Appi oach to stool microscopy

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IBi, W n I t P K l U W e r

Approach to stool microscopy

Author
Danny A Milner. Jr, MD

Section Editor
Edward T Ryan, MD, DTMH

Deputy Editor
Elinor L Baron, MD. DTMH

Al! topics are updated a s new evidence becomes available and our peer review process is complete.
Literatura review current through: S e p 2015. | This topic last updated: Oct 15, 2014.
INTRODUCTION Stool microscopy is a diagnostic tool for identification of parasitic organisms including
protozoa and helminths; it is also usefui for quantification of fecal leul<^cytes.
Issues related to stool microscopy will be reviewed here; clinicai issues,C3lated to evaluation of patients with
gastrointestinal symptoms are discussed separately. ( S e e related topic|.)
CLINICAL A P P R O A C H Analysis of stool for parasites has its highest clinicai utility when caring for
patients with diarrhea or intestinal symptomatology lasting two w e e k s or longer or during outbreaks of
intestinal infection. Most intestinal infections resolve within a few days of onset, and microscpio evaluation
of stool for parasites in such short-term enteritis is usually unrevealing.
The approach to organism identification via stool microscopy should be guided by the clinicai presentation,
including geographic exposure, age, and nature of clinicai symptoms. T h e differential diagnosis of intestinal
parasitic infection also includes bacterial and virai pathogens; in such cases, usefui diagnostic tests include
cjjlure, antigen detion, fecal leukocyte count, serology, and/or specialized testing for yiral pathogens
(See "Approach to the adult with acute diarrhea in resource-rich countries". sctionn 'Diagnostic
approach'.)
Rapid antigen testing is more usefui than microscopy for diagnosis of Giardia,
Entamoeba

Cryptosporidium,

and

infection. In general, the presence of diarrhea that is watery and nonbloody that occurs in

association with camping, animal contact, 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 clinicai specimens tailored to clinicai symptoms, such a s
bronchoalveolar lavage fluid, cerebrospinal fluid, and/or urine.
Most helminths do not cause diarrhea; for c a s e s in which helminth infection is suspected, both gross
evaluation (for proglottids) and microscpio evaluation (for eggs and larvae) are appropriate.
The diagnosis of pinworm {Enterobius

vermicularis)

is established using alternative techniques such a s the

"scotch tape test" on anal mucosa, a s pinworms are not routinely found on stool examination.
SPECIIMEN C O L L E C T I O N AND PREPARATION Ideally, fresh stool samples should be sent to the
laboratory a s soon a s possible after collection; stool should not be refciaerated prior to recei|3t by the
laboratory, A fresh stool sample, if stilt 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 clinicai settings this is not feasible.
Sample collection into Cary-Blair media is a preferred method [2]. B e c a u s e prompt receipt by the laboratory

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Appr oach to stool microscopy

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is not always possible, preservation solution should be utilized for any sample with delayed review (eg,
home collection, outpatient clinics, off-site laboratories, etc), Evaluation of stool following use of preservation
solution is usually sufficient for the diagnosis of most 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 thoroughiy 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.
Clinicai laboratory procedures can be variable regarding number and types of specimens accepted, In the
setting of clinicai 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],
M I C R O S C O P Y T E C H N I Q U E S Microscopy techniques for stool examination include wet mount
preparation and permanently stained slides, An ocular micrometer on the microscope is usefui 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. T h e slide is first examined at low
power (lOx 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, T h e
slide should then be examined at high power (40x objective) for protozoan cysts and trophozoites.
For short-term storage, the coversiip can be sealed with fingernail polish or a 1:1 solution of paraffin and
petroleum jeily, which requires heating to 70C,
Stained slides Stained slide preparations of stool can be prepared depending on the pathogen(s)
suspected, based on clinicai history,

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

Acid-fast staining allows visualization of Cryptosporidium,

Optical brightening allows visualization of microsporidia by distinguishing organism structure from

Cyciospora,

and

isospora.

background material.
ORGANISM IDENTIFICATION
Nematodes Nematode eggs recognized on wet mount include Ascaris,

Trictiuris, hookworm,

Enterobius,

and Capinaria (picture 1), Ascaris eggs are large with rough surfaces and a dark, dense center Trictiuris
eggs have distinctive mucus plugs at either end a s well a s 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"), Capiiiaria eggs are similar to
Trichuris but are smaller with more flattened ends,
Nematode larvae recognized on wet mounts for include hookworm and Strongyioides.

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Approach to stool microscopy

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examined carefully to determine species when possible; Strongyioides

larva may be distinguished from

hookworm larva by the presence of a short buccal cavity and primordial genitlia midway down the body.
Strongyioides

may also be diagnosed using an agar plate method in which stool is placed on media and

observed for the presence of larvae.

Trematodes Trematode eggs recognized on wet mount with iodine include Sctiistosoma
Sciiistosoma

haematobium,

(picture 2). Schistosoma

Schistosoma japonicum,

terminal (S. haematobium

S. mel<ongi). Cionorchis and Opisthorchis

and Fasciolopsis

Opisthorchis,

Fascioia, and

mansoni,
Paragonimus

eggs are the largest of the helminths and can be distinguished by their spines,

which are lateral (S. mansoni),

Fascioia

Cionorchis,

and S . intercaiatum),

or vestigial (S. japonicum

or

eggs have an operculum and small remnant at the opposite end.

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,
nana, Diphyilobothrium

Iatum, Hymenolepis

Hymenolepis

diminuta, and Dipylidium caninum (picture 3). T h e eggs can be

usefui 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 a s they progress from
immature to gravid (picture 4). Taenia, Dipylidium, and Diphyilobothrium
in length), while Hymenolepis

proglottids are large (0.5 to 1.5 cm

species are much smaller (0.5 mm x 1.0 mm). Distinguishing these species

requires carefui gross and macroscopic examination. T h e scolices (the anchor) are quite small (<1 mm in
diameter) but can be examined under a dissecting or light microscope to identify hookiets and suckers for
speciation.
The proglottids of Taenia have >12 ovarian branches ( I saginata) and <12 ovarian branches {T. solium). T.
solium also has a scolex with hookiets (picture 4). T h e eggs of Taenia are indistinguishable.
Diphyilobothrium

Iatum 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 a s 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
Cryptosporidium,

Cystoisospora

(formerly Isospora),

Cyciospora,

histolytica, Giardia

and Balantidium

lamblia,

coli (picture 5). E.

histolytica may appear a s a cyst form with chromatid bar Giardia lamblia has distinctive flagella.
Cryptosporidium

is small with acid-fast staining. Cystoisospora

has an obiong shape, one ortwo nuclei,

natural fluorescence, and acid-fast positivity. Cyciospora

is similar but larger than Cryptosporidium,

natural fluorescence and acid-fast positivity. Balantidium

coli is the largest protozoan infecting humans and

with

the only ciliate.

SUMMARY

Stool microscopy is a diagnostic tool for identification of parasitic organisms, including protozoa and
helminths. ( S e e '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. ( S e e 'Clinicai approach' above.)

In the setting of clinicai 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
a s soon a s possible after collection. ( S e e 'Soecimen collection and preoaration' above.)

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Approach to stool microscopy

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

Nematode eggs recognized on wet mount include Ascaris, Trictiuris, hookworm, Enterobius,

and

Capinaria (picture 1). ( S e e 'Organism identification' above.)

Trematode eggs recognized on wet mount with iodine include Schistosoma

haematobium,

Schistosoma japonicum,

Cionorchis,

Opisthorchis,

mansoni,

Schistosoma

Fascioia, and Paragonimus

(picture

2)- ( S e e 'Organism identification' above.)

Cestode eggs recognized on wet mount include Taenia, Diphyilobothrium

Hymenolepis

Iatum, Hymenolepis

nana,

diminuta, and Dipylidium caninum (picture 3). Proglottids are large, macroscopic

structures passed in stool; they vary greatly in size a s they progress from immature to gravid (picture
4). ( S e e 'Organism identification' above.)

Protozoa recognized on microscopy include Entamoeba

Cystoisospora,

Cyciospora,

and Balantidium

histolytica, Giardia lamblia,

Cryptosporidium,

co//(picture 5). ( S e e 'prqanisrn identification' above.)

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