subsequently, these should be examined on higher magnification for identification/confirmation. The slide should then be examined at high power (40x objective) for protozoan cysts and trophozoites.

For short-term storage, the coverslip can be sealed with fingernail polish or a 1:1 solution of paraffin and petroleum jelly, which requires heating to 70°C of the mixture before application to the coverslip edges.

Kato-Katz technique — For quantification of helminth eggs (eg, Ascaris, Trichuris, hookworm, and schistosomiasis) in regions with high prevalence (>20 percent), the Kato-Katz technique permits quantification of eggs per gram of stool [6]. The technique is described in the table (table 1).

Stained slides — Stained slide preparations of stool can be prepared depending on the pathogen(s) suspected, based on clinical history.

Trichrome staining allows visualization of protozoa, yeast, and human cells.

Acid-fast staining allows visualization of Cryptosporidium, Cyclospora, and Cystoisospora.

Optical brightening allows visualization of microsporidia by distinguishing organism structure from background material.

Auramine O stains all DNA and is helpful for detection of microsporida [7] and other parasites [8]. Two of the coccidian parasites (Cyclospora and Cystoisospora) naturally fluoresce under ultraviolet (UV) microscopy.

ORGANISM IDENTIFICATION

Nematodes — Nematode eggs recognized on wet mount include Ascaris, Trichuris, hookworm, Enterobius, and Capillaria (picture 1). Ascaris eggs are large with rough surfaces and a dark, dense center. Trichuris eggs have distinctive mucus plugs at either end as well as a "tea tray" appearance. Hookworm eggs are optically clear at the edges with a dense center composed of one or more cells. Enterobius eggs have a distinctive oval shape with slight concavity (best seen on "scotch tape test"). Capillaria eggs are similar to Trichuris but are smaller with more flattened ends.

Worms passed in stool may be submitted to the microbiology laboratory for examination. It is important to distinguish between human pathogens (Ascaris and Anisakis worms) and nonpathogenic worms (figure 1).

Nematode larvae recognized on wet mounts include hookworm and Strongyloides. Larvae should be examined carefully to determine species when possible; Strongyloides larva may be distinguished from hookworm larva by the presence of a short buccal cavity and primordial genitalia midway down the body. Strongyloides may also be diagnosed using an agar plate method in which stool is placed on media and observed for the presence of larvae (migrating).

Trematodes — Trematode eggs recognized on wet mount with iodine include Schistosoma mansoni, Schistosoma haematobium, Schistosoma japonicum, Clonorchis/Opisthorchis, Fasciola/Fasciolopsis, and Paragonimus (picture 2). Schistosoma eggs are the largest of the helminths and can be distinguished by their spines, which are lateral (S. mansoni), terminal (S. haematobium and S. intercalatum), or vestigial (S. japonicum or S. mekongi). Clonorchis and Opisthorchis eggs have an operculum and small remnant at the opposite end. Fasciola and Fasciolopsis are also operculated but smooth. Paragonimus eggs are also operculated but have a thick, almost pointed opposite end.

Cestodes — Cestode eggs recognized on wet mount include Taenia solium/Taenia saginata, Hymenolepis nana, Dibothriocephalus latus, Hymenolepis diminuta, and Dipylidium caninum (picture 3). The eggs can be useful for diagnosis, although the primary diagnostic forms are the mature (gravid) proglottids and the scolex.

Proglottids are large, macroscopic structures passed in stool; they vary greatly in size as they progress from immature to gravid (picture 4). Taenia, Dipylidium, and Dibothriocephalus proglottids are large (0.5 to 1.5 cm in length), while Hymenolepis species are much smaller (0.5 mm x 1.0 mm). Distinguishing these species requires careful gross and macroscopic examination. The scolices (the anchor) are quite small (≤1 mm in diameter) but can be examined under a dissecting or light microscope to identify hooklets (T. solium) and suckers (Taenia species) for speciation.

The proglottids of Taenia have >14 ovarian branches (T. saginata) and <14 ovarian branches (T. solium). T. solium also has a scolex with hooklets (picture 4). The eggs of Taenia are indistinguishable. D latus has a distinctive proglottid containing a "central rosette" that is visible with or without ink infusion preparation. Hymenolepis nana and H. diminuta can be distinguished by size of the eggs as well as the presence of polar filaments in the eggs of H. diminuta; the proglottids are very similar.

Protozoa — Protozoa recognized on microscopy include Entamoeba histolytica, Giardia lamblia, Cryptosporidium, Cystoisospora (formerly Isospora), Cyclospora, and Balantidium coli (picture 5). E. histolytica may appear as a cyst form with chromatid bar. Giardia lamblia has distinctive flagella. Cryptosporidium is small with acid-fast staining. Cystoisospora has an oblong shape, one or two nuclei, natural fluorescence, and acid-fast positivity. Cyclospora is similar but larger than Cryptosporidium, with natural fluorescence and acid-fast positivity. Balantidium coli is the largest protozoan infecting humans and the only ciliate.

SUMMARY

Stool microscopy is a diagnostic tool for identification of parasitic organisms, including protozoa and helminths. (See 'Introduction' above.)

Stool microscopy evaluation is appropriate in the setting of bloody diarrhea, eosinophilia, HIV infection, bacteremia with an enteric organism, and/or exposure to a region where parasitic infections are endemic. (See 'Clinical approach' above.)

In the setting of clinical suspicion for intestinal parasitic infection, three stool specimens should be collected on separate days over a 10-day period. Fresh stool samples should be sent to the laboratory as soon as possible after collection. (See 'Specimen collection and preparation' above.)

Microscopy techniques for stool examination include wet mount preparation and permanently stained slides. (See 'Microscopy techniques' above.)

Nematode eggs recognized on wet mount include Ascaris, Trichuris, hookworm, Enterobius, and Capillaria (picture 1). (See 'Organism identification' above.)

Trematode eggs recognized on wet mount with iodine include Schistosoma mansoni, Schistosoma haematobium, Schistosoma japonicum, Clonorchis, Opisthorchis, Fasciola, and Paragonimus (picture 2). (See 'Organism identification' above.)

Cestode eggs recognized on wet mount include Taenia, Dibothriocephalus latus, Hymenolepis nana, Hymenolepis diminuta, and Dipylidium caninum (picture 3). Proglottids are large, macroscopic structures passed in stool; they vary greatly in size as they progress from immature to gravid (picture 4). (See 'Organism identification' above.)

Protozoa recognized on microscopy include Entamoeba histolytica, Giardia lamblia, Cryptosporidium, Cystoisospora, Cyclospora, and Balantidium coli (picture 5). (See 'Organism identification' above.)

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  3. Koneman EW. ColorAtlas and Textbook of Diagnostic Microbiology, 6th ed, Lippincott Williams & Wilkins, Philadelphia p.1249, 2005.
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  6. World Health Organization. Bench Aids for the diagnosis of intestinal parasites. WHO, Geneva 2012. http://apps.who.int/iris/bitstream/10665/37323/1/9789241544764_eng.pdf?ua=1 (Accessed on April 10, 2017).
  7. Conteas CN, Sowerby T, Berlin GW, et al. Fluorescence techniques for diagnosing intestinal microsporidiosis in stool, enteric fluid, and biopsy specimens from acquired immunodeficiency syndrome patients with chronic diarrhea. Arch Pathol Lab Med 1996; 120:847.
  8. Hanscheid T, Cristino JM, Salgado MJ. Screening of auramine-stained smears of all fecal samples is a rapid and inexpensive way to increase the detection of coccidial infections. Int J Infect Dis 2008; 12:47.
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