Protozoa Reference

Entamoeba histolytica (True Pathogen - Amebiasis)
Organism:
This organism belongs to the amebae, is a true pathogen, and
causes amebiasis. Both the trophozoite (usual size, 12-60 m)
and cyst forms (usual size, 10-20 m) can be found in clinical
specimens.
Entamoeba histolytica
Note: ingested RBCs in troph
Entamoeba histolytica/E. dispar
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Life Cycle:
Large bowel, organisms passed in feces, AND/OR
Large bowel, dissemination via blood (liver, lung, brain,
pericardium, other tissues).
Acquired:
Fecal-oral transmission via cyst form; contaminated food and
water
Epidemiology:
Worldwide, primarily human-to-human transmission
Clinical Features:
Intestinal: Diarrhea, dysentery or asymptomatic; it is estimated
that only a small proportion (2 to 8%) of infected individuals will
have invasive disease beyond the lumen of the bowel. Also,
organisms may be spontaneously eliminated with no disease
symptoms.
Extraintestinal: Right upper quadrant pain, fever. Blood flow
draining the intestine tends to return to the liver, most commonly
the upper right lobe. The organisms present in the submucosa
can therefore be carried via the bloodstream to the liver. Onset of
symptoms may be gradual or sudden; upper right abdominal pain
with fever from 38 to 39C is the most consistent finding.
Weakness, weight loss, cough, and sweating are less commonly
seen. There tends to be hepatomegaly with tenderness; however,
liver function tests may be normal or slightly abnormal, with
jaundice being very rare. There may be changes at the base of
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the right lung owing to the elevated diaphragm. The abscess can
be visualized radiologically, sonically, or by radionuclear scan,
and the majority of patients have a single abscess in the right
lobe of the liver. The most common complication is rupture of the
abscess into the pleural space. An abscess can also extend into
peritoneum and through the skin. Hematogenous spread to the
brain, as well as to the lung, pericardium, and other sites, is
possible.
Clinical Specimen:
Intestinal: Stool, sigmoidoscopy specimens
Extraintestinal: Liver aspirate, biopsy specimen, serum for
antibody
Laboratory Diagnosis:
Intestinal: Ova and Parasite examination (concentration,
permanent stained smear); fecal immunoassay to detect antigen
of Entamoeba histolytica/E. dispar group or the true pathogen, E.
histolytica.
Extraintestinal: Trichrome, PAS stains, routine histology (H&E
stain); test for antibody. E. histolytica cysts and trophozoites are
found in the stools of only a few patients with liver abscess. About
60% of these patients have no intestinal symptoms or any history
of dysentery.
Organism Description:
Trophozoite: Evenly arranged nuclear chromatin, central
karyosome, fine cytoplasm (may contain ingested RBCs)
Cyst: May contain chromatoidal bars with smooth, rounded
edges; mature cyst contains 4 nuclei (rarely more seen).
Laboratory Report:
Without confirmation using the specific immunoassay to detect
the true pathogen, E. histolytica, the report must
indicate: Entamoeba histolytica/E. dispar group (indicate
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cysts and/or trophozoites); this report indicates that the organism

morphology cannot be used to differentiate the true pathogen,E.
histolytica, vs. the nonpathogen, E. dispar.
Report Comment: Submit a fresh stool if you want
confirmation of the true pathogen (Entamoeba
histolytica). The laboratory will then test the fresh stool (fresh,
frozen, some acceptable in Cary-Blair) for the presence of the true
pathogen, Entamoeba histolytica, antigen. If confirmation of E.
histolytica is not performed, then the physician will usually treat if
the patient is symptomatic.
Treatment:
Metronidazole
Control:
Improved hygiene, adequate disposal of fecal waste, adequate
washing of contaminated fruits and vegetables
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Entamoeba hartmanni (Nonpathogen)

Organism:
This organism belongs to the amebae, is a nonpathogen, and
causes no disease. Both the trophozoite (usual size, 4-12 m)
specimens.
Trophozoite <12 microns
Cyst <10 microns
Life Cycle:
Large bowel, organisms passed in feces
Acquired:
water
Epidemiology:
Clinical Features:
None
Clinical Specimen:
Intestinal: Stool
permanent stained smear); E. hartmanni will be identified based
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on size differences (differentiate from the Entamoeba

histolytica/E. dispar group).
Trophozoite: Evenly arranged nuclear chromatin, central
karyosome, fine cytoplasm (will normally not contain ingested
RBCs)
Cyst: Usually contains chromatoidal bars with smooth, rounded
edges, mature cyst contains 4 nuclei (often two-nucleated cysts
seen).
Laboratory Report:
Entamoeba hartmanni (indicate trophozoites and/or cysts).
Treatment:
None
Control:
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Entamoeba coli (Nonpathogen)

Organism:
specimens.
Trophozoites
Cysts
Life Cycle:
Intestine, organisms passed in feces
Acquired:
water
Epidemiology:
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Clinical Features:
None
Clinical Specimen:
Intestinal: Stool
permanent stained smear); identification based on morphology
Trophozoite: Unevenly arranged nuclear chromatin, eccentric
karyosome, messy cytoplasm (may contain lots of debris)
Cyst: May contain chromatoidal bars with sharp, jagged edges;
mature cyst contains 8 nuclei (rarely more seen).
Laboratory Report:
Entamoeba coli (indicate cysts and/or trophozoites)
Treatment:
None
Control:
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Entamoeba gingivalis
Entomoeba gingivalis is a protozoan endoparasite, residing in the
tartar and puspockets of teeth of pyorrhoea infected human
beings. It is the first parasitic amoeba known to human beings. E.
gingivalis was first of all observed by Gros in 1849 in the tartar of
teeth but its detail description was given by Von Prowazak in
1904. Smith and Barrett (1915) described it as causative agent of
pyorrhoea alveolaris.
Geographical distribution:
E. gingivalis is cosmopolitan in distribution. It is estimated that in
India more than 70 per cent population is infected by this
parasite. With advancing age, the percentage of individuals
suffering from the infection of E. gingivalis increases.
Life cycle:
E. gingivalis is a monogenetic parasite. Human beings are their
only host, however occasionally the parasite has also been
reported from the mouth of dogs, cats, horses and captive
monkeys. Only trophozoite stage exists during the life cycle. The
size of the trophozoite ranges from 5 to 30 diameters however,
the usual size is from 10 to 20 .
The single celled trophozoite is differentiated into outer clear

ectoplasm and inner granular endoplasm. During non motile
condition, ectoplasm is hardly visible but during motile stage it
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appears as a thick layer comprising about one half of the volume

of the actively motile animal.
The endoplasm is granular, vacuolated and usually crowded with
floating food particles. Food vacuoles contain dark-staining
rounded bodies largely derived from the nuclei of degenerated
epithelial cells, lymphocytes and occasionally leukocytes. The
organism also, ingests bacteria but do not take red blood cells. E.
gingivalis is basically a scavenger of disintegrated cells. Bacteria
are a minor source of nourishment.
The endoplasm contains a single, small nucleus. The nucleus is
spherical and m unstained condition is usually inconspicuous. The
moderately thick nuclear membrane contains irregularly
distributed small masses of chromatin. A central or eccentric
karyosome is present inside the nucleus from which delicate
radiating fibrils extends up to peripheral ring.
The parasite moves by forming ectoplasmic pseudopodia. During
resting stage several pseudopodia may be seen extruding in
various directions but during directional progress one more large
pseudopodia ma, be extended. E. gingivalis reproduoes by binary
fission. Cysticsrnge have not been seen during the life cycle.
Trophozoite donot survive outside the host body.
Mode of infection:
Transmission of E. gingivalis from one person to another occurs by
mouth to mouth kissing. It may be transmitted due to coughing
by infected cook at the time of dishing.
Pathology:
The actual pathogenicity of these protozoa is not well as certain
as there is no convincing evidence that the organism causes oral
disease. The parasite lies in pyorrhoea alveolaris and was
previously supposed to cause pyorrhoea but nowadays, few
workers have that suggested though this protozoon actually
thrives as a commensal in the gingival tissues of man, it purely
prepares the teeth for pyorrhoea and its purely commensal
relationship with man is in doubt.
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Treatment:
Treatment of abnormal oral conditions or disease is the best way
to eliminate the parasite. No specific drug or medicine is
prescribed to kill the organism.
Prophylaxis:
Since, the organism is not directly causing harm to the host; there
is no specific prophylaxtic measure to prevent its occurrence.
Proper hygiene may reduce the incidence of E. gingivalis.
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Endolimax nana (Nonpathogen)

Organism:
specimens.
Endolimax nana trophozoites
Endolimax nana cysts

Life Cycle:
Acquired:
water
Epidemiology:
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Clinical Features:
None
Clinical Specimen:
Intestinal: Stool
Trophozoite: No nuclear chromatin, large karyosome, relatively
clean cytoplasm (may contain some debris); tremendous nuclear
variation (can mimic Entamoeba hartmanni, Dientamoeba
fragilis and Iodamoeba btschlii).
Cyst: May contain linear structures (pale), mature cyst contains 4
nuclei (rare to see two-nucleated stage).
Laboratory Report:
Endolimax nana (indicate cysts and/or trophozoites)
Treatment:
None
Control:
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Iodamoeba btschlii (Nonpathogen)

Organism:
specimens.
Trophozoite
Cysts, stained
Cysts in iodine
Life Cycle:
Acquired:
water
Epidemiology:
Clinical Features:
None
Clinical Specimen:
Intestinal: Stool
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Trophozoite: No nuclear chromatin, large karyosome, relatively
messy cytoplasm (may contain some debris and vacuoles); (can
mimic Endolimax nana).
Cyst: Will contain large glycogen vacuole (may collapse on itself),
mature cyst contains 1 nucleus; peripheral nuclear chromatin
often seen on one side basket nucleus nucleus contains large
karyosome that appears a brown in an iodine stain.
Laboratory Report:
Iodamoeba btschlii (indicate cysts and/or trophozoites)
Treatment:
None
Control:
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Naegleria fowleri (True Pathogen Primary Amebic

Meningoencephalitis/PAM)
Organism:
Infections of the central nervous system (CNS) caused by
free-living amebae have been recognized only since the
mid-1960s, and our understanding of this disease process is still
incomplete. One type of meningoencephalitis (PAM) is a fulminant
and rapidly fatal disease that affects mainly children and young
adults. The disease closely resembles bacterial meningitis but is
caused by N. fowleri, an organism found in moist soil and
freshwater habitats. Close to 200 cases of PAM have occurred
worldwide, and approximately 90 of those cases have been
reported from the United States. Until recently, it was believed
that this infection was limited to humans; however, infections
have also been reported in other animals.
Naegleria fowleri trophozoites Trophozoite
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Cyst from
culture
Trophozoit Flagell
e wet
ated
mount
stage
Naegleria trophozoites in tissue (cysts are

not seen in tissue)
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Life Cycle:
The trophozoites can occur in two forms, ameboid and flagellate.
Motility can be observed in hanging-drop preparations from
cultures of cerebrospinal fluid (CSF); the ameboid form (the only
form recognized in humans) is elongate with a broad anterior end
and tapered posterior end. The size ranges from 7 to 35 m. The
diameter of the rounded forms is usually 15 m. There is a large,
central karyosome and no peripheral nuclear chromatin. The
cytoplasm is somewhat granular and contains vacuoles. The
ameboid-form organisms change to the transient, pear-shaped
flagellate form when they are transferred from culture or teased
from tissue into water and maintained at a temperature of 27 to
37C. The change may occur very quickly (within a few hours) or
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may take as long as 20 h. The flagellate form has two flagella at

the broad end. Motility is typical, with either spinning or jerky
movements. These flagellate forms do not divide, but when the
flagella are lost, the ameboid forms resume reproduction.
Cysts from nature and from agar cultures look the same and have
a single nucleus almost identical to that seen in the trophozoite.
They are generally round, measuring from 7 to 15 m, and there
is a thick double wall.
Acquired:
The amebae may enter the nasal cavity by inhalation or
aspiration of water, dust, or aerosols containing the trophozoites
or cysts. The organisms then penetrate the nasal mucosa,
probably through phagocytosis of the olfactory epithelium cells,
and migrate via the olfactory nerves to the brain. Data suggest
that N. fowleri directly ingests brain tissue by producing food cups
or amebostomes, in addition to producing a contact-dependent
cytolysis which is mediated by a heat-stable hemolytic protein,
heat-labile cytolysis, and/or phospholipase enzymes. Cysts of N.
fowleri are generally not seen in brain tissue.
Epidemiology:
Nearly 200 presumptive or proven cases of PAM have been
reported in the literature, including cases from the United States,
Ireland, England, Belgium, Czechoslovakia, Australia, New
Zealand, Brazil, and Zambia. Clinical patient histories indicate
exposure to the organism via freshwater lakes or swimming pools
(not in the US due to chlorination) shortly before onset; patients
had been previously healthy with no specific underlying problems.
PathogenicNaegleria organisms have also been isolated from
nasal passages of individuals with no history of water exposure,
thus suggesting the possibility of airborne exposure.
Clinical Features:
Early symptoms include vague upper respiratory distress,
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headache, lethargy, and occasionally olfactory problems. The

acute phase includes sore throat, stuffy blocked or discharging
nose, and severe headache. Progressive symptoms include
pyrexia, vomiting, and stiffness of the neck. Mental confusion and
coma usually occur approximately 3 to 5 days prior to death. The
cause of death is usually cardiorespiratory arrest and pulmonary
edema.
PAM can resemble acute purulent bacterial meningitis,
and these conditions may be difficult to differentiate,
particularly in the early stages. The CSF may have a
predominantly polymorphonuclear leukocytosis, increased
protein, and decreased glucose concentration like that
seen with bacterial meningitis. Unfortunately, if the CSF
Gram stain is interpreted incorrectly (identification of
bacteria as a false positive), the resulting antibacterial
therapy has no impact on the amebae and the patient will
usually die within several days.
Clinical Specimen:
CSF: Spinal fluid placed on a slide with coverslip (do not use
counting chamber organisms will look like WBCs).
Laboratory Diagnosis: THIS REQUEST IS ALWAYS A STAT!
Specimens should never be refrigerated prior to examination.
When centrifuging the CSF, low speeds (250 g) should be used
so that the trophozoites are not damaged. Although bright-field
microscopy with reduced light is acceptable, phase microscopy, if
available, is recommended. Use of smears stained with Giemsa or
Wright's stain or a Giemsa-Wright's stain combination can also be
helpful. If N. fowleri is the causative agent, trophozoites only are
normally seen. If the infecting organism isAcanthamoeba spp.,
cysts may also be seen in specimens from CNS infection.
Unfortunately, most cases are diagnosed at autopsy; confirmation
of these tissue findings must include culture and/or special
staining with monoclonal reagents in indirect fluorescent antibody
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procedures. Organisms can also be cultured on nonnutrient agar

plated with Escherichia coli.
In cases of presumptive pyogenic meningitis in which no bacteria
are identified in the CSF, the computed tomography appearance
of basal arachnoiditis (obliteration of basal cisterns in the
precontrast scan with marked enhancement after the
administration of intravenous contrast medium) should alert the
staff to the possibility of acute PAM.
The amebae can be identified in histologic preparations by
indirect immunofluorescence and immunoperoxidase techniques.
The organism in tissue sections looks very much like
an Iodamoeba btschlii trophozoite, with a very large karyosome
and no peripheral nuclear chromatin; the organisms can also be
seen with routine histologic stains.
Trophozoite: the ameboid form (the only form recognized in
humans) is elongate with a broad anterior end and tapered
posterior end. The size ranges from 7 to 35 m. The diameter of
the rounded forms is usually 15 m. There is a large, central
karyosome and no peripheral nuclear chromatin. The cytoplasm is
somewhat granular and contains vacuoles. The ameboid-form
organisms change to the transient, pear-shaped flagellate form
when they are transferred from culture or teased from tissue into
water and maintained at a temperature of 27 to 37C. The change
may occur very quickly (within a few hours) or may take as long
as 20 h. The flagellate form has two flagella at the broad end.
Motility is typical, with either spinning or jerky movements. These
flagellate forms do not divide, but when the flagella are lost, the
ameboid forms resume reproduction.
Cyst: The cyst typically has a double wall and can be seen in
culture, but not in tissue or the CSF.
Culture: The most effective approach uses nonnutrient agar
plates with Page's saline and an overlay growth of Escherichia
coli on which the amebae feed. There is also evidence that
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phosphate-buffered saline can be used. Specimens transported in

ameba-saline (5.0 ml) and filtered through 13-mm,
0.22-m-pore-size cellulose acetate and nitrate filters (Millipore
Corp., Bedford, Mass.) have also been acceptable for organism
recovery. The filter is then placed in the center of the nonnutrient
agar plate seeded with E. coli. Tissue stains are also effective,
and cysts can be stained with Gomori's silver methenamine,
periodic acid-Schiff, and calcofluor white. Both cysts and
trophozoites can be recovered from culture.
Laboratory Report:
Naegleria fowleri seen and/or cultured
Treatment:
Many antimicrobial and antiparasitic drugs have been screened
for in vitro and in vivo activity against N. fowleri. Although N.
fowleri is very sensitive to amphotericin B in vitro, only a few
patients have recovered after receiving intrathecal and
intravenous injections of this drug alone or in combination with
miconazole. One case within California in which the patient was
successfully treated with amphotericin B, miconazole, and
rifampin has been documented. Delay in diagnosis and the
fulminant nature of PAM result in few survivors; this is unfortunate
since N. fowleri is quite sensitive to the antifungal agent
Amphotericin B.
Control:
General preventive measures include public awareness of
potential hazards of contaminated water. It has been
recommended that warm discharge water not be used for sports
and recreational purposes (20), particularly since DNA restriction
fragment profiles of environmental strains and human isolates
were homogeneous. N. fowleri cysts produced in the warm
summer months may survive the winter and are capable of
growth during the following summer.
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Acanthamoeba spp. (True Pathogen Granulomatous

Amebic Encephalitis/GAE) (Keratitis, Cutaneous Infections)
Organism:
The most characteristic feature of this genus is the presence of
spine-like pseudopods called acanthapodia. Several species
of Acanthamoeba (A. culbertsoni, A. castellanii, A. polyphaga, A.
astronyxis, A. healyi, and A. divionensis) cause granulomatous
amebic encephalitis (GAE), primarily in immunosuppressed,
chronically ill, or otherwise debilitated persons. These patients
tend to have no relevant history involving exposure to
recreational freshwater. Acanthamoeba spp. also cause amebic
keratitis and corneal ulcerations, as well as cutaneous infections.
Acanthamoeba cysts
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Tropho
Acantham
zoite
oeba in
wet
brain
mount
Keratitis
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Cutane
ous
lesion
Life Cycle:
Unlike N. fowleri, Acanthamoeba spp. do not have a flagellate
stage in the life cycle, only the trophozoite and cyst.
Acquired:
The amebae may enter through the lower respiratory tract or
through ulcerated or broken skin, causing GAE, particularly those
who are immunocompromised. With keratitis, two factors are
often involved: trauma and contaminated water.
Epidemiology:
Mitochondrial DNA fingerprinting of Acanthamoeba spp. from the
American Type Culture Collection and environmental sources has
confirmed that approximately half of the 35 isolates displayed
fingerprints similar to those of clinical isolates. Comparisons with
other published mitochondrial DNA fingerprints indicated that two
groups have counterparts in Europe and Japan and in Europe and
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Australia. These data provide strong evidence that the most

common clinical isolates do have counterparts that are
geographically widespread and can be isolated from the
environment. Acanthamoeba cysts can remain viable in water at
4C for 24 years. In animal studies, it has been shown that some
of the isolates completely lost their virulence only after 8 years of
in vitro cultivation. On the basis of these results, one can assume
that cyst viability may be as long as 25 years and that these cysts
can maintain their invasive properties.
Clinical Features:
GAE. Meningoencephalitis caused by Acanthamoeba spp. may
present as an acute suppurative inflammation of the brain and
meninges like that seen with N. fowleri infection.
However, Acanthamoeba spp. are generally reported to cause a
more chronic protracted slowly progressive form of
meningoencephalitis (GAE). The incubation period of GAE is
unknown; several weeks or months are probably necessary to
establish the disease. The clinical course tends to be subacute or
chronic and is usually associated with trauma or underlying
disease, not swimming. GAE is characterized by confusion,
dizziness, drowsiness, nausea, vomiting, headache, lethargy, stiff
neck, seizures, and sometimes hemiparesis. Within the CNS, the
cerebral hemispheres are the most likely tissue to be involved.
There may be edema and hemorrhagic necrosis within the
temporal, parietal, and occipital lobes. A chronic inflammatory
exudate can be seen over the cortex, mainly of
polymorphonuclear leukocytes and mononuclear cells. Unlike in
PAM caused by N. fowleri, both trophozoites and cysts are found
throughout the tissue. Granulomatous inflammation necrosis and
thrombosed vessels are seen in the brain. Multinucleated giant
cells forming granulomas can be seen in immunocompetent
patients, while in immunocompromised individuals, granuloma
formation is generally poor or lacking. It is unknown whether
brain necrosis is caused by direct tissue destruction caused by the
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organisms, by inflammatory cytokines such as interleukin-1 or

tumor necrosis factor, or both. Also, dissemination to other
tissues such as the liver, kidneys, trachea, and adrenals can occur
in immunocompromised individuals; more unusual sites also
include the ear and necrotic bone from a bone graft of the
mandible.
Keratitis. Keratitis, uveitis, and corneal ulceration have been
associated withAcanthamoeba spp. Infections have been seen in
both hard- and soft-lens wearers, and particular attention has
been paid to soft-lens disinfection systems, including home-made
saline solutions. Heat disinfection overall appears to be more
effective than cold disinfection methods in
killingAcanthamoeba trophozoites and cysts. The onset of corneal
infection withAcanthamoeba spp. can vary tremendously;
however, two factors often appear to be involved: trauma and
contaminated water. When corneal abrasions occur, the disease
process is usually more rapid, with ulceration, corneal infiltration,
iritis, scleritis, severe pain, hypopyon (pus in the anterior
chamber), and loss of vision. When this process occurs in an
individual who wears contact lenses, the onset is more gradual,
but the results are often the same. A contact lens can act as a
mechanical vector for transport of amebae present in the storage
case onto the cornea. Subsequent multiplication and invasion of
the tissue may occur.
Cutaneous lesions. Cutaneous infections are more common in
patients with AIDS, regardless of the presence or absence of CNS
involvement. The disease includes the presence of hard
erythematous nodules or skin ulcers. The early lesions appear as
firm papulonodules that drain purulent material; these lesions
then develop into nonhealing indurated ulcers. Although
disseminated skin lesions may be the first sign
of Acanthamoeba infection, it is unclear whether the skin lesions
represent a primary focus or may result from hematogenous
spread from other body sites. Although the mortality rate for
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these individuals without CNS involvement is around 75%, it

increases to 100% if cutaneous infection is accompanied by CNS
disease
Clinical Specimen:
Tissue or Scans: In the differential diagnosis, other
space-occupying lesions of the CNS (tumor, abscess, fungal
infection, etc.) must also be considered. Some of the predisposing
factors have included Hodgkin's disease, diabetes, alcoholism,
pregnancy, and steroid therapy. Organisms have also been found
in the adrenal gland, brain, eyes, kidneys, liver, pancreas, skin,
spleen, thyroid gland, and uterus.
Eye: Decreased corneal sensation has contributed to the
misdiagnosis ofAcanthamoeba keratitis as herpes simplex
keratitis; this mistake can also be attributed to the presence of
irregular epithelial lesions, stromal infiltrative keratitis, and
edema, which are commonly seen in herpes simplex
keratitis.Acanthamoeba keratitis may be present as a secondary
or opportunistic infection in patients with herpes simplex keratitis.
Unfortunately, as a result, treatment can be delayed for 2 weeks
to 3 months. The presence of nonhealing corneal ulcers and the
presence of ring infiltrates are also clinical signs that alert the
ophthalmologist to the possibility of amebic infection.
Specimens should never be refrigerated prior to examination.
helpful. If N. fowleri is the causative agent, trophozoites only are
normally seen. If the infecting organism isAcanthamoeba spp.,
cysts may also be seen in specimens from CNS infection.
Unfortunately, most cases are diagnosed at autopsy; confirmation
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of these tissue findings must include culture and/or special

staining with monoclonal reagents in indirect fluorescent antibody
procedures. Organisms can also be cultured on nonnutrient agar
plated with Escherichia coli.
The most effective approach uses nonnutrient agar plates with
Page's saline and an overlay growth of Escherichia coli on which
the amebae feed. Tissue stains are also effective, and cysts can
be stained with Gomori's silver methenamine, periodic acid-Schiff,
and calcofluor white.
The amebae can be identified in histologic preparations by
indirect immunofluorescence and immunoperoxidase techniques.
The organism in tissue sections looks very much like
an Iodamoeba btschlii trophozoite, with a very large karyosome
and no peripheral nuclear chromatin; the organisms can also be
seen with routine histologic stains.
Trophozoite Motile organisms have spine-like pseudopods;
however, progressive movement is usually not very evident.
There is a wide range (25 to 40 m), with the average diameter of
the trophozoites being 30 m. The nucleus has the typical large
karyosome, like that seen in N. fowleri. This morphology can be
seen on a wet preparation.
Cyst: The cysts are usually round with a single nucleus, also
having the large karyosome seen in the trophozoite nucleus. The
double wall is usually visible, with the slightly wrinkled outer cyst
wall and what has been described as a polyhedral inner cyst wall.
This cyst morphology can be seen in organisms cultured on agar
plates. Cyst formation occurs under adverse environmental
condition; they are resistant to biocides, chlorination, and
antibiotics. The cysts can also survive low temperatures (0 to
2C).
Culture: The most effective approach uses nonnutrient agar
plates with Page's saline and an overlay growth of Escherichia
coli on which the amebae feed. There is also evidence that
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phosphate-buffered saline can be used. Specimens transported in

ameba-saline (5.0 ml) and filtered through 13-mm,
0.22-m-pore-size cellulose acetate and nitrate filters (Millipore
Corp., Bedford, Mass.) have also been acceptable for organism
recovery. The filter is then placed in the center of the nonnutrient
agar plate seeded with E. coli. Tissue stains are also effective,
and cysts can be stained with Gomori's silver methenamine,
periodic acid-Schiff, and calcofluor white. Both cysts and
trophozoites can be recovered from culture.
Laboratory Report:
Acanthamoeba spp.seen and/or cultured
Treatment:
Trophozoites and cysts of Acanthamoeba isolates vary in their
sensitivity to antimicrobial agents. They are sensitive in vitro to
ketoconazole, pentamidine, hydroxystilbamidine, paromomycin,
5-fluorocytosine, polymyxin, sulfadiazine, trimethoprimsulfamethoxazole, azithromycin, and extracts of medicinal plants
and, especially, to combinations of these drugs. Increased
awareness of these infections and early therapy play a large role
in patient outcomes. The use of drug combinations helps address
resistance that may exist or occur during treatment. In vitro
testing confirms strain and species differences in sensitivity, so
that no single drug is effective against all amebae.
Because amebic keratitis may be misdiagnosed and/or therapy
delayed, total blindness may result in the infected eye(s).
However, if the infection is correctly diagnosed early and the
epithelium alone is involved, debridement may be sufficient for
cure. This approach not only removes organisms, but enhances
the delivery of topical medications. However, if treatment is
delayed and the organisms invade the cornea or tissues below the
cornea, therapy may need to be continued for many months to a
year or longer. These patients must continually be monitored
because the cysts tend to be quite resistant to therapy; treatment
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failures tend to be fairly common. In vitro susceptibility testing

may be very helpful in these cases
Control:
General preventive measures are similar to those for N.
fowleri. Several disinfectants have been tested against
both Naegleria spp. andAcanthamoeba spp. The conclusion is
that Naegleria spp. are generally susceptible to swimming pool
levels of chlorine but Acanthamoeba spp. are more resistant. The
recovery of Acanthamoeba spp. in nasal isolates and pharyngeal
swabs may indicate human introduction of the organisms into
swimming pools. With the increasing reports of infection in
wearers of soft contact lenses, it will be important to carefully
consider the effectiveness of various contact lens disinfection
systems. A number of factors probably play a role in the
increased incidence of infection: a large number of HIV-infected
individuals and more patients undergoing cancer chemotherapy
or immunosuppressive therapy for organ transplantation.
31 | P a g e
Giardia lamblia (duodenalis, intestinalis) (Pathogen)

Organism:
This organism belongs to the flagellates, is a true pathogen, and
causes giardiasis. Both the trophozoite (usual size, 10-20 m long
and 5-15 m wide) and cyst forms (usual size, 11-14 m long and
7-10 m wide) can be found in clinical specimens.
Life Cycle:
Intestine (duodenum, occasionally gallbladder and rarely
bronchoalveolar lavage fluid), organisms passed in feces
Acquired:
water. Various studies have published data suggesting that
zoonotic transmission between humans and animals is, at times,
more likely or less likely to occur.
Epidemiology:
32 | P a g e
Clinical Features:
Intestinal: Diarrhea, epigastric pain, flatulence, increased fat and
mucus in stool, gallbladder colic and jaundice. The incubation
time for giardiasis ranges from approximately 12 to 20 days.
Because the acute stage usually lasts only a few days, giardiasis
may not be recognized as the cause but may mimic acute viral
enteritis, bacillary dysentery, bacterial or other food poisonings,
acute intestinal amebiasis, or "traveler's diarrhea"
(toxigenic Escherichia coli). However, the type of diarrhea plus the
lack of blood, mucus, and cellular exudate is consistent with
giardiasis.
The acute phase is often followed by a subacute or chronic phase.
Symptoms in these patients include recurrent, brief episodes of
loose, foul-smelling stools; there may be increased distention and
foul flatus. Between passing the mushy stools, the patient may
have normal stools or may be constipated. Abdominal discomfort
continues to include marked distention and belching with a
rotten-egg taste. Chronic disease must be differentiated from
amebiasis; disease caused by other intestinal parasites such
asDientamoeba fragilis, Cryptosporidium spp., Cyclospora
cayetanensis,Isospora belli, Strongyloides stercoralis; and
inflammatory bowel disease and irritable colon. On the basis of
symptoms such as upper intestinal discomfort, heartburn, and
belching, giardiasis must be differentiated from duodenal ulcer,
hiatal hernia, and gallbladder and pancreatic disease.
Clinical Specimen:
Intestinal: Stool, duodenal aspirates, EnteroTest capsule, biopsy
Extraintestinal: Fluids
Laboratory Diagnosis*:
permanent stained smear); fecal immunoassay to
detect Giardia (antigen and/or actual organism using FA
immunoassay); two fecal immunoassays are required prior to
33 | P a g e
reporting a negative antigen (due to shedding patterns).

stain)
Trophozoite: Teardrop shaped from the front, curved portion of a
spoon from the side; two nuclei, curved median bodies, linear
axonemes
Cyst: Round to oval, will contain 4 nuclei and more median
bodies and axonemes; cysts are very three-dimensional; they
may also appear to be shrunk within the cyst membrane.
Laboratory Report:
Giardia lamblia (indicate trophozoites and/or cysts)
Treatment:
In the absence of a parasitologic diagnosis, the treatment of
suspected giardiasis is a common question with no clear-cut
answer. The approach depends on the alternatives and the degree
of suspicion of giardiasis, both of which will vary among patients
and physicians. However, it is not recommended that treatment
be given without a good parasitologic workup, particularly since
relief of symptoms does not allow a retrospective diagnosis of
giardiasis; the most commonly used drug, metronidazole, targets
other organisms besides G. lamblia.
Another question involves treatment of asymptomatic
patients. Generally, it is recommended that all cases of proven
giardiasis be treated because the infection may cause subclinical
malabsorption, symptoms are often periodic and may appear
later, and a carrier is a potential source of infection for others.
Certainly, in areas of the world where infection rates, as well as
the prospect of reinfection, are extremely high, the
benefit-per-cost ratio would also have to be examined.
34 | P a g e
Control:
washing of contaminated fruits and vegetables. Most experts
agree that the single most effective practice that prevents the
spread of infection in the child care setting is thorough
handwashing by the children, staff, and visitors. Rubbing the
hands together under running water is the most important part of
washing away infectious organisms. Premoistened towelettes or
wipes and waterless hand cleaners should not be used as a
substitute for washing hands with soap and running water. These
guidelines are not limited to giardiasis but include all potentially
infectious organisms.
Because of the potential for wild animal and possibly domestic
animal reservoir hosts, measures in addition to personal hygiene
and improved sanitary measures have to be considered. Iodine
has been recommended as an effective disinfectant for drinking
water, but it must be used according to directions. Filtration
systems have also been recommended, although they have
certain drawbacks, such as clogging.
35 | P a g e
Dientamoeba fragilis (Pathogen)

Organism:
This organism belongs to the flagellates, is a true pathogen, and
causes symptoms. There is only a trophozoite stage (usual size,
9-12 m); no cyst stage has been identified.
Trophozoites with single nucleus

Beginning to fragment (left),
fragmented (right)
Trophozoites with two fragmented nuclei

Life Cycle:
Intestine, organisms passed in feces; trophozoites thought to be
transmitted inside of helminth eggs (Ascaris lumbricoides,
Enterobius vermicularis).
Acquired:
Fecal-oral transmission via trophozoite form; contaminated food
and water
Epidemiology:
36 | P a g e
Clinical Features:
Intestinal: Intermittent diarrhea, abdominal pain, nausea,
anorexia, malaise, fatigue, poor weight gain, unexplained
eosinophilia. The most common symptoms in patients infected
with this parasite appear to be intermittent diarrhea and fatigue.
In some patients, both the organism and the symptoms persist or
reappear until appropriate treatment is initiated. Clinicians should
include infection with D. fragilis in their differential diagnosis in
patients presenting with abdominal pain, diarrhea, unexplained
flatulence, nausea, and vomiting.
Clinical Specimen:
Intestinal: Stool
permanent stained smear); permanent stained smear mandatory
for identification. It is particularly important that permanently
stained smears of stool material be examined with an oil
immersion objective (100). These organisms have been
recovered in formed stool; therefore a permanent stained smear
must be prepared for every stool sample submitted for a parasite
examination.
Trophozoite: Round, 1 (20-40%) nucleus or 2 nuclei (60-80%);
nuclei tend to fragment into 3-5 granules. Cytoplasm often filled
with ingested debris; size range of trophozoites is tremendous,
even on a single slide.
Laboratory Report:
Dientamoeba fragilis (indicate trophozoites)
Treatment:
Metronidazole
Control:
37 | P a g e
38 | P a g e
Chilomastix mesnili (Nonpathogen)

Organism:
This organism belongs to the flagellates, is a nonpathogen, and
causes no disease. Both the trophozoite (usual size, 6-24 m long
and 4-8 m wide) and cyst forms (usual size, 6-10 m long and 46 m wide) can be found in clinical specimens.
Trophozoites
Cyst in iodine
Cyst,
permanent
stain
Life Cycle:
Acquired:
water
Epidemiology:
Clinical Features:
None.
Clinical Specimen:
Intestinal: Stool
permanent stained smear)
39 | P a g e
Trophozoite: Pear shaped, 1 nucleus and distinct oral groove
(cytostome); flagella are rarely seen without special stains.
Cyst: Pear or lemon shaped, will contain 1 nucleus; the
cytostomal fibril is curved and is called the shepherds Crook.
Laboratory Report:
Chilomastix mesnili (indicate trophozoites and/or cysts)
Treatment:
None
Control:
40 | P a g e
Trichomonas vaginalis (Pathogen)

Organism:
This organism belongs to the flagellates, is a pathogen, and
causes disease. The trophozoite (usual size, 7-23 m long and 515 m wide) can be found in clinical specimens such as urine,
urethral discharge, and vaginal smears.
Trichomonas vaginalis trophozoites

Note: Undulating membrane goes about half way down the body,
not to the bottom.
Life Cycle:
Urinary-genital system; no known cyst form.
Acquired:
Although the exact mode of transmission is not known, it is
assumed to be by sexual transmission or rarely contaminated
towels, underpants, etc. Humans are the only natural host for T.
vaginalis, and normal body sites for these organisms include the
vagina and prostate. Apparently, the organisms feed on the
mucosal surface of the vagina, where bacteria and leukocytes are
found. The preferred pH for good growth in females is slightly
alkaline or acidic (6.0 to 6.3 optimal), not the normal pH of the
healthy vagina.
Epidemiology:
Worldwide. It is estimated that 5 million women and 1 million
men in the United States have trichomoniasis, with an annual
incidence estimated to be 7.4 million new cases. The annual
41 | P a g e
incidence of trichomoniasis worldwide is estimated to be more

than 170 million cases, which does not include the number of
asymptomatic cases that are not treated. In North America, more
than eight million new cases are reported yearly, with an
estimated rate of asymptomatic cases being as high as 50%.
Trichomoniasis is now the primary nonviral sexually transmitted
disease worldwide. Infection with T. vaginalis has major health
consequences for women, including complications in pregnancy,
association with cervical cancer, and predisposition to HIV
infection.
Clinical Features:
T. vaginalis is site specific and usually cannot survive outside the
urogenital system. Although the incubation period is not welldefined, in vitro studies provide a range of 4 to 28 days. After
introduction, proliferation begins, with resulting inflammation and
large numbers of trophozoites in the tissues and the secretions.
As the infection becomes more chronic, the purulent discharge
diminishes, with a decrease in the number of organisms. The
onset of symptoms such as vaginal or vulval pruritus and
discharge is often sudden and occurs during or after menstruation
as a result of the increased vaginal acidity. In acute infections,
diffuse vulvitis is seen and is due to copious leukorrhea. The
discharge is frothy, yellow or green, and mucopurulent; however,
only about 10 to 12% of women exhibit this frothy discharge.
Small punctate hemorrhagic spots can be seen on the vaginal and
cervical mucosa; this has been called a "strawberry appearance"
and is seen in about 2% of patients. In general, symptoms
include vaginal discharge (42%), odor (50%), and edema or
erythema (22 to 37%). Other complaints include dysuria and
lower abdominal pain.
In chronic infections, symptoms may be very mild with pruritus
and some pain during sexual intercourse, while the vaginal
secretion may be scanty and mixed with mucus. Individuals with
these symptoms are the major source of transmission. From 25 to
50% of infected women may be asymptomatic and have a normal
vaginal pH of 3.8 to 4.2 and normal vaginal flora. Although there
is a carrier form, about 50% of these women will develop clinical
42 | P a g e
symptoms during the following six months.

T. vaginalis may also cause neonatal pneumonia.
Clinical Specimen:
Urogenital System: Vaginal and urethral discharges, prostatic
secretions. Since the morphology of nonpathogenic P.
hominis from stool samples is very similar to that of pathogenic T.
vaginalis, it is important to ensure that the specimen is not
contaminated with fecal material.
Urogenital System: Diagnosis is based on the recovery of the
organisms from the appropriate clinical specimen. Wet mounts,
stained smears, culture, and antigen detection are available.
Recombinant DNA techniques are becoming more widely
available.
Trophozoite: The trophozoite is pear-shaped, has a jerky, rapid
motility; the undulating membrane extends half the length of the
trophozoite with no free posterior flagellum; the axostyle is seen
to protrude through the bottom of the organism
Laboratory Report:
Trichomonas vaginalis (indicate trophozoites)
Treatment:
Metronidazole
Control:
Infection is acquired primarily through sexual intercourse,
hence the need to diagnose and treat asymptomatic males. The
organism can survive for some time in a moist environment such
as damp towels and underclothes; however, this mode of
transmission is thought to be very rare. Evidence continues to
accumulate implicating T. vaginalis as a potential contributor to
poor outcomes in both women and men. In women, this infection
may play a role in development of cervical neoplasia,
43 | P a g e
postoperative infections, and potential problems with pregnancy.

It is also seen as a factor in atypical pelvic inflammatory disease
and infertility. In men, trichomoniasis causes nongonoccocal
urethritis and contributes to male infertility.
44 | P a g e
Balantidium coli (Pathogen)

Organism:
This organism belongs to the ciliates, is a true pathogen, and
causes balantidiasis. Both the trophozoite (usual size, 50-100 m
long and 40-70 m wide) and cyst forms (usual size, 5-70 m in
diameter) can be found in clinical specimens.
Tropho
Trop
zoites
hozoi Cyst
in
te
tissue
Life Cycle:
Large bowel, trophozoites and cysts passed in stool AND/OR
Large bowel, tissue invasion (mucosal ulcers, abscess formation)
Acquired:
water
Epidemiology:
Worldwide, primarily human-to-human transmission, also pig-tohuman transmission. Particularly susceptible to infection are
persons working as pig farmers or in slaughterhouses (28%
infection in New Guinea). Human infection is fairly rare in
temperate areas, although once the infection is established, it can
develop into an epidemic, particularly where poor environmental
sanitation and personal hygiene are found. This situation has
been seen in mental hospitals in the United States.
45 | P a g e
Clinical Features:
Intestinal: Some individuals with B. coli infections are totally
asymptomatic, whereas others have symptoms of severe
dysentery similar to those seen in patients with amebiasis.
Symptoms usually include diarrhea or dysentery, tenesmus,
nausea, vomiting, anorexia, and headache. Insomnia, muscular
weakness, and weight loss have also been reported. The diarrhea
may persist for weeks to months prior to the development of
dysentery. There may be tremendous fluid loss, with a type of
diarrhea similar to that seen in cholera or in some coccidial or
microsporidial infections.
Extraintestinal: B. coli have the potential to invade tissue. On
contact with the mucosa, B. coli may penetrate the mucosa with
cellular infiltration in the area of the developing ulcer. Some of the
abscess formations may extend to the muscular layer. The ulcers
may vary in shape, and the ulcer bed may be full of pus and
necrotic debris. Although the number of cases is small,
extraintestinal disease has been reported (peritonitis, urinary
tract, inflammatory vaginitis).
Clinical Specimen:
Intestinal: Stool, examination of mucosal ulcers (scrapings,
biopsy)
Extraintestinal: Fluids
permanent stained smear); organisms are so large, they may be
missed on permanent stain (resemble helminth eggs or debris);
the concentration sediment is the most appropriate way to
visualize the organisms.
stain)
Trophozoite: Large, oval shaped; two nuclei (bean-shaped
macronucleus easily visible, very small micronucleus often not
seen); cilia easily seen around the periphery of the trophozoite.
46 | P a g e
Cyst: Round, will contain the same 2 nuclei as seen in the

trophozoite; cilia more difficult to see within the cyst wall.
Laboratory Report:
Balantidium coli (indicate trophozoites and/or cysts)
Treatment:
Tetracyclines, Metronidazole,
Control:
washing of contaminated fruits and vegetables; prevention of
human-pig contact, hygienic rearing of pigs.
47 | P a g e
Sarcocystis spp. (Pathogen)

Organism:
This organism belongs to the coccidia. The organism once known
asIsospora hominis is now recognized as being a part of the life
cycle ofSarcocystis spp. Two well-described species
are Sarcocystis bovihominis(cattle) and S. suihominis (pigs). When
uncooked meat from these infected animals is ingested by
humans, gamogony can occur within the intestinal cells, with the
eventual production of the sporocysts in stool. Some publications
refer to S. bovihominis as S. hominis. In this case, humans who
have ingested meat containing the mature sarcocysts serve as
the definitive hosts
Sarcocysts in muscle
Sporocysts;
oocyst wall
appears to be
missing
Life Cycle:
Intestine,
Species of Sarcocystis have an obligatory 2-host life
cycle. Intermediate hosts such as herbivores and omnivores
become infected through ingestion of sporocysts excreted in the
feces of the definitive host such as carnivores and omnivores.
The definitive hosts become infected through ingestion of mature
cysts found in muscles of the intermediate hosts. In some
intermediate hosts, such as cattle and sheep, all adult animals
may be infected.
Acquired:
Fecal-oral transmission via oocyst form; contaminated poorly
cooked meat
48 | P a g e
Epidemiology:
Worldwide, human-to-human transmission, food-borne and
waterborne transmission
Clinical Features:
Muscle: When humans accidentally ingest oocysts from other
animal stool sources, the sarcocysts that develop in human
muscle apparently do little if any harm (schizogony). The
prepatent period in humans is from 9 to 10 days. There is
essentially no inflammatory response to these stages in the
muscle and no conclusive evidence of pathogenicity of the mature
sarcocyst. However, symptoms have been seen in some patients,
probably associated with disintegration of the sarcocysts. Painful
muscle swellings, measuring 1 to 3 cm in diameter, initially
associated with erythema of the overlying skin in various parts of
the body, occur periodically and last for 2 days to 2 weeks. Some
patients also have fever, diffuse myalgia, muscle tenderness,
weakness, eosinophilia, and bronchospasm.
Intestine: When humans serve as definitive hosts, prevention
involves adequate cooking of beef and pork; when humans are
intermediate hosts, preventive measures involve careful disposal
of animal feces that may contain the infective sporocysts. This
may be impossible in the wilderness areas, where wild animals
may serve as reservoir hosts for many of the different types of
organisms that have been grouped under the
termSarcocystis "lindemanni." However, this name is no longer
used.
Clinical Specimen:
Muscle: Biopsy. Intact sarcocysts in skeletal or cardiac muscle of
humans measure up to 100 by 325 m and are usually not
accompanied by an inflammatory reaction. Each sarcocyst
contains many bradyzoites, approximately 7 to 16 m long.
Inflammation follows disintegration of the cysts and death of the
intracystic bradyzoites. Vasculitis is seen in the muscle and
subcutaneous tissues. Histologic findings include myositis with
vasculitis and sometimes myonecrosis.
Intestinal: Stool.
49 | P a g e
Muscle: Routine histology
Stool: Ova and Parasite examination (concentration most
important method)
Sarcocyst: Sarcocysts measure up to 100 by 325 m; each
sarcocysts contains many bradyzoites, approximately 7 to 16 m
long.
Oocyst: Oocysts measure 15-19 by 8-10 m; oocyst wall appears
to be missing; cant differentiate S. hominis from S. suihominis.
Laboratory Report:
Sarcocystis oocysts or sarcocysts from tissue
Treatment:
No specific therapy is known for the muscle stages; however,
corticosteroids should reduce the allergic inflammatory reactions
occurring after cyst rupture.
50 | P a g e
Other Genera and Species (Pathogen, Microsporidia)

Organism:
The microsporidia are obligate intracellular parasites that have
been recognized in a variety of animals, particularly
invertebrates; they have been reclassified with the Fungi in the
Phylum Microsporidia. Typical sizes range from 1.5 to 5 m wide
and 2 to 7 m long; unfortunately, the organisms found in
humans tend to be quite small, ranging from 1.5 to 2 m. Until
recently, awareness and understanding of human infections have
been marginal; only with increased understanding of AIDS within
the immunosuppressed population has attention been focused on
these organisms.
Spore
Extruded
with
Polar
Polar
Tubule
Tubule
Silver
stain
51 | P a g e
Spores in
corneal
tissue
button
Silver
PAS stain,
stain, eye
eye tissue
tissue
Life Cycle:
Infection occurs with the introduction of infective sporoplasm
through the polar tubule into the host cell. The microsporidia
multiply extensively within the host cell cytoplasm; the life cycle
includes repeated divisions by binary fission (merogony) or
multiple fission (schizogony) and spore production (sporogony).
Both merogony and sporogony can occur in the same cell at the
same time. During sporogony, a thick spore wall is formed, thus
providing environmental protection for this infectious stage of the
parasite. An example of infection potential is illustrated
by Enterocytozoon bieneusi, an intestinal pathogen. The spores
are released into the intestinal lumen and are passed in the stool.
These spores are environmentally resistant and can then be
ingested by other hosts. There is also evidence for inhalation or
spores and evidence in animals that suggests that human
microsporidiosis may also be transmitted via the rectal route.
Currently, there are a number of genera that have been
recognized as human pathogens: the more
common, Encephalitozoon and Enterocytozoon, and the less
common, Nosema,
Anncaliia (Brachiola), Pleistophora,Trachipleistophora, Vittaforma,
and "Microsporidium," a catch-all genus for organisms that have
not yet been classified (or may never be classified due to a lack of
specimen). Classification criteria include spore size, configuration
of the nuclei within the spores and developing forms, the number
of polar tubule coils within the spore, and the relationship
between the organism and host cell.
Acquired:
Fecal-oral transmission via infective spores; contaminated food
and water
Epidemiology:
Clinical Features (Other Microsporidia):
Organisms have been recovered from the eye, CNS, urine,
sinuses, conjunctivae, nasal epithelium, respiratory tract,
myocardium, diaphragm, arterial walls, kidney tubules, adrenal
cortex, liver, lungs, and muscle fibers.
52 | P a g e
Clinical Specimen:
Intestinal: Stool, examination of mucosal surface (biopsy);
dissemination to kidneys, lower airways and biliary tract appears
to occur via infected macrophages.
Extraintestinal: Fluids, biopsy specimens
Laboratory Diagnosis: Note: ID to the species level is not
possible from special stains.
Intestinal: Ova and Parasite examination (concentration ONLY);
from concentrate sediment, (500 x g for 10 min) modified
trichrome stains are performed. Some fecal immunoassays are
available in Europe, but are not FDA approved for use within the
United States. Multiple fecal examinations may be required to
recover the organisms, particularly if the stools are formed; there
is a direct relationship between the stool consistency and the
number of spores present (diarrhea = more spores).
Stool preparations must be very thin, the staining time is 90 min,
and the slide must be examined at x 1,000 (or higher)
magnification. Unfortunately, there are many objects within stool
material that are oval, stain pinkish with trichrome, and measure
approximately 1.5 to 3 m. If this stain is used for the
identification of microsporidia in stool, positive control material
should be available for comparison. Additional modifications of
this method include the use of heat and a shorter staining time.
There is also some evidence to indicate that pretreatment of fecal
specimens (1:1) with 10% KOH may provide a better quality
smear to examine when using the modified trichrome stains.
Another approach involves the use of chemofluorescent agents
(optical brightening agents) such as Calcofluor, Fungi-Fluor, or
Uvitex 2B. These reagents are sensitive, but nonspecific; objects
other than microsporidial spores will also fluoresce. This is a
particular problem when examining stool specimens; both false
positive and false negative results have been seen.
Extraintestinal: Modified trichrome stains
Tissue: Tissue stains such as PAS, Silver, tissue Gram stains and
others are specifically recommended for the microsporidial
spores. Microsporidial infections can be misdiagnosed in tissues
and can be confused withCryptococcus neoformans infections.
53 | P a g e
Mucus granules in goblet cells can take up stain and can be very
confusing. Good preservation and thin tissue sections (1 m) that
have been resin-embedded enhance the resolution of cellular
detail. Demonstration of the coiled polar tube within spores is
diagnostic for microsporidial infection. Encephalitozoon
intestinalis is not confined to epithelial cells, but is seen in
macrophages in the lamina propria. Although the primary site
appears to be the small bowel, these organisms can disseminate
to other sites, including duodenum, jejunum, ileum, colon, kidney,
liver, and gallbladder. Although electron microscopy is very
specific, the sensitivity is not that high.
Spore: Oval spores, containing a coiled polar tubule. However,
the polar tubule is not visible in every spore; when seen, it
appears as a horizontal or diagonal line across the spore. Without
seeing the polar tubule, it is not possible to definitively ID the
structures as polar tubules/microsporidial spores (Microsporidial
spores present.)
Tissue: Developing spores (groups) can be seen within the tissue
cells.
Laboratory Report:
Microsporidialspores present; if in stool, the two most likely
species areEnterocytozoon bieneusi and Encephalitozoon
intestinalis. If from tissues, other genera and species are also
possibilities.
Treatment:
Although a number of drugs have been tested, few are
totally effective. This species responds well to albendazole,
whereasEnterocytozoon bieneusi does not. Although apparently
static rather than cidal effects are seen with E.
bieneusi infections, treatment with albendazole results in
reduction of symptoms in as many as 50% of patients infected
with this organism. Albendazole as a systemic agent is
recommended when the organisms have been confirmed in urine
or nasal smears.
Fumagillin (soluble salt Fumidil B) has activity against
microsporidia, and solutions applied topically have been used in
54 | P a g e
corneal infections. The effects of this drug are static rather than
cidal, and relapses of infection occur when the treatment has
been discontinued. In one study, the efficacy of fumagillin was
measured by clearance of E. bieneusi from stools and intestinal
biopsy specimens; four patients who received fumagillin remained
free of E. bieneusiafter a mean follow-up of 10 months.
Itraconazole can also be recommended to treat ocular, nasal, and
paranasal sinus infection caused by E. cuniculi parasites when
albendazole fails.
55 | P a g e
Sappinia spp. (True Pathogen Granulomatous Amebic

Encephalitis/GAE)
Organism:
The genus Sappinia with the single species Sappinia pedata was
established for an amoeba with two nuclei and pedicellate "cysts"
by Dangeard in 1896. In 1912, Alexeieff transferred an also
double nucleated, but apparently sexually reproducing amoeba to
this genus as Sappinia diploidea that had been described as
Amoeba diploidea by Hartmann and Ngler in 1908. Molecular
analyses have confirmed the differentiation between S.
pedata andS. diploidea; however, the genus splits into more than
two well separated clusters. The genus Sappinia is now classified
as a member of the Thecamoebidae and, moreover, as potentially
pathogenic. Subacute or chronic CNS infections due
to Acanthamoeba spp, Balamuthia mandrillaris,
and Sappinia spp., which occasionally cause cerebral abscess, are
termed granulomatous amebic encephalitis (GAE). In 2001,
Gelman and colleagues reported a case of severe encephalitis in
an immunocompetent young man caused by Sappinia. Another
case of GAE and abscess formation was reported from Turkey in
2009; in this case, the patient died.
Sappinia trophozoite andcyst

56 | P a g e
Trophozoites in tissue (note the appearance of 2 nuclei)
57 | P a g e
Life Cycle:
The life cycle is similar to that seen with Acanthamoeba spp.;
likeAcanthamoeba spp., Sappinia does not have a flagellated
stage in its life cycle as do organisms classified as N. fowleri.
Acquired:
The amebae may enter through the lower respiratory tract or
through ulcerated or broken skin, causing GAE, particularly those
who are immunocompromised.
Epidemiology:
The amoebae have a cosmopolitan distribution in soil and water,
providing multiple opportunities for contacts with humans and
animals, as evidenced by antibody titers in surveyed human
populations. The numbers of infections caused by these amoebae
are low in comparison to other protozoal parasitic infections.
Pathogenic free-living amebae can be isolated from freshwater
lakes, thermally polluted waters, sediment, thermal springs,
swimming pools, soil, air conditioning vents, air, and the domestic
water supply. In addition to causing human disease, these
organisms also can harbor intracellular pathogenic bacteria such
as Legionella pneumophila and may serve as vectors of bacterial
infections in humans.
Clinical Features:
GAE. In one of the cases of Sappinia amoebic encephalitis that
has been reported, a sinus infection occurred prior to the onset of
symptoms. The individual developed nausea, vomiting, bifrontal
headache, photophobia, and blurry vision. A loss of consciousness
occurred for a brief. A successful outcome in this patient was
reported after surgical excision of a tumor-like mass in the brain
and treatment using azithromycin, intravenous pentamidine,
itraconazole, and flucytosine.
Clinical Specimen:
Tissue or Scans: In the differential diagnosis, other
58 | P a g e
space-occupying lesions of the CNS (tumor, abscess, fungal

infection, etc.) must also be considered. A solitary tumor-like
mass without an abscess wall can be seen via MRI.
PCR: Multiplex PCR studies can distinguish Acanthamoeba,
Balamuthia, and Naegleria from Sappinia for simultaneous
detection of the four genera.
CSF specimens should never be refrigerated prior to examination.
helpful.
Culture on nonnutrient agar with an overlay of bacteria and on
tissue culture cells is also an option.
Trophozoite: Sappinia amoebae can be distinguished from other
free-living amebae by the presence of a distinctive double nucleus
in which the 2 nuclei are closely apposed with a central flattening.
Two nucleoli are found in the double nucleus. These structures can
be observed in paraffin sections stained with H & E, Giemsa, or
Periodic Acid Schiff. The amoebae are readily observed in cryostat
sections stained with H & E. Brain tissue can be fixed in
glutaraldehyde and prepared for transmission electron
microscopy to visualize the amoebae in tissue.
Laboratory Report:
Sappinia confirmed (confirmation to genus/species is available
through CDC).
Free-living amebae present.
59 | P a g e
Treatment:
Treatment has included surgery, as well as azithromycin,
intravenous pentamidine, itraconazole, and flucytosine.
60 | P a g e
Cystoisospora (Isospora) belli (Pathogen)

Organism:
This organism belongs to the coccidia, is a true pathogen, and
causes isosporiasis. The oval oocysts containing one or two
immature sporonts, measure 20 to 33 by 10 to 19 m, and are
found in fecal specimens.
Top Row:
Immature
oocysts
Bottom
Row,
Calcofluor
White,
MAF
Matu
re
Mat
oocy ure
st, oocy
wet st,
mou MAF
nt
MAF = Modified Acid-Fast Stain (1% Acid Rinse)

Life Cycle:
Intestine, oocysts passed in feces, are not infectious (require
further development outside of the body), survive in the
environment, and are transmitted via contaminated food and/or
water. Rare extraintestinal infections have been documented in
severely immunocompromised patients.
61 | P a g e
Acquired:
Fecal-oral transmission via oocyst form; contaminated food and
water
Epidemiology:
Worldwide, human-to-human transmission, food-borne and
waterborne transmission
Clinical Features:
Intestinal: Diarrhea which last for months to years (watery, 6-10
per day), weight loss, abdominal colic, and fever.
Immunosuppressed tend to have profuse diarrhea with an
abnormal mucosa.
Extraintestinal: At autopsy, microscopic findings associated
with I. belliinfection were seen in lymph nodes and walls of the
intestine.
Chronic infections develop in some patients, and oocysts can be
shed for several months to years. In one particular case, an
immunocompetent individual had symptoms for 26 years and C.
belli was recovered in stool a number of times over a 10-year
period. Eosinophilia is found in many patients, recurrences are
quite common, and the disease is more severe in infants and
young children.
Clinical Specimen:
Intestinal: Stool
Extraintestinal: Biopsy specimens, routine histology
Intestinal: Concentration sediment wet preparation; modified
acid-fast stains, calcofluor white, auramine-rhodamine
Extraintestinal: Routine histology
Oocyst: Oval oocysts (measure 20-33 m by 10-19 m,
containing 1 or 2 immature sporonts. Continued development
outside the body required for infectivity
Tissue: Developing stages seen within the cells of the intestine
62 | P a g e
Laboratory Report:
Cystoisospora (Isospora) belli oocysts
Treatment:
Trimethoprim-sulfamethoxazole (TMP-SMX)
Control:
63 | P a g e
Cryptosporidium spp. (Pathogen)

Organism:
This organism belongs to the coccidia, is a true pathogen,
and causes cryptosporidiosis. The round oocysts measure 4-6 m
and are most commonly found in fecal specimens. What was
previously calledCryptosporidium parvum and was thought to be
the primary Cryptosporidiumspecies infecting humans is now
classified as two separate species, C. parvum (mammals,
including humans) and C. hominis (primarily humans).
Differentiation of these two species based on oocyst morphology
is not possible.
Life Cycle:
Intestine, oocysts passed in feces, are immediately infectious,
survive in the environment, and are transmitted via contaminated
food and/or water. Extraintestinal infections can occur in the gall
bladder, lungs, liver, and pancreas, primarily in severely
immunocompromised patients.
Internal autoinfection occurs, particularly in the compromised
patient; immunocompetent patients tend to self cure over a
period of a few weeks.
Acquired:
Fecal-oral transmission via oocyst form; contaminated food and
water
Epidemiology:
Worldwide, primarily human-to-human transmission, also animalto-human transmission
64 | P a g e
Clinical Features:
Intestinal: Intermittent diarrhea (5-10 watery, frothy bowel
movements per day), nausea, low-grade fever, abdominal
cramps, anorexia; some may have relatively few symptoms.
Extraintestinal: Immunocompromised patients cannot self-cure;
the illness is chronic and becomes progressively worse, and the
sequelae may be a major factor leading to death.
Once the primary infection has been established, the immune
status of the host plays a very important role in determining the
length and severity of the illness.
Clinical Specimen:
Intestinal: Stool, examination of mucosal surface (biopsy)
Extraintestinal: Fluids, biopsy specimens
from concentrate sediment, (500 x g for 10 min) modified acidfast stains are performed. Fecal immunoassays are also available
(FA, EIA, immunochromatographic cartridges). Multiple fecal
examinations may be required to recover the organisms,
particularly if the stools are formed; there is a direct relationship
between the stool consistency and the number of oocysts present
(diarrhea = more oocysts).
Extraintestinal: Modified acid-fast stains
Tissue: Found at all levels of the intestinal tract, with the jejunum
being the most heavily infected site. Routine H&E staining is
sufficient to demonstrate the organisms. Under regular light
microscopy, the organisms are visible as small (~1 3 m) round
structures aligned along the brush border (intracellular, but
extracytoplasmic and found in parasitophorous vacuoles).
Oocyst: Round oocysts, containing 4 sporozoites. However,
sporozoites are not always seen in every oocyst; the oocysts are
immediately infectious when passed (even if sporozoites are not
visible).
Tissue: Oocysts (~1 3 m) can be seen aligned along the brush
border (intracellular, but extracytoplasmic and found in
parasitophorous vacuoles).
65 | P a g e
Laboratory Report:
Cryptosporidium spp.oocysts
Treatment:
Garcia, L.S. 2007. Diagnostic Medical Parasitology, 5th ed., ASM
Press, Washington, D.C.
Although a number of drugs have been tested, none are totally
effective.
Control:
washing of contaminated fruits and vegetables; prevention of
human-animal contact, particularly cattle
Cyclospora cayetanensis (Pathogen)
Organism:
This organism belongs to the coccidia, is a true pathogen, and
causes cyclosporiasis. The round oocysts measure 8-10 m
(twice the size ofCryptosporidium spp.) and are found in fecal
specimens.
Autofluorescence
Modified Acid-Fast Stains (Acid-Fast
Variable)
Safranin Stain
Low Magnification
Life Cycle:
Intestine, oocysts passed in feces, are NOT immediately
infectious, survive in the environment, and are transmitted via
contaminated food and/or water.
66 | P a g e
Acquired:
Fecal-oral transmission via oocyst form; outbreaks linked to
contaminated water and various types of fresh produce
(raspberries, basil, baby lettuce leaves, snow peas) have been
reported.
Epidemiology:
Worldwide, primarily human-to-human transmission, animal-tohuman transmission not documented
Clinical Features:
Intestinal: Nausea, low-grade fever, fatigue, anorexia, up to 7
bowel movements per day; relapses common. Developmental
stages of C. cayetanensis usually occur within epithelial cells of
the jejunum and lower portion of the
duodenum. Cyclospora infection reveals characteristics of a small
bowel pathogen, including upper gastrointestinal symptoms,
malabsorption of D-xylose, weight loss, and moderate to marked
erythema of the distal duodenum. Histopathology in small bowel
biopsy specimens reveals acute and chronic inflammation, partial
villous atrophy, and crypt hyperplasia. In patients with AIDS,
symptoms may persist for as long as 12 weeks; biliary disease
has also been reported in this group.
Clinical Specimen:
Intestinal: Stool
from concentrate sediment, (500 x g for 10 min) modified acidfast stains are performed. Some oocysts stain deep red with a
mottled appearance but no internal organization, while unstained
organisms appear as glassy, wrinkled spheres. Oocysts are
considered acid-fast variable, with colors ranging from clear to
deep red; this is unlike Cryptosporidium where almost all the
67 | P a g e
oocysts stain using modified acid-fast stains. In order to maintain

color in Cyclospora, use only a 1-3% acid decolorizer (1%
recommended) for good staining of all the coccidia
(Cryptosporidium, Cyclospora, Cystoisospora). These oocysts also
autofluoresce green (450to 490 DM excitation filter) or blue (365
DM excitation filter).
Tissue: Found at all levels of the intestinal tract, with the jejunum
being the most heavily infected site. Routine H&E staining is
sufficient to demonstrate the organisms. Under regular light
microscopy, the organisms are visible within a vacuole at the
luminal end of the enterocyte rather than at the brush border
like Cryptosporidium.
Oocyst: Round oocysts, containing no internal structure
(immature when passed not infectious).
Tissue: Organisms are visible within a vacuole at the luminal end
of the enterocyte rather than at the brush border
like Cryptosporidium.
Laboratory Report:
Cyclospora cayetanensis oocysts
Treatment:
Disease self-limiting with weeks, but trimethoprimsulfamethoxazole drug of choice.
Control:
washing of contaminated fruits and vegetables; primary food
sources identified have been fresh berries (imported from
Guatemala), basil, baby lettuce leaves as salad (mesclun)
Plasmodium falciparum (True Pathogen Malignant Tertian
Malaria)
68 | P a g e
Organism:
This organism belongs to the phylum Apicomplexa, is a true
pathogen, and causes malaria. In general, the ring forms, the
gametocytes, and occasionally a mature schizont can be found in
clinical specimens (thick and thin blood films). Other forms are
sequestered in the deep tissue capillaries.
Plasmodium falciparum ring

Plasmodium
forms
falciparumgametocyte
Life Cycle:
Within an hour of infection, sporozoites from the mosquito are
carried via the blood to the liver, where they penetrate
parenchymal cells, thus initiating the preerythrocytic or primary
exoerythrocytic cycle. The sporozoites become round or oval and
begin dividing, resulting in large numbers of liver merozoites. The
merozoites leave the liver and invade the red blood cells (RBCs),
initiating the erythrocytic cycle.
P. falciparum tends to invade all ages of RBCs, and the proportion
of infected cells may exceed 50%. Schizogony occurs in the
internal organs (spleen, liver, bone marrow, etc.) rather than in
the circulating blood. Ischemia caused by the plugging of vessels
within these organs by masses of parasitized RBCs will produce
various symptoms, depending on the organ involved. It has been
suggested that a decrease in the ability of the RBCs to change
shape when passing through capillaries or the splenic filter may
lead to the plugging of the vessels.
Acquired:
Bite: female anopheline mosquito; blood, shared needles,
congenital infections
Epidemiology:
69 | P a g e
Tropics, mosquito to human, human to human transmission

Clinical Features:
Early Infection: Onset of a P. falciparum malaria attack occurs
from 8 to 12 days after infection and is preceded by 3 to 4 days of
vague symptoms such as aches, pains, headache, fatigue,
anorexia, or nausea. The onset is characterized by fever, a more
severe headache, and nausea and vomiting, with occasional
severe epigastric pain. There may be only a feeling of chilliness at
the onset of fever. Periodicity of the cycle will not be established
during the early stages, and the presumptive diagnosis may be
totally unrelated to a possible malaria infection. If the fever does
develop a synchronous cycle, it is usually a cycle of somewhat
less than 48 h. An untreated primary attack of P.
falciparum malaria usually ends within 2 to 3 weeks. True relapses
from the liver do not occur, and after a year, recrudescences are
rare.
Complications: Severe or fatal complications of P.
falciparum malaria can occur at any time and are related to the
plugging of vessels in the internal organs, the symptoms
depending on the organ(s) involved. Severe complications may
not correlate with the parasitemia seen in the peripheral blood.
Clinical Specimen:
Blood: Multiple draws (EDTA). Malaria is one of the few
parasitic infections considered to be immediately
life-threatening, and a patient with the diagnosis of P.
falciparum malaria should be considered a medical
emergency because the disease can be rapidly fatal. Any
laboratory providing the expertise to identify malarial
parasites should do so on a 24-h basis, 7 days/week.
Although malaria is no longer endemic within the United States,
this infection is life threatening, and laboratory requests for blood
smear examination and organism identification should be treated
as STAT requests. Frequently, for a number of different reasons,
organism recovery and identification may be more difficult than
the textbooks imply. It is very important that this fact be
recognized, particularly when one is dealing with a possibly fatal
infection with P. falciparum.
70 | P a g e
Both thick and thin blood films should be prepared on

admission of the patient (clinic, emergency room, in-house),
and at least 300 oil immersion fields should be examined on the
thick and thin film before a negative report is issued. Since one
set of negative films will not rule out malaria, additional blood
specimens should be examined over a 36 h time frame. Although
Giemsa stain is used for parasitic blood work; the organisms can
also be seen with other blood stains such as Wrights, WrightGiemsa, or any of the rapid blood stains. Blood collected with
EDTA anticoagulant is acceptable; however, if the blood remains
in the tube for any length of time, true stippling may not be
visible within the infected RBCs (P. vivax as an example),
organisms will change their morphology, and some of the
parasites will disintegrate (after 4-6 h). Also, it is important to
remember that the proper ratio between blood and anticoagulant
is necessary for good organism morphology.
Ring Forms: Although ring forms of P. falciparum have been
described as small, when the blood is collected in EDTA
(venipuncture), the rings continue to grow; thus their size may
mimic the other species.
Gametocyte: The gametocytes have been described as crescentshaped; however, in patients with an early infection, the
gametocytes have not yet appeared in the blood. This is a critical
feature of immunologically nave patients (travelers with no prior
exposure to malaria) who present to the ER with symptoms, but
very light parasitemias and no gametocytes in the blood.
Laboratory Report:
A number of reports can be relevant remember to add the
appropriate report comments.
Report 1: No Parasites Seen: The submission of a single blood
specimen will not rule out malaria; submit additional bloods every
4-6 hours for 3 days if malaria remains a consideration.
Report 2: Plasmodium spp. Seen: Unable to rule out Plasmodium
falciparumor Plasmodium knowlesi
Report 3: Plasmodium spp., possible mixed infection: Unable to
rule outPlasmodium falciparum or Plasmodium knowlesi
Report 4: Negative for parasites using automated hematology
71 | P a g e
instruments. Automated Hematology instruments will not detect

low malaria parasitemias seen in immunologically nave patients
(travelers)
Treatment:
Control:
Improved mosquito control, impregnated bed nets, potential
vaccines, prophylaxis
72 | P a g e
Plasmodium knowlesi (True Pathogen Monkey Malaria)

Organism:
pathogen, and causes malaria. All forms of the human life cycle
will appear in the thick and thin blood films.
Plasmodium knowlesi ring

P. knowlesi band
forms
forms
NOTE: Early infection mimics P. falciparum; late infection
mimics P. malariae.
As early as 1999, a fifth malaria has been implicated in human
disease. Plasmodium knowlesi, a malaria parasite of long-tailed
macaque monkeys has been confirmed in a number of human
cases from endemic areas such as Malaysian Borneo, Thailand,
Myanmar, and the Philippines. Cases have also been found in
travelers returning from these areas. Although it is well known
that under laboratory conditions some monkey malarias can be
transmitted to humans, it is now well established that P.
knowlesi is emerging as an important zoonotic human pathogen.
Life Cycle:
begin dividing, resulting in large numbers of liver merozoites. P.
knowlesi is closely related to P. vivax. However, important
differences in P. knowlesi include host blood cell preference
(normal size RBCs), absence of a dormant liver stage (the
73 | P a g e
hypnozoite), and length of the asexual cycle (24 h). In P.

vivax and P. ovale, a secondary or dormant schizogony occurs
from organisms that remain quiescent in the liver until a later
time; they are called hypnozoites. Delayed schizogony does not
occur in P. falciparum, P. malaria, or P. knowlesi.
P. knowlesi infects all ages of RBCs, and the parasitemia can equal
that seen in cases of P. falciparum. Splenomegaly occurs during
the first few weeks, and the spleen will progress from being soft
and palpable to hard during a chronic infection. If therapy is given
early, the spleen will return to normal size. Leukopenia is seen;
leukocytosis may be present during the febrile episodes. Total
plasma proteins are unchanged, although the albumin may be low
and the globulin fraction may be elevated due to antibody
development. Serum potassium may also be increased.
Acquired:
Epidemiology:
Forested areas of Southeast Asia (Malaysian Borneo, Thailand,
Myanmar, Philippines).
Clinical Features:
Early Infection: Patients may be asymptomatic, or may exhibit
chills, minor headaches and a daily low-grade fever, while other
patients may present with high fevers, mild abdominal problems,
leukopenia and thrombocytopenia. During the first few days, the
patient may not exhibit a typical paroxysm pattern but rather
have a steady low-grade fever or an irregular remittent fever
pattern. However, most patients with P. knowlesi malaria have
febrile illness and associated symptoms that are indistinguishable
from those caused by other malaria species. P. knowlesi requires
24 hours to complete its asexual erythrocytic cycle, which results
in a unique quotidian type of fever pattern different from that for
the other 4 human malaria species. However, this fever pattern
has no practical value for presumptive diagnosis because
characteristic fever variations would not be observed during the
early phase of infections, and mixed species infections would
further complicate pattern recognition related to febrile
symptoms.
74 | P a g e
Complications: Severe complications can be seen in P.

knowlesi infections, and coma and sudden death or other
symptoms of cerebral involvement have been reported. Autopsy
findings are very similar to those seen in fatal P.
falciparum malaria, including the cerebral pathology.
Clinical Specimen:
Prepare thick and thin blood films immediately after
receipt of the blood.
infection with P. falciparum or P. knowlesi. Both thick and thin
blood films should be prepared on admission of the
patient (clinic, emergency room, in-house), and at least 300 oil
immersion fields should be examined on the thick and thin film
before a negative report is issued. Since one set of negative films
will not rule out malaria, additional blood specimens should be
examined over a 36 h time frame. Although Giemsa stain is used
for parasitic blood work; the organisms can also be seen with
other blood stains such as Wrights, Wright-Giemsa, or any of the
rapid blood stains. Blood collected with EDTA anticoagulant is
acceptable; however, if the blood remains in the tube for any
length of time, true stippling may not be visible within the
infected RBCs (P. vivax as an example), organisms will change
their morphology, and some of the parasites will disintegrate
(after 4-6 h). Also, it is important to remember that the proper
75 | P a g e
ratio between blood and anticoagulant is necessary for good

organism morphology.
NOTE: P. knowlesi infection should be considered in patients with
a travel history to forested areas of Southeast Asia, especially if P.
malariae is diagnosed, unusual forms are seen with microscopy,
or if a mixed infection with P. falciparum/P. malariae is diagnosed.
Since the disease is potentially fatal, proper identification to the
species level is critical.
Ring Forms: Ring forms of P. knowlesi tend to mimic those of P.
falciparum(headphones, multiple rings/cell, and various sizes of
rings). As the rings grow, the infection begins to mimic
morphology seen with P. malariae (normal size to small RBCs,
band forms, mature schizont like daisy head).
Gametocyte: The gametocytes are round and tend to fill the
entire RBC. Exflagellation of the male gametocytes can occur in
the blood if the blood cools and becomes aerated (cap removed
lag time between cooling/cap removal and preparation of the
thick and thin blood films.
Laboratory Report:
(travelers)
Treatment:
76 | P a g e
Control:
77 | P a g e
Plasmodium malariae (True Pathogen Quartan Malaria)

Organism:
Plasmodium
P. malariae band P. malariae mature
malariae ring form form
schizont
NOTE:
Normal to small infected RBCs, thick ring, band form,
daisy head mature schizont
Life Cycle:
initiating the erythrocytic cycle. In P. vivax and P. ovale, a
secondary or dormant schizogony occurs from organisms that
remain quiescent in the liver until a later time; they are called
hypnozoites. Delayed schizogony does not occur in P.
falciparum, P. malariae, or P. knowlesi.
P. malariae primarily invades the older RBCs, so the number of
infected cells is somewhat limited. The cycle is typically 72 hours,
thus it begins again on the 4th day. Splenomegaly occurs during
the first few weeks, and the spleen can progress from being soft
and palpable to hard during a chronic infection. If therapy is given
early, the spleen will return to normal size. Leukopenia is seen;
leukocytosis may be present during the febrile episodes. Total
78 | P a g e
plasma proteins are unchanged, although the albumin may be low

and the globulin fraction may be elevated due to antibody
development. Serum potassium may also be increased.
Acquired:
Epidemiology:
Sporadic distribution, mosquito to human, human to human
transmission
Clinical Features:
Early Infection: The incubation period between infection and
symptoms may be much longer than that seen with P. vivax or P.
ovale malaria, ranging from about 27 to 40 days. Parasites can be
found in the bloodstream several days before the initial attack,
and the prodromal symptoms may resemble those ofP.
vivax malaria. A regular periodicity of 72 hours is seen from the
beginning, with a more severe paroxysm, including a longer cold
stage and more severe symptoms during the hot stage. Collapse
during the sweating phase is not uncommon.
Complications: Proteinuria is common in P. malariae infections
and in children may be associated with clinical signs of the
nephrotic syndrome. It has been suggested that kidney problems
may result from deposition within the glomeruli of circulating
antigen-antibody complexes in an antigen-excess situation seen
with a chronic infection. Apparently, the nephrotic syndrome
associated with P. malariae infections is unaffected by the
administration of steroids. A membranoproliferative type of
glomerulonephritis with relatively sparse proliferation of
endothelial and mesangial cells is the most common type of
lesion seen in quartan malaria. Using immunofluorescence,
granular deposits of IgM, IgG, and C3 are seen. Since chronic
glomerular disease associated with P. malariae infections is
usually not reversible with therapy, genetic and environmental
factors may play a role in the nephrotic syndrome. The infection
may end with spontaneous recovery, or there may be a
recrudescence or series of recrudescences over a long period of
years.These patients are left with a latent infection and
79 | P a g e
persisting low-grade parasitemia for many, many

years (can be >40 years).
Clinical Specimen:
80 | P a g e
Ring Forms: Ring forms of P. malariae tend to be thick, compact
(non-ameboid), and may mimic a signet ring with a large stone
(nucleus); however, the ring forms can also mimic those of other
species. True stippling (Schffners dots) is not seen in P.
malariae. It is important to remember than the infected RBCs tend
to be normal to small size, often smaller than the uninfected
RBCs. Band forms are also very typical in these infections.
Mature Schizont: The mature schizont has been described like a
daisy head with 8-10 merozoites arranged around the excess
pigment.
Gametocyte: The gametocytes are round to oval and tend to fill
the entire RBC (smaller than normal infected RBC). Exflagellation
of the male gametocytes can occur in the blood if the blood cools
and becomes aerated (cap removed lag time between
cooling/cap removal and preparation of the thick and thin blood
films.
Laboratory Report:
(travelers)
Treatment:
Control:
81 | P a g e
82 | P a g e
Plasmodium ovale (True Pathogen Ovale Malaria)

Organism:
Plasmodium ovale ring forms NOTE:

Enlarged RBCs, non-ameboid rings, stippling
Life Cycle:
falciparum, P. malariae, or P. knowlesi.
P. ovale infects only the reticulocytes and the parasitemia is
usually limited to 2 to 5% of the available RBCs. Splenomegaly
occurs during the first few weeks, and the spleen will progress
from being soft and palpable to hard during a chronic infection. If
therapy is given early, the spleen will return to normal size.
Leukopenia is seen; leukocytosis may be present during the
febrile episodes. Total plasma proteins are unchanged, although
the albumin may be low and the globulin fraction may be
83 | P a g e
elevated due to antibody development. Serum potassium may

also be increased.
Acquired:
Epidemiology:
Central West Africa and some South Pacific islands, mosquito to
human, human to human transmission
Clinical Features:
Early Infection: The primary clinical attack usually occurs from 7
to 10 days after infection, although there are strain differences,
with a much longer incubation period being possible. In some
patients, symptoms such as headache, photophobia, muscle
aches, anorexia, nausea, and sometimes vomiting may occur
before organisms can be detected in the bloodstream. In other
patients, the parasites can be found in the bloodstream several
days before symptoms appear. During the first few days, the
pattern. Once the typical paroxysms begin, after an irregular
periodicity, a regular 48-h cycle is established. An untreated
primary attack may last from 3 weeks to 2 months or longer. The
paroxysms become less severe and more irregular in frequency
and then stop altogether. In some cases, relapses may occur after
weeks, months, or up to 5 or more years. P. ovale malaria is
usually less severe than P. vivax, tends to relapse less frequently,
and usually ends with spontaneous recovery, often after no more
than 6 to 10 paroxysms. The incubation period is similar to P.
vivaxmalaria, but the frequency and severity of the symptoms are
much less, with a lower fever and a lack of typical rigors.
Complications: Complications are less common in P. ovale, and
the infection often ends with spontaneous recovery.
Clinical Specimen:
84 | P a g e

Ring Forms: Ring forms of P. ovale tend to be round, compact
(non-ameboid), and may mimic ring forms of the other species.
Stippling (Schffners dots) appears immediately and is not
delayed as seen in P. vivax. However, if the blood in EDTA stands
for >one hour, the stippling may not be seen, regardless of the pH
of the buffer. Often the RBCs are somewhat oval, tend to have
sharp, spiky edges (fimbriated edges), and the ring forms are
85 | P a g e
compact rather than spread out.

Gametocyte: The gametocytes are round to oval and tend to fill
the entire RBC. Exflagellation of the male gametocytes can occur
in the blood if the blood cools and becomes aerated (cap removed
Laboratory Report:
(travelers)
Treatment:
Control:
86 | P a g e
Plasmodium vivax (True Pathogen Tertian Malaria)

Organism:
Plasmodium vivax ring forms schizont Plasmodium

NOTE: Enlarged RBC's, ameboid rings, vivaxmature
stippling
Thick Blood Film
Life Cycle:
falciparum, P. malaria, or P. knowlesi.
P. vivax infects only the reticulocytes and the parasitemia is
usually limited to 2 to 4% of the available RBCs. Splenomegaly
occurs during the first few weeks, and the spleen will progress
from being soft and palpable to hard during a chronic infection. If
therapy is given early, the spleen will return to normal size.
Leukopenia is seen; leukocytosis may be present during the
febrile episodes. Total plasma proteins are unchanged, although
the albumin may be low and the globulin fraction may be
87 | P a g e
elevated due to antibody development. Serum potassium may

also be increased.
Acquired:
Epidemiology:
P. vivax may account for 80% of the infections; tropics, subtropics,
and temperate zones, mosquito to human, human to human
transmission
Clinical Features:
Early Infection: The primary clinical attack usually occurs from 7
to 10 days after infection, although there are strain differences,
with a much longer incubation period being possible. In some
patients, symptoms such as headache, photophobia, muscle
aches, anorexia, nausea, and sometimes vomiting may occur
before organisms can be detected in the bloodstream. In other
patients, the parasites can be found in the bloodstream several
days before symptoms appear. During the first few days, the
pattern. Once the typical paroxysms begin, after an irregular
periodicity, a regular 48-h cycle is established. An untreated
primary attack may last from 3 weeks to 2 months or longer. The
paroxysms become less severe and more irregular in frequency
and then stop altogether. In 50% of the cases, relapses may occur
after weeks, months, or up to 5 or more years.
Complications: Severe complications are less common in P.
vivax infections, although coma and sudden death or other
symptoms of cerebral involvement have been reported. Severe
sequelae can be seen in cases of primaquine-tolerant or
primaquine-resistant cases.
Clinical Specimen:
88 | P a g e

Ring Forms: Ring forms of P. vivax tend to be delicate, spread out
(ameboid), and may mimic ring forms of the other species.
Stippling (Schffners dots) do not appear immediately as they do
in P. ovale, but appear a few hours later. However, if the blood in
EDTA stands for >one hour, the stippling may not be seen,
regardless of the pH of the buffer.
Mature Schizont : Often quite large containing 18+ merozoites.
89 | P a g e
Gametocyte: The gametocytes are round and tend to fill the

entire RBC. Exflagellation of the male gametocytes can occur in
the blood if the blood cools and becomes aerated (cap removed
Laboratory Report:
(travelers)
Treatment:
Control:
90 | P a g e
Malaria characteristics with fresh blood or blood collected

using EDTA with no extended lag time (preparation of
thick and thin blood films within < 60 min of collection)
Plasmodium vivax (benign tertian malaria)
1.
48-hour cycle
2.
Tends to infect young cells
3.
Enlarged RBCs
4.
Schffner's dots (true stippling) after 8-10 hours
5.
Delicate ring
6.
Very ameboid trophozoite
7.
Mature schizont contains 12-24 merozoites
Plasmodium malariae (quartan malaria)
1.
72-hour cycle (long incubation period)
2.
Tends to infect old cells
3.
Normal size RBCs
4.
No stippling
5.
Thick ring, large nucleus
6.
Trophozoite tends to form "bands" across the cell
7.
Mature schizont contains 6-12 merozoites
Plasmodium ovale
1.
48-hour cycle
2.
Tends to infect young cells
3.
Enlarged RBCs with fimbriated edges (oval)
4.
Schffner's dots appear in the beginning (in RBCs with very
young ring forms in contrast to P. vivax)
5.
Smaller ring than P. vivax
6.
Trophozoite less ameboid than that of P. vivax
7.
Mature schizont contains average 8 merozoites
Plasmodium falciparum (malignant tertian malaria)
1.
36-48-hour cycle
2.
Tends to infect any cell regardless of age, thus very heavy
infection may result
3.
All sizes of RBCs
91 | P a g e
4.
No Schffner's dots (Maurer's dots: may be larger, single

dots, bluish)
5.
Multiple rings/cell (only young rings, gametocytes, and
occasional mature schizonts are seen in peripheral blood)
6.
Delicate rings, may have two dots of chromatin/ring,
appliqu or accol forms
7.
Crescent-shaped gametocytes
Plasmodium knowlesi (simian malaria)*
1.
24-hour cycle
2.
Tends to infect any cell regardless of age, thus very heavy
infection may result
3.
All sizes of RBCs, but most tend to be normal size
4.
No Schffners dots (faint, clumpy dots later in cycle)
5.
Multiple rings/cell (may have 2-3)
6.
Delicate rings, may have two or three dots of
chromatin/ring, appliqu forms
7.
Band form trophozoites commonly seen
8.
Mature schizont contains 16 merozoites, no rosettes
9.
Gametocytes round, tend to fill the cell
*Early stages mimic P. falciparum; later stages mimic P. malariae
92 | P a g e
Toxoplasma gondii (Toxoplasmosis) (Pathogen)

Organism:
This organism is closely related to other coccidia and has
similarities to malarial parasites, is a true pathogen, and causes
toxoplasmosis. Both the tachyzoites (crescent shaped, 2-3 m
wide by 4-8 m long) and cyst forms containing the bradyzoites
(may contain few to hundreds of organisms, may reach 100 m
long and 7-10 m wide) can be found in clinical specimens.
Tachyzoites
Bradyzoites
Tachyzoites in
Bone Marrow
Life Cycle:
Oocysts in cat feces transmitted to mammals/birds; trophozoites,
cysts in meat; sexual forms again develop in GI cells of the cat.
Acquired:
Fecal-oral transmission via oocyst form from members of
the Felidae family (house cat, most common); poorly cooked
meats and water contaminated with oocysts. Tissue
transplantation, blood transfusion, and congenital infections are
known, as well.
Epidemiology: Worldwide
Human infection can be acquired through ingestion or handling of
infected meat or through ingestion of infective oocysts, which can
remain viable within cool, moist soil for a year or longer. Certainly,
hand washing is highly recommended when there has been
potential exposure to the oocysts. It is recommended that meat
93 | P a g e
be cooked so that the internal temperature reaches 150F (66C).

The question of the house cat always arises; indoor cats fed on
dry, canned, or boiled food are unlikely to be infected. However,
cats that go outside and have access to other infected animals
(birds or mice) may shed oocysts in their feces; therefore, other
preventive measures, including changing the litter box daily and
disinfecting the pan with boiling water, have been recommended.
Also, the feces should not be placed onto the soil but, rather,
should be disposed of either in the toilet or within bags.
Clinical Features:
Large numbers of people who are serologically active suggest the
majority of infections are benign, with few or no symptoms (e.g.,
cold or light case of the flu). The most severe symptoms are seen
with congenital, transplacental infections or infections in the
compromised patient. Congenital infections (acquired in first or
second trimester) include: retinochoroiditis, cerebral calcification,
and possibly hydrocephalus or microcephaly. CNS involvement
may not appear until several years later. Infections acquired
postnatally can be categorized as: (1) lymphadenitis, fever,
headache, myalgia, (2) typhus-like exanthemous form with
myocarditis, meningoencephalitis, atypical pneumonia, and
possibly death, (3), CNS involvement, usually fatal, and (4)
retinochoroiditis, which may be severe. Most common symptoms
in adults are local or generalized lymphadenopathy (nodes of the
neck). In immunocompromised patients, CNS symptoms are
common.
Chorioretinitis in immunocompetent patients is generally due to
an earlier congenital infection. Patients may be asymptomatic
until the second or third decade; at that point, cysts may rupture
with lesions then developing in the eye. The number of people
who develop chorioretinitis later in life is unknown but may
represent over two-thirds. Also, up to 30% of patients relapse
after treatment. Chorioretinitis is usually bilateral in patients with
congenitally acquired infection, and is generally unilateral in
patients with recently acquired infection.
Clinical Specimen:
Various serologic procedures are recommended (very complex
94 | P a g e
diagnostic algorithms serology results may be difficult to

interpret; biopsy specimens, buffy coat cells, CSF, BAL
Serologies; can also use PCR, biopsy specimens, buffy coat cells,
CSF fluid, or isolation in tissue culture
Tachyzoite: Crescent shaped may resemble smaller gametocyte
ofPlasmodium falciparum.
Cyst: Cysts containing bradyzoites may be somewhat elongate.
Laboratory Report: Toxoplasma gondii serology results are
reported
Treatment:
Control:
Improved cooking of meats, adequate disposal of cat fecal waste,
adequate washing of contaminated litter boxes, hand washing
when potentially exposed to oocysts
95 | P a g e
Blastocystis hominis (Pathogen)

Organism:
This organism belongs to the amebae (may be reclassified into its
own group), is a potential pathogen in some patients, and can
cause disease. The central body form is most commonly seen
(usual size, 6-40 m) and can be found in clinical
specimens. What is called Blastocystis hominis is a
group of strains or species, about half of which are
pathogenic and half are nonpathogenic. Pathogenicity
differences cannot be determined on the basis of
morphology.
Permanent stained slide: Central Body form

Body form
Iodine: Central
Life Cycle:
Acquired:
Fecal-oral transmission via central body form; contaminated food
and water
Epidemiology:
Clinical Features:
Infection with B. hominis may be the cause of diarrhea, cramps,
nausea, fever, vomiting, and abdominal pain and may require
therapy. The incidence of this organism appears to be higher
than suspected in stools submitted for parasite examination; it is
considered the most common protozoan worldwide (review of
96 | P a g e
published literature). In symptomatic patients in whom no other

etiologic agent has been identified, B. hominis should certainly be
considered the possible pathogen. When a symptomatic B.
hominis infection responds to therapy, the improvement may also
represent elimination of some other undetected pathogenic
organism (E. histolytica, G. lamblia, D. fragilis).
Clinical Specimen:
Intestinal: Stool
permanent stained smear); identification based on morphology;
fecal immunoassay currently under development.
Central body form: Central clear area (can be clear, or stain
red/green/blue with trichrome stain); small nuclei are around the
outside of the clear area.
Laboratory Report:
Blastocystis hominis (indicate quantity: rare, few, mod, many);
quantity may be linked to presence or absence of symptoms;
quantitation should be performed on the permanent stained slide.
Report Comment:
The name Blastocystis hominis contains approximately 10
different organisms, none of which can be differentiated on the
basis of organism morphology; some are pathogenic and some
are non-pathogenic. If no other pathogens are found, B.
hominis may be the cause of patient symptoms
AND
Other organisms capable of causing diarrhea should also be ruled
out.
Treatment:
Garcia, L.S. 2007. Diagnostic Medical Parasitology, 5th ed., ASM
Press, Washington, D.C.
97 | P a g e
Control:
98 | P a g e
If amebic trophozoites measuring 13 m and containing red blood cells

within the cytoplasm are seen in a permanent stained smear, the correct
report would indicate the presence of:
A. Entamoeba coli trophozoites

B. Entamoeba hartmanni trophozoites
C. Entamoeba histolytica trophozoites
D. Entamoeba polecki trophozoites
ANSWER: The amebic trophozoites described above would be
identified as Entamoeba histolytica (the presence of ingested RBCs
allows this identification to be made). If no RBCs are seen in the
cytoplasm of the trophozoites, the correct report would be:
Entamoeba histolytica/E. dispar trophozoites seen. The correct
answer is c.
Intestinal protozoa that do not have a cyst stage in the life cycle include
the following:
A. Dientamoeba fragilis and Entamoeba hartmanni

B. Entamoeba hartmanni and Iodamoeba btschlii
C. Iodamoeba btschlii and Pentatrichomonas hominis
D. Pentatrichomonas hominis and Dientamoeba fragilis
99 | P a g e
ANSWER: Of the organisms listed above, those that do not have a

cyst stage in the life cycle include: Dientamoeba fragilis and
Pentatrichomonas hominis. The correct answer is d.
An amebic cyst measuring 9 m and containing four nuclei and
chromatoidal bars with smooth, rounded ends is most likely:
A. Endolimax nana
B. Entamoeba hartmanni
C. Iodamoeba btschlii
D. Entamoeba histolytica
ANSWER: The amebic cyst described above would be identified as
Entamoeba hartmanni (size and chromatoidal bars with smooth,
rounded ends). The correct answer is b.
If a patient has watery diarrhea, the stage in the life cycle of the intestinal
protozoa that is most likely to be seen in the permanent stained smear is
the:
A. Cyst
B. Precyst
C. Trophozoite
D. Pretrophozoite
ANSWER: When a patient has diarrhea, the GI tract contents are
moving through the system rapidly, thus there is no time for cyst
formation. It is very likely the protozoan stage most likely to be seen
will be the trophozoite. The term "pretrophozoite" is not a correct
term. The correct answer is c.
If amebic trophozoites measuring 14 m, containing debris and a single
nucleus with evenly arranged chromatin and a small, compact karyosome
are seen in a permanent stained smear, they should be reported as:
100 | P a g e
A. Entamoeba histolytica/E. dispar trophozoites

B. Entamoeba hartmanni trophozoites
C. Entamoeba dispar trophozoites
D. Entamoeba histolytica trophozoites
ANSWER: These trophozoites have morphologic characteristics that
are consistent with Entamoeba histolytica/E. dispar. Without the
specific presence of ingested RBCs in the cytoplasm or the use of
specific immunoassay reagents, it will be impossible to identify
these organisms as the pathogen, E. histolytica or the nonpathogen,
E. dispar. The morphology of both trophozoites are basically
identical (without the presence of ingested RBCs). The correct
answer is a.
The presence of Charcot-Leyden (C-L) crystals in the permanent stained

smear indicates:
A. Blood in the stool

B. Excess yeast cells
C. An immune response
D. Nothing
101 | P a g e
ANSWER: Charcot-Leyden crystals are formed from the breakdown

products of eosinophils, thus the correct answer is (c). These
crystals are an indication of an immune response, which may or may
not be linked to a parasitic infection.
A small lemon-shaped cyst is present in the permanent stained smear,
and it has a single nucleus and a curved fibril that has been called "the
shepherd's crook" - this organism is most likely:
A. Pentatrichomonas hominis
B. Enteromonas hominis
C. Retortamonas intestinalis
D. Chilomastix mesnili
ANSWER: Chilomastix mesnili is a non-pathogenic flagellate that
can be found in the feces. The description above accurately
describes the cyst form of this organism (lemon-shaped, single
nucleus, curved fibril called the shepherd's crook.. The correct
answer is d.
The presence of nonpathogenic protozoa in the intestinal tract indicates:
A. The patient has ingested something contaminated with fecal

material containing infective cysts
B. The patient is likely to become symptomatic within a few days
to two weeks
C. The patient will remain asymptomatic for at least a month
D. The patient also has pathogenic protozoa in the intestinal tract
if additional stools are examined within 10 days
ANSWER: The presence of nonpathogenic protozoa in the intestinal
tract indicates the patient has ingested something contaminated
with fecal material containing infective cysts. It does not necessarily
102 | P a g e
mean pathogens are also present, nor does it mean the patient will
become symptomatic. The correct answer is a.
Intestinal protozoa that are considered nonpathogenic include:
A. Blastocystis hominis and Entamoeba histolytica

B. Endolimax nana and Entamoeba coli
C. Giardia lamblia and Iodamoeba btschlii
D. Entamoeba hartmanni and Dientamoeba fragilis
ANSWER: Nonpathogenic intestinal protozoa include Endolimax
nana, Entamoeba coli, Iodamoeba btschlii, and Entamoeba
hartmanni. Pathogenic protozoa include: Blastocystis hominis,
Entamoeba histolytica, Giardia lamblia, and Dientamoeba fragilis.
Therefore, the correct answer is b.
A protozoan cyst that contains four nuclei, median bodies and axonemes
should be identified as:
A. Giardia lamblia
B. Trichomonas vaginalis
C. Dientamoeba fragilis
D. Pentatrichomonas hominis
ANSWER: A protozoan cyst that contains four nuclei, median
bodies, and axonemes can be identified as Giardia lamblia. None of
the other intestinal flagellates mentioned have a cyst in the life
cycle. The correct answer is a.
103 | P a g e
Characteristics of Dientamoeba fragilis include all of the following except:
A. This organism is a flagellate with internal flagella

B. Transmission is thought to occur through the ingestion of
specific helminth eggs
C. This organism is pathogenic and tends to be more commonly
found in children
D. Trophozoites contain a single nucleus, while the cysts contain
two nuclei
ANSWER: All of the responses (except d) contain correct
information regarding the characteristics of Dientamoeba fragilis.
Therefore, the correct answer is d, this organism does not have a
cyst form in the life cycle.
Cryptosporidium parvum, Cyclospora cayetanensis, and Isospora belli are

considered to be:
A. Metazoa
B. Microsporidia
C. Sporozoa
D. Coccidia
ANSWER: The three organisms mentioned above are protozoa
within the coccidia group. Therefore, the correct answer is d.
Which of the following organisms are most likely to be seen in a direct
wet mount from fresh stool?
104 | P a g e
A. Entamoeba dispar cysts

B. Blastocystis hominis trophozoites
C. Entamoeba coli cysts
D. Giardia lamblia trophozoites
ANSWER: Protozoan cysts are not motile, therefore of the answers
above, both (a) and (c) are eliminated. Blastocystis hominis does not
move by pseudopods, flagella or cilia, so that answer is eliminated.
Therefore, the correct answer is d; flagellate trophozoites move
using their flagella for motility.
Which of the following organisms are most likely to be recovered from
duodenal aspirate material?
A. Isospora belli and hookworm

B. Giardia lamblia and Strongyloides stercoralis
C. Cryptosporidium parvum and Enterobius vermicularis
D. Microsporidia and Ascaris lumbricoides
ANSWER: Of all the choices listed above, those organisms that
might be recovered from the examination of duodenal aspirate
would include: Giardia lamblia, Strongyloides stercoralis,
Cryptosporidium parvum, and the microsporidia. Therefore, the
correct answer is b.
It is generally accepted that the most commonly recovered protozoan
within the United States is:
A. Entamoeba dispar
B. Entamoeba coli
C. Giardia lamblia
105 | P a g e
D. Balantidium coli
ANSWER: Of all the protozoa recovered from human fecal
specimens, the most common is probably Blastocystis hominis;
however, not all laboratories maintain statistics on this organism.
So, the most common and most accurate response of those listed
above would be Giardia lamblia. Therefore, the correct answer is c.
Microsporidial spores are generally described as looking like:
A. Coccidian oocysts
B. Amebic cysts
C. Fungal spores
D. Bacteria
ANSWER: Microsporidial spores in human intestinal infections
generally measure approximately 1 to 2.5 microns, are oval, and
tend to stain pink with the modified trichrome stains. This
appearance tends to mimic bacteria or very small yeast. Therefore,
the correct answer is d.
In the human, microsporidian spores generally measure approximately:
A. 1 to 4 m
B. 4 to6 m
C. 8 to10 m
D. None of the above
ANSWER: Microsporidial spores in human infections (all body sites)
generally measure approximately 1 to 4.0 microns. Therefore, the
correct answer is a.
The horizontal "stripe" or "bar" seen in microsporidial spores is actually
the:
106 | P a g e
A. Feeding groove fibril

B. Polar tubule
C. Vacuole outline
D. Axostyle support rod
ANSWER: The polar tubule is coiled within the microsporidial spore;
evidence of this internal polar tubule is often seen in stained spores
as a horizontal or diagonal "stripe" or "bar" that provides evidence
that the object is a true microsporidial spore and not artifact.
Occasionally, structures can be seen within the Cryptosporidium parvum
oocysts that are stained with the modified acid-fast stain; they are:
A. Sporozoites
B. Gametocytes
C. Blastocysts
D. Sporoblasts
ANSWER: Cryptosporidium oocysts measure approximately 4 to 6
microns, are immediately infective when passed in the stool and
contain four sporozoites. Although these sporozoites are not visible
within every oocyst, the oocyst is still infective. Therefore, the
correct answer is a.
The following organisms are immediately infectious when passed in the
stool (regardless of the stool consistency):
A. Entamoeba histolytica cysts and Isospora belli oocysts

B. Giardia lamblia trophozoites and Cyclospora cayetanensis
oocysts
C. Microsporidian spores and Endolimax nana trophozoites
107 | P a g e
D. Cryptosporidium parvum oocysts and Entamoeba coli cysts

ANSWER: Although protozoan cysts are generally infectious when
passed, trophozoites are not. Neither Isospora or Cyclospora oocysts
are infective when passed. Therefore, the correct answer is d.
Which of the following infections are more likely to cause major
symptoms in the immunocompromised patient?
A. Entamoeba dispar
B. Enterocytozoon bieneusi
C. Pentatrichomonas hominis
D. Giardia lamblia
ANSWER: It has been well documented that microsporidial
infections (Enterocytozoon bieneusi) in the compromised host can
cause major symptoms/disease. Both Entamoeba dispar and
Pentatrichomonas hominis are nonpathogenic, and, although Giardia
lamblia is pathogenic, the infection in the compromised host may
not be that different from that seen in the immunocompetent host.
Which of the following should be quantitated (rare, few, moderate, many,
packed) on the laboratory report form?
A. White blood cells (WBCs) and Blastocystis hominis

B. Red blood cells (RBCs) and Entamoeba histolytica
C. Budding yeast cells and Giardia lamblia
D. Charcot-Leyden crystals and microsporidian spores
ANSWER: Generally, it is not recommended that intestinal protozoa
be quantitated on the report form; however, there is one exception
and that is Blastocystis hominis. There may be a relationship
between numbers and symptoms, so the recommendation is to
108 | P a g e
report and quantitate this particular organism. It is also

recommended that WBCs, RBCs, and budding yeast cells should be
quantitated when reported. Therefore, the correct answer is a.
If Entamoeba spp. trophozoites are seen in a permanent stained smear
(no ingested RBCs, 13 m, nucleus with evenly arranged chromatin and
small, compact karyosome), they should be reported as:
A. Entamoeba histolytica trophozoites

B. Entamoeba coli trophozoites
C. Entamoeba dispar trophozoites
D. Entamoeba histolytica/E. dispar trophozoites
ANSWER: If Entamoeba spp. trophozoites with the above
characteristics are seen in the permanent stained smear, the correct
identification should be Entamoeba histolytica/E. dispar
trophozoites. Unless RBCs were seen in the cytoplasm of the
trophozoite, one could not identify the organism as a true pathogen,
Entamoeba histolytica. Entamoeba coli would tend to have uneven
nuclear chromatin with a large, eccentric karyosome. The correct
answer is d.
Balantidium coli trophozoites and cysts are best seen in the:
A. Permanent stained smear

B. Direct wet preparation
C. Concentration wet preparation
D. Agar plate culture
ANSWER: Because Balantidium coli trophozoites and cysts are so
large, they tend to overstain on the permanent stained smear and
can be confused with helminth eggs and/or artifacts. The best
approach to identification is the examination of the concentration
109 | P a g e
wet preparation, where the morphology (including cilia on the

trophozoite) can be easily seen. Therefore, the correct answer is c.
All but which one of the following organisms tends to cause a severe
watery diarrhea, particularly in the compromised patient?
A. Cryptosporidium parvum
B. Enterocytozoon bieneusi
C. Balantidium coli
D. Trichomonas vaginalis
ANSWER: Trichomonas vaginalis is a pathogenic flagellate that can
be found in the urinary-genital tract; it is not found in the stool and
does not cause diarrhea. The correct answer is d.
In a patient with diarrhea, occasionally Entamoeba histolytica/E. dispar
four nucleated cysts (no chromatoidal bars) are identified as being present;
however, these cells that have been misdiagnosed as protozoa are really
A. Macrophages
B. Polymorphonuclear leukocytes
C. Epithelial cells
D. Eosinophils
ANSWER: When a patient has diarrhea, the intestinal contents
move through the system very quickly; consequently, there is no
time for protozoan cyst formation. When polymorphonuclear
leukocytes (PMNs) are in the stool for some time, the lobed nuclei
come apart and can resemble protozoan cysts with four separate
"nuclei" - however, these "protozoan cysts" are really human cells.
Charcot-Leyden crystals in human clinical material are frequently
associated with an immune response and are thought to be the breakdown
products of
110 | P a g e
A. Neutrophils
B. Eosinophils
C. Monocytes
D. Lymphocytes
ANSWER: Charcot-Leyden (CL) crystals are formed from the
breakdown products of eosinophils. Therefore, the correct answer is
b.
Parasitic organisms that may be sexually transmitted include
A. Pentatrichomonas hominis
B. Dientamoeba fragilis
C. Trichomonas vaginalis
D. Enteromonas hominis
ANSWER: Trichomonas vaginalis is known to cause a sexually
transmitted disease, trichomoniasis. The other organisms are found
in the intestinal tract. Therefore, the correct answer is c.
The specimen that is LEAST LIKELY to provide recovery of Trichomonas
vaginalis is
A. Urine
B. Urethral discharge
C. Prostatic discharge
D. Feces
ANSWER: Trichomonas vaginalis is found in the urinary-genital tract
and does not inhabit the intestinal tract. Therefore, the specimen
111 | P a g e
that is least likely to provide recovery of this organism is the stool

specimen. The correct answer is d.
The recommended technique for the recovery of Dientamoeba fragilis
from stool is the
A. Formalin concentrate
B. Trichrome stained smear
C. Modified acid-fast stained smear
D. Giemsa stain
ANSWER: Dientamoeba fragilis is a pathogenic flagellate that has
no known cyst form in the life cycle. Therefore, the most important
method for the identification of this organism is the permanent
stained smear (trichrome, iron-hematoxylin, etc.); without using this
approach, the majority of infections using wet mount examination
only will be missed. Therefore, the correct answer is b.
112 | P a g e

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Entamoeba histolytica (True Pathogen - Amebiasis)

Entamoeba histolytica/E. dispar

cysts and/or trophozoites); this report indicates that the organism

Entamoeba hartmanni (Nonpathogen)

Trophozoite <12 microns

Cyst <10 microns

on size differences (differentiate from the Entamoeba

Entamoeba coli (Nonpathogen)

The single celled trophozoite is differentiated into outer clear

appears as a thick layer comprising about one half of the volume

Endolimax nana (Nonpathogen)

Endolimax nana trophozoites

Endolimax nana cysts

Iodamoeba btschlii (Nonpathogen)

Naegleria fowleri (True Pathogen Primary Amebic

Naegleria fowleri trophozoites Trophozoite

Naegleria trophozoites in tissue (cysts are

may take as long as 20 h. The flagellate form has two flagella at

headache, lethargy, and occasionally olfactory problems. The

procedures. Organisms can also be cultured on nonnutrient agar

phosphate-buffered saline can be used. Specimens transported in

Acanthamoeba spp. (True Pathogen Granulomatous

Australia. These data provide strong evidence that the most

organisms, by inflammatory cytokines such as interleukin-1 or

these individuals without CNS involvement is around 75%, it

of these tissue findings must include culture and/or special

phosphate-buffered saline can be used. Specimens transported in

failures tend to be fairly common. In vitro susceptibility testing

Giardia lamblia (duodenalis, intestinalis) (Pathogen)

reporting a negative antigen (due to shedding patterns).

Dientamoeba fragilis (Pathogen)

Trophozoites with single nucleus

Trophozoites with two fragmented nuclei

Chilomastix mesnili (Nonpathogen)

Trichomonas vaginalis (Pathogen)

Trichomonas vaginalis trophozoites

incidence of trichomoniasis worldwide is estimated to be more

symptoms during the following six months.

postoperative infections, and potential problems with pregnancy.

Balantidium coli (Pathogen)

Cyst: Round, will contain the same 2 nuclei as seen in the

Sarcocystis spp. (Pathogen)

Other Genera and Species (Pathogen, Microsporidia)

Sappinia spp. (True Pathogen Granulomatous Amebic

Sappinia trophozoite andcyst

Trophozoites in tissue (note the appearance of 2 nuclei)

space-occupying lesions of the CNS (tumor, abscess, fungal

Cystoisospora (Isospora) belli (Pathogen)

MAF = Modified Acid-Fast Stain (1% Acid Rinse)

Cryptosporidium spp. (Pathogen)

oocysts stain using modified acid-fast stains. In order to maintain

Plasmodium falciparum ring

Tropics, mosquito to human, human to human transmission

Both thick and thin blood films should be prepared on

instruments. Automated Hematology instruments will not detect

Plasmodium knowlesi (True Pathogen Monkey Malaria)

Plasmodium knowlesi ring

hypnozoite), and length of the asexual cycle (24 h). In P.

Complications: Severe complications can be seen in P.

ratio between blood and anticoagulant is necessary for good

Plasmodium malariae (True Pathogen Quartan Malaria)

plasma proteins are unchanged, although the albumin may be low

persisting low-grade parasitemia for many, many

Plasmodium ovale (True Pathogen Ovale Malaria)