Protozoa

JOSEFA G. HILADO MD
LEVEL II RESIDENT

BLOOD AND TISSUE
PROTOZOA
Malaria
Babesiosis
Hemoflagellates
Trypanosoma
Leishmania
Toxoplasma gondii
Opportunistic Free-Living Amebae
INTESTINAL AND UROGENITAL
PROTOZOA
Amebae and Blastocystis hominis
Entamoeba histolytica
Nonpathogenic Amebae
Blastocystis hominis
Flagellates
Dientamoeba fragilis
Giardia lamblia
Chilomatix mesnili
Pentatrichomonas hominis
Trichomonas vaginalis
Other Flagellates
Ciliates
Balantidium coli
Coccidia
Cystoisospora belli
Sarcocystis spp.,
Cryptosporidium spp.,
Cyclospora cayetanensis
Microsporidia

1. MALARIA
Plasmodium vivax
Plasmodium ovale
Plasmodium malariae
Plasmodium falciparum
-From Italian mal’ aria
Female Anopheles mosquito
4

1. MALARIA
- >70% is caused by P.
falciparum
- <30% is caused by P.
vivax
- <1% by P. malariae
- 1 reported case of P.
ovale in Palawan (1960s)
5

MEET THE OTHER BANDITS:
- Anopheles minimus var. flavirostris
- principal malaria vector, abound in the foothill
areas
- Anopheles litoralis
- transmission in coastal areas of Mindanao
- Anopheles maculates and Anopheles flavirostris
- transmission in higher altitudes
- Anopheles mangyans
- abound in forest fringes
- Tranfusion malaria
- Congenital malaria
6

1. MALARIA
 The defining clinical features of a malarial
attack or paroxysm consist of (in order):
 shaking chills (COLD STAGE)
 fever (up to 40°C or higher) (HOT STAGE OR
FLUSH PHASE)
 generalized diaphoresis (SWEATING STAGE)
 resolution of fever.
7

1. MALARIA
 The paroxysm occurs over 6-10 hours and is
initiated by the synchronous rupture of
erythrocytes with the release of new
infectious blood stage forms known as
merozoites.
8

1. MALARIA
 Life Cycle
 Malarial parasites undergo
a :
 sexual phase (sporogony) in
Anopheles mosquitoes that
results in the production of
infectious sporozoites.
 asexual stage (schizogony) in
humans that results in the
production of schizonts and
merozoites.
9

ASEXUAL
SCHIZOGONY
SEXUAL
SPOROGONY
10

ASEXUAL
SCHIZOGONY
SEXUAL
SPOROGONY
11

ASEXUAL
SCHIZOGONY
SEXUAL
SPOROGONY
12

ASEXUAL
SCHIZOGONY
SEXUAL
SPOROGONY
8 to 21 DAYS
13

ASEXUAL
SCHIZOGONY
SEXUAL
SPOROGONY
14

ASEXUAL
SCHIZOGONY
SEXUAL
SPOROGONY
CLINICAL
SYMPTOMS
15

1. MALARIA
 NOTE:
 P. vivax and P. ovale differ from P.
falciparum and P. malariae in that
true disease relapses of the
former species may occur weeks
to months following subsidence
of previous attacks.
 Due to renewed exoerythrocytic
and, eventually, erythrocytic
schizogony from latent hepatic
sporozoites, which are known as
hypnozoites
16

1. MALARIA
 NOTE:
 Recurrences of disease due to P.
falciparum or P. malariae, called
recrudescences, arise from an
increase in numbers of persisting
blood stage forms to clinically
detectable levels, not from
persisting liver stage forms.
17

1. MALARIA
 Liver cells are infected only by
sporozoites from the mosquito;
thus, transfusion-acquired P.
vivax or P. ovale infection does not
relapse.
18

1. MALARIA
 P. vivax and P. ovale parasites primarily infect
young erythrocytes
 P. malariae infects older erythrocytes
 P. falciparum infects erythrocytes of all ages.
19

THE MORPHOLOGY
 Morphologic stages seen in erythrocytes
include
 trophozoites (growing forms)
 schizonts (dividing forms)
 gametocytes (sexual forms) .
20

 Figure 61-2 Plasmodium vivax.
 1, Normal-size erythrocyte with marginal ring form
trophozoite.
 2, Young signet ring form of trophozoite in macrocyte.
 3, Slightly older ring form trophozoite in erythrocyte
showing basophilic stippling.
 4, Polychromatophilic erythrocyte containing young tertian
parasite with pseudopodia.
 5, Ring form of trophozoite showing pigment in cytoplasm
of an enlarged cell containing Schüffner's stippling. This
stippling does not appear in all cells containing the growing
and older forms of Plasmodium vivax, but it can be found
with any stage from the fairly young ring form onward.
 6 and 7, Very tenuous medium trophozoite forms.
 8, Three ameboid trophozoites with fused cytoplasm.
 9-13, Older ameboid trophozoites in process of
development.
 10, Two ameboid trophozoites in one cell.
 14, Mature trophozoite.
 15, Mature trophozoite with chromatin apparently in
process of division.
 16–19, Schizonts showing progressive steps in division
(presegmenting schizonts).
 20, Mature schizont.
 21 and 22, Developing gametocytes.
 23, Mature microgametocyte.
 24, Mature macrogametocyte.
21

 Figure 61-3 Plasmodium malariae.
 1, Young ring form trophozoite of
quartan malaria.
 2–4, Young trophozoite forms of the
parasite showing gradual increase of
chromatin and cytoplasm.
 5, Developing ring form of trophozoite
showing pigment granule.
 6, Early band form of trophozoite –
elongated chromatin, some pigment
apparent.
 7–12, Some forms that the developing
trophozoite of quartan malaria may
take.
 13 and 14, Mature trophozoites – one a
band form.
 15–19, Phases in the development of
the schizont (presegmenting
schizonts).
 20, Mature schizont.
 21, Immature microgametocyte.
 22, Immature macrogametocyte.
 23, Mature microgametocyte.
 24, Mature macrogametocyte. 22

Figure 61-4 Plasmodium falciparum.
 1, Very young ring-form trophozoite.
 2, Double infection of single cell with young trophozoites, one
a ‘marginal form,’ the other ‘signet ring’ form.
 3 and 4, Young trophozoites showing double chromatin dots.
 5–7, Developing trophozoite forms.
 8, Three medium trophozoites in one cell.
 9, Trophozoite showing pigment, in a cell containing Maurer's
dots.
 10 and 11, Two trophozoites in each of two cells, showing
variation of forms that parasites may assume.
 12, Almost mature trophozoite showing haze of pigment
throughout cytoplasm. Maurer's dots in the cell.
 13, Estivo-autumnal ‘slender forms.’
 14, Mature trophozoite, showing clumped pigment.
 15, Parasite in the process of initial chromatin division.
 16–19, Various phases of the development of the schizont
(presegmenting schizonts).
 20, Mature schizont.
 21–24, Successive forms in the development of the
gametocyte – usually not found in the peripheral circulation.
 25, Immature macrogametocyte.
 26, Mature macrogametocyte.
 27, Immature microgametocyte.
 28, Mature microgametocyte. (Courtesy of National Institute
of Health, USPHS.)
23

 Figure 61-5 Plasmodium
ovale.
 1, Young ring-shaped
trophozoite.
 2–5, Older ring-shaped
trophozoites.
 6–8, Older ameboid
trophozoites.
 9, 11, and 12, Doubly infected
cells, trophozoites.
 10, Doubly infected cell, young
gametocytes.
 13, First stage of the schizont.
 14–19, Schizonts, progressive
stages.
 20, Mature gametocyte.
24

CLINICAL DIFFERENTIATION
P. falciparum P. vivax P. ovale P. malariae
Other names Malignant tertian Benign tertian Benign tertian Quartan
Prepatent period
(Sporozoite injection
to detection in blood
11-14 days 11-15 days 14-26 days 3-4 weeks
Incubation period
(Sporozoite injection
to symptomatology)
8-15 days 12-20 days 11-16 days 18-40 days
Erythrocytic cycle (hr) 48 48 48 72
Age of infected RBCs All stages Young RBCs Young RBCs Aging RBCs
Persistent EE stages No Yes Yes No
Parasitemia (mm3
)
Average
Maximum
50 000-500 000
Up to 2 500 000
20 000
50 000
9000
30 000
6000
20 000
Duration of untreated
infection (year)
0.5-2.0 1.5-4.0 1.5-4.0 1-30
26

2. BABESIA
Etiologic agents are apicomplexan protozoa found
worldwide that infect erythrocytes, often
producing febrile illness of variable severity.
Babesia microti - responsible for infection.
Ixodes scapularis - usual tick vector.
Babesia parasites multiply in erythrocytes by schizogony but
do not produce gametocytes.
B. microti trophozoite usually appear as delicate ring forms
that may be easily confused with those of malarial
parasites, especially P. falciparum (Fig. A)

2. BABESIA
 Trophozoites can be differentiated from those
of malarial parasites by:
 presence of multiple rings in one cell that may form a
tetrad (Maltese cross) and the absence of large,
growing trophozoites and gametocytes;
 Babesia-infected cells lack hemozoin pigment,
which is present in Plasmodium-infected cells.
 History of residence in or travel to endemic
areas, or of a recent tick bite, might suggest
Babesia infection.

3. HEMOFLAGELLATES
 Order Kinetoplastida
 Characterized by:
 presence of a large mitochondrion known as a kinetoplast,
which contains enough DNA to be seen by light microscopy
when treated with Giemsa stain.
 Two genera important in human disease:
 Trypanosoma
 Leishmania
 both are transmitted by arthropod vectors and have
animal hosts that serve as reservoirs.

3. HEMOFLAGELLATES
 Kinetoplastida assume different morphologic forms
depending on their presence in vertebrate hosts, including
humans, or in their insect vectors (Fig. 62-7).

3. HEMOFLAGELLATES
 Amastigote stage
 spherical, is 2–5 µm in diameter
 nucleus and kinetoplast.
 By definition, an external flagellum is lacking
 But with axoneme (the intracellular portion of the
flagellum) at the ultrastructural level.
 In human or animal hosts infected with T. cruzi or
Leishmania spp., where they multiply exclusively
within cells.

3. HEMOFLAGELLATES
 Promastigote
 elongated and slender organism
 central nucleus
 anteriorly located kinetoplast and axoneme
 free flagellum extending from the anterior end.
 in the insect vectors of Leishmania
 stage detected in culture.

3. HEMOFLAGELLATES
 Epimastigote
 similar to the promastigote, but the kinetoplast is
found closer to the nucleus
 small undulating membrane that becomes a free
flagellum.
 All species of Trypanosoma that infect humans
assume an epimastigote stage in the insect vector
or in culture.

3. HEMOFLAGELLATES
 Trypomastigote
 kinetoplast is found at the posterior end
 flagellum forms an undulating membrane that
extends the length of the cell, emerging as a free
flagellum at the anterior end.
 bloodstream of mammalian hosts infected with
various Trypanosoma spp.

3. HEMOFLAGELLATES
 Infectious stages found in appropriate insect
vectors following transformation from the
epimastigote form are known as metacyclic
trypomastigotes.

3.1 TRYPANOSOMA
 Trypanosoma brucei
 African or Old World trypanosomiasis
 T. cruzi
 American or New World trypanosomiasis, or
Chagas’ disease

3.1 TRYPANOSOMA
 Bloodstream trypomastigotes of the T. brucei group (see Fig. 62-6, B)
are up to 30 µm long with graceful curves and a small kinetoplast.

3.1 TRYPANOSOMA
T. cruzi are shorter (20 µm), assume S and C
shapes on stained blood films, and display a
larger kinetoplast.
In equatorial Africa, T. brucei group infect both
animals and humans - tsetse flies in the genus
Glossina.
Multiplication of organisms at the bite site 
transient chancre.

3.1 TRYPANOSOMA
 East African trypanosomiasis
 T. brucei rhodesiense
 Rapidly progressive acute febrile illness with
lymphadenopathy.
 Patients die before central nervous system
(CNS) involvement is prominent.

3.1 TRYPANOSOMA
 Western Africa
 T. brucei gambiense - classic African sleeping sickness.
 more chronic course that begins with intermittent
fevers, night sweats, and malaise.
 Lymphadenopathy, especially of the cervical lymph
nodes (Winterbottom’s sign), may be pronounced.
 Involvement of the CNS
 Somnolence, confusion, and fatigue progress, leading to
stupor, coma, and eventual death.

3.1 TRYPANOSOMA
 Humans - primary reservoir of T. brucei
gambiense.
 diagnosis - suspected on the basis of geographic
history and clinical findings.
 High total IgM levels in blood and cerebrospinal
fluid (CSF).
 Pleocytosis occurs with 50–500 mononuclear
cells per microliter in CSF.

3.1 TRYPANOSOMA
 diagnosis - parasites on thick and thin films of
peripheral blood, buffy coat preparations, or
aspirates of lymph nodes or bone marrow, or
in spun CSF that is stained with Giemsa.

3.1 TRYPANOSOMA
 American trypanosomiasis
 Chagas’ disease
 T. cruzi.
 Mexico and Central and South America
 kissing bugs of the family Reduviidae.

3.1 TRYPANOSOMA
 At the time of feeding, the reduviid bug
defecates.
 The bug feces contain infective
trypomastigotes that, as a result of
scratching or rubbing, enter the body at the
bite site or through intact mucosa of the
mouth or conjunctiva.

3.1 TRYPANOSOMA
 Infective forms actively enter nearby tissue cells,
where they transform into dividing amastigotes.
 When the infected cell is filled with amastigotes,
transformation to trypomastigotes occurs,
followed by cell rupture.
 Trypomastigotes are released into the peripheral
blood and reach distant tissues, where they can
start the reproductive cycle de novo.

3.1 TRYPANOSOMA
 Chagas’ disease
 acute
 chronic
 Acute disease
 common in children younger than 5 years of age
 characterized by malaise, chills, fever,
hepatosplenomegaly, and myocarditis.
 Swelling of the tissues around the eye (Romaña’s
sign) - inoculation of the organisms occurs on the
face.

3.1 TRYPANOSOMA
 Swelling of tissues at other locations
following the bite of an infected reduviid -
chagoma.
 older individuals
 acute course is milder and often asymptomatic
 patient remains infected for life.

3.1 TRYPANOSOMA
 Chronic manifestations
 megaesophagus, megacolon, and alterations in
the conduction system of the heart
 Latter is due to destruction of the effector cells of
the parasympathetic system by autoantibodies.
 Infection can be transmitted by blood
transfusion

3.1 TRYPANOSOMA
 Diagnosis in the acute stage
 parasite on thick and thin blood films, in buffy
coat smears, or in aspirates of chagomas or
enlarged lymph nodes.
 Aspirates, blood, and biopsy specimens can
also be cultured using Novy-MacNeal-Nicolle
medium.

3.1 TRYPANOSOMA
 In endemic areas, xenodiagnosis (examination
of the gut contents of laboratory-raised reduviids
that have been allowed to feed on a patient) may
be used.
 In the chronic stage, serodiagnosis is the
method of choice.
 EIA, IFA, and CF tests are available
 cannot differentiate between acute and chronic
disease
 cross-reactions may occur in patients with
leishmaniasis.

3.2 LEISHMANIA
 A disease of the reticuloendothelial system
 caused by kinetoplastid protozoa of the genus
Leishmania.
 infect humans and have animal reservoirs
 transmitted by sandflies belonging to the genera
Phlebotomus in the Old World and Lutzomyia in
the New World.
 assume the amastigote form in mammalian
hosts and the promastigote form in insect
vectors.

3.2 LEISHMANIA
 Species of Leishmania cannot be
differentiated by examination of amastigotes
or promastigotes.
 clinical forms
 Cutaneous
 Mucocutaneous
 visceral diseases

3.2 LEISHMANIA
 Cutaneous Leishmaniasis or Old World
cutaneous leishmaniasis (oriental sore)
 southern Europe, northern and eastern Africa, the
Middle East, Iran, Afghanistan, India, and
southern Russia.
 caused by:
 Leishmania tropica
 Leishmania major
 Leishmania aethiopica
 L. Donovani
 Leishmania infantum

3.2 LEISHMANIA
 L. tropica
 urban or dry ulcer
 L. major.
 rural or wet ulcer
 Ulcers develop on an exposed area of the
body and heal spontaneously.
 Infection produces long-lasting immunity.

3.2 LEISHMANIA
 L. aethiopica
 more aggressive cutaneous infection
 in some individuals, they metastasizes to produce
mucosal lesions or diffuse cutaneous
leishmaniasis, the latter of which is characterized
by multiple skin nodules resembling lepromatous
leprosy.

3.2 LEISHMANIA
 Cutaneous leishmaniasis of the New World
 caused by:
 Leishmania mexicana
 Leishmania braziliensis
 Leishmania amazonensis
 Leishmania venezuelensis
 Leishmania garnhami
 Leishmania pifanoi
 Leishmania peruviana
 Leishmania panamensis
 Leishmania guyanensis

3.2 LEISHMANIA
 L. mexicana
 earlobe (chiclero ulcer)
 self-limiting, and are not known to metastasize to
the mucosa.
 L. mexicana and L. amazonensis may produce
diffuse cutaneous lesions similar to those
produced by L. aethiopica.

3.2 LEISHMANIA
 L. Peruviana
 western slopes of the Peruvian Andes
 causes an infection called uta, a benign cutaneous
lesion that occurs predominantly in children.
 L. peruviana
 acquired usually at home
 main reservoirs are domestic dogs.

3.2 LEISHMANIA
 Mucocutaneous Leishmaniasis (espundia)
 L. braziliensis and related species
 produce typical cutaneous lesions that generally are
more aggressive, last longer, and often disseminate to
mucous membranes, especially in the nasal, oral, or
pharyngeal areas.
 In these locations, they may produce disfiguring
lesions secondary to erosion of soft tissues and
cartilage.
 L. braziliensis is distributed in Mexico and
Central and South America.

3.2 LEISHMANIA
 Visceral Leishmaniasis / Visceral leishmaniasis
of the Old World
 occurs sporadically over a wide geographic area
 L. donovani or by L. infantum.
 L. donovani
 predominates in Africa, India, and Asia
 L. infantum
 predominates in the Mediterranean region and
the Middle East, although overlapping ranges
occur.

3.2 LEISHMANIA
 New World visceral leishmaniasis
 L. chagasi
 occurs sporadically throughout Central and South
America.
 The infection is usually benign and often
subclinical
 young children and malnourished individuals,
have marked involvement of the viscera,
especially liver, spleen, bone marrow, and
lymph nodes.

3.2 LEISHMANIA
 The infection is called kala-azar in India, in
reference to the darkening of the skin.
 Also is an opportunistic infection in
individuals with concurrent human
immunodeficiency virus (HIV), and the
condition responds poorly to therapy in such
circumstances.

3.2 LEISHMANIA
 Diagnosis:
 visualization of amastigotes in smears, imprints,
or biopsies, or by growth of promastigotes in
culture.
 In integumentary leishmaniasis, the border of
the most active lesion should be biopsied,
and the fresh biopsy should be used to make
imprints.

3.2 LEISHMANIA
 A smear should be prepared by making a 2–3-
mm incision at the border of the ulcer and
recovering small amounts of tissue from the
cut surfaces with the scalpel blade.
 Both the imprint and the smear should be
treated with Giemsa stain.

3.2 LEISHMANIA
 Specimens that may be submitted when
visceral leishmaniasis is suspected include:
 Buffy coat preparations
 lymph node and bone marrow aspirates
 spleen and liver biopsies.

3.2 LEISHMANIA
 culture
 desirable because it is more sensitive
 allows determination of the species or subspecies,
to help in clinical management of the patient.
 Biopsy or aspirate specimens collected
aseptically are cultured in Novy-MacNeal-
Nicolle medium or in Schneider’s Drosophila
medium supplemented with fetal calf serum.
 Cultures usually begin to show promastigotes
in 2–5 days but should be held for 4 weeks.

3.2 LEISHMANIA
 Amastigotes found in imprints, smears, and tissue sections
are recognized by their size (2–4 µm) and the presence of
delicate cytoplasm, a nucleus, and a kinetoplast (see Fig.
62-6, C).

3.2 LEISHMANIA
 Amastigotes must be differentiated from
other intracellular organisms, including yeast
cells of Histoplasma capsulatum and
trophozoites of T. gondii.
 Leishmania spp. have a kinetoplast and do not
have a cell wall.
 In contrast, Histoplasma lack the kinetoplast,
and the cell wall stains with periodic acid–
Schiff (PAS) and methenamine silver stains.

4. TOXOPLASMA GONDII
 protozoan parasite of the phylum Apicomplexa
 worldwide distribution in humans and in
domestic and wild animals, especially carnivores.
 Infection in immunocompetent persons is
generally asymptomatic or mild
 immunocompromised patients may experience
serious complications.
 Infection in utero may result in serious
congenital infection with sequelae or stillbirth

 Sexual stage
 completed in the intestinal epithelium of cats and
other felines, which serve exclusively as definitive
hosts.
 During this enteroepithelial cycle, asexual
schizogony and sexual gametogony occur 
development of immature oocysts  passed in
the feces.
 Oocysts mature to the infective stage (which
contain two sporocysts with four sporozoites
each) in the environment in 2–21 days.

Ingestion of infective oocysts  infection of a
wide variety of susceptible vertebrate hosts in
which actively growing trophozoites
(tachyzoites) may infect any nucleated cells.
Proliferation of tachyzoites  cell death and
injury to the host during acute infection.

Once immunity has developed, the organisms form
tissue cysts that may eventually contain
hundreds or thousands of slowly growing
bradyzoites.
The presence of tissue cysts is characteristic of
chronic infection.
All stages of the life cycle occur in felines, but only
trophozoite and cyst stages occur in humans and
other intermediate hosts.

 Humans acquire infection by ingestion of
inadequately cooked meat, especially lamb or
pork, that contains tissue cysts or by
ingestion of infective oocysts from material
contaminated by cat feces.

 Transmission via blood transfusion and
through organ transplantation also can occur.
 Most acute infections are asymptomatic or
mimic other infectious diseases in which fever
and lymphadenopathy are prominent.

 Congenital infection may occur when the
mother develops acute infection during
gestation.
 Risk of infection to the neonate is unrelated
to the presence or absence of symptoms in
the mother, but severity of infection depends
on the stage of gestation at which it is
acquired.

 Intrauterine death, microcephaly, or
hydrocephaly with intracranial calcifications may
develop if infection is acquired in the first half of
pregnancy.
 Infections in the second half of pregnancy are
usually asymptomatic at birth, although fever,
hepatosplenomegaly, and jaundice may appear.
 Chorioretinitis, psychomotor retardation, and
convulsive disorders may appear months or
years later.

 In immunosuppressed individuals, especially
those with AIDS, infection with T. gondii
usually presents with CNS involvement .
 Other possible clinical and pathologic
manifestations include pneumonitis,
myocarditis, retinitis, pancreatitis, or orchitis
(Luft, 1989; Schnapp, 1992).

 Toxoplasmosis may be difficult to diagnose
clinically and is often discovered at autopsy
(Gutierrez, 2000).
 These infections usually result from
reactivation of a latent infection, acquired
months or years before, but occasionally
result from a primary infection.

 Diagnosis :
 examination of tissues, blood, or body fluids.
 Demonstration of tachyzoites or tissue cysts is
definitive but may prove difficult to demonstrate
in H&Estained sections; fluorescent or
immunoperoxidase stains, if available, are useful.

 Giemsa is good for staining smears of body
fluids and tissue imprints.
 Organisms may be demonstrated by
inoculating appropriate material into tissue
culture or uninfected mice.

 Isolation of organisms from blood or body
fluid serves as evidence of acute infection,
 Recovery from tissues may reflect chronic
infection.

 In smears, tachyzoites are crescent-shaped or
oval, measuring approximately 3 × 7 µm
 cysts measure up to 30 µm in diameter and
are usually spherical, except in muscle fibers,
where they appear elongate (see Fig. 62-6, D
through F).

 PCR
 highly sensitive and specific in detecting
toxoplasmic encephalitis, disseminated disease,
and intrauterine infection
 Serology
 primary approach in establishing a diagnosis of
toxoplasmosis

 Sabin-Feldman dye test and the IFA test
 standards against which other methods are
compared
 former is performed in only a few centers.
 EIA tests
 commercially available and generally provide
results similar to those of IFA.
 Antibodies appear in 1–2 weeks
 titers peak at 6–8 weeks.

 Tests for IgM-specific antibodies
 useful for diagnosis of congenital and acute
infection
 However, knowledge of test limitations,
specifically the occurrence of false-positive
reactions, is extremely important.

 Interpretation of IgG and IgM antibody titers
varies by test method and by manufacturer.
 The laboratory performing the test should
provide the necessary interpretive criteria

5. OPPORTUNISTIC FREE-LIVING
AMOEBAE
 Naegleria
 Acanthamoeba
 Balamuthia
 Sappinia

5. OPPORTUNISTIC FREE-
LIVING AMOEBAE
 inhabitants of soil, water, and other
environmental substrates.
 All 4 are associated with opportunistic infection
of the CNS, and Acanthamoeba causes keratitis.
 Primary amebic meningoencephalitis
 caused by the ameboflagellate Naegleria fowleri
 children and young adults who have been
swimming or diving in warm, freshwater lakes or
pools.

LIVING AMOEBAE
 The ameboflagellate enters the brain via the
cribriform plate and olfactory bulbs and reaches
the frontal lobes, where it produces an acute
hemorrhagic meningoencephalitis that is usually
fatal within 1 week of onset of symptoms.
 extremely poor prognosis, despite vigorous
therapeutic intervention.
 Diagnosis
 established at autopsy examination by the finding of
trophozoites (cysts are rarely seen) in tissue sections (Fig.
62-8, A).

LIVING AMOEBAE

LIVING AMOEBAE
 Antemortem diagnosis
 made occasionally by identifying typical
trophozoites in CSF on direct wet mounts, in
stained preparations, or in culture.
 Trophozoites
 10–35 µm
 large, round, central karyosomes
 if exposed to warm distilled water, convert to
flagellated forms in 1–2 hours.

LIVING AMOEBAE
 Cysts
 Spherical
 7–15 µm in diameter
 Culture usually is performed on nonnutrient agar
plates (1.5% agar, 0.5% sodium chloride, pH 6.6–7.0)
seeded with a lawn of heat-killed or living Escherichia
coli.
 Amebae ingest the bacteria, leaving tracks in
the bacterial lawn, which may be seen under
low-power magnification using reduced light
(Fig. 62-8, B).

LIVING AMOEBAE
 Granulomatous amebic
meningoencephalitis (GAE)
 may be caused by several species of
Acanthamoeba, including Acanthamoeba
castellani, Acanthamoeba culbertsoni,
Acanthamoeba polyphaga, and Acanthamoeba
astronyxis, among others.
 usually a subacute or chronic opportunistic
infection of chronically ill, debilitated, and
immunosuppressed individuals, leading to death
weeks to months following onset of symptoms.

LIVING AMOEBAE
 spread hematogenously from primary foci in
skin, pharynx, or the respiratory tract.
 Systemic infections occur in individuals with
AIDS and may present as ulcerative skin
lesions, subcutaneous abscesses, or
erythematous nodules (Fig. 62-8, C).

LIVING AMOEBAE
 Exposure to fresh water is not necessary
because cysts of Acanthamoeba readily
become airborne and may be recovered from
the throat and nasal passages.
 The pathologic reaction in tissues is
granulomatous, with trophozoites
predominating in viable tissue, and cysts
predominating in areas of necrosis.

LIVING AMOEBAE
 Diagnosis
 established at autopsy
 organisms may be recognized in brain biopsies or
recovered using the culture technique described
for Naegleria.
 Acanthamoeba trophozoites
 somewhat larger than Naegleria, measuring 15–45
µm
 display needle-like filamentous projections from
the cell known as acanthopodia.

LIVING AMOEBAE
 Cysts measure 10–25 µm and are double-walled, displaying
a wrinkled outer wall (ectocyst) and a polygonal, stellate, or
round inner wall (endocyst) (Fig. 62-8, D).

LIVING AMOEBAE
 Currently, genotyping is the preferred
approach used in differentiating types of
Acanthamoeba.
 Immunofluorescence and immunoperoxidase
techniques may prove useful in identifying
and differentiating species and are available
from the CDC.

LIVING AMOEBAE
 GAE may also be caused by leptomyxid
amebae, specifically Balamuthia
mandrillaris.
 Morphologically, Balamuthia cannot be
differentiated from Acanthamoeba by
routine histology, although differences may
be detected at the ultrastructural level.

LIVING AMOEBAE
 These organisms are antigenically distinct
and may be identified using specific
monoclonal or polyclonal antisera in DFA or
immunoperoxidase assays.
 Balamuthia do not grow on agar plates used
for Naegleria and Acanthamoeba, but they
have been recovered in tissue culture using
mammalian cell lines.

LIVING AMOEBAE
 Acanthamoeba keratitis
 increasingly recognized painful infection of the
cornea
 persons who use daily-wear or extended-wear soft
contact lenses or who have experienced trauma to
the cornea.
 Incomplete or infrequent disinfection and use of
homemade saline and multipurpose solutions are
known risk factors for acquiring the infection.

LIVING AMOEBAE
 characterized by development of a paracentral
ring infiltrate of the corneal stroma, which
progresses to ulceration and possible perforation,
with loss of the eye.
 The infection may be confused with fungal,
bacterial, or herpetic keratitis but is
characteristically refractory to commonly used
antimicrobials.

LIVING AMOEBAE
 Keratoplasty
 used routinely in management of this disease.
 Diagnosis
 established by demonstrating amebic
trophozoites or cysts in corneal scrapings or
biopsies (Fig. 62-8, E).

LIVING AMOEBAE
 A variety of permanent stains can be used:
 Giemsa
 PAS
 trichrome.
 fluorochrome Calcofluor white is especially
helpful in recognizing amebic cysts (Fig. 62-8, F)

1.1 ENTAMOEBA HISTOLYTICA
 Cause:
 amebic dysentery,
 amebic colitis
 liver abscesses
 Analysis of isoenzyme patterns (zymodemes)
has shown that only certain strains can cause
invasive disease and that most infections remain
undetected.

 Genetic and biochemical differences between
invasive and noninvasive strains have been
identified, and it has been proposed that
nonpathogenic strains should be named
Entamoeba dispar.
 Amebic dysentery
 acute disease characterized by bloody diarrhea
with abdominal cramping.
 Invasion of the intestinal mucosa occurs, producing
ulceration that may lead to perforation and
peritonitis.

 amebic colitis
 mimic ulcerative colitis and other forms of
inflammatory bowel disease.
 Symptoms
 less severe than in amebic dysentery
 nonbloody diarrhea
 Constipation
 abdominal cramping
 weight loss
 Small, pinpoint mucosal ulcerations may develop and
expand within the submucosa to form flask-shaped
ulcers.

 Amebic liver abscess
 most common form of extraintestinal amebiasis
 occurring in approximately 5% of patients with a
history of intestinal amebiasis.
 Symptoms include fever and right upper quadrant
pain.
 diagnosed by radiographic scans, ultrasound, and
serologic tests.

 Amebae are present in the stool in less than half
of patients at the time liver abscess is manifest.
 Amebic hepatitis
 characterized by an enlarged, tender liver in
someone with intestinal amebiasis.
 Its pathogenesis is poorly understood.

 Rarely, amebic abscesses appear in other organs,
such as the lung, brain, or skin, by
hematogenous spread from the intestine or by
contiguous spread from a liver abscess.
 Masses of granulomatous tissue, known as
amebomas, may form in response to the
presence of amebae, which in the intestine may
cause a so-called napkin ring lesion that could
be mistaken for a carcinoma.

 Diagnosis
 Examination of a series of stool specimens.
 If the patient has been given antibiotics or
contrast media, the amebic infection may be
masked for a period of time.

 Aspirated material from liver abscesses
 examined microscopically to detect trophozoites.
 The last material aspirated is most likely to
contain trophozoites and may be examined by
direct microscopic examination or permanently
stained slides.

 If tissue is available, sections may show
organisms that stain prominently with PAS (Fig.
62-11, C).

 Culture procedures
 are not widely used for diagnosis
 useful for research and are essential for
determining pathogenicity based on zymodemes.
 EIA antigen detection tests
 specific, sensitive, and able to differentiate E.
histolytica from E. dispar are commercially
available (see Table 62-5)

 Serologic tests (see Table 62-6)
 most useful for diagnosis of extraintestinal
infection because approximately 95% of patients
with amebic liver abscess are seropositive.
 This decreases to 70% for patients with active
intestinal infection and to 10% in asymptomatic
carriers.
 Detectable titers may persist for months or years
after successful treatment .

 Trophozoites of E. Histolytica
 10–60 µm in diameter
 commensal forms usually 15–20 µm
 invasive forms greater than 20 µm in greatest
dimension (Table 62-9; Figs. 62-9 through 62-12).

 direct wet mounts
 trophozoites show progressive motility via rapidly
formed hyaline pseudopodia that demonstrate a
sharp demarcation between endoplasm and
ectoplasm
 unstained nuclei are not visible.
 invasive disease
 some trophozoites contain ingested erythrocytes
(see Fig. 62-11, C), a feature diagnostic of E.
histolytica infection.

 stained preparations
 peripheral nuclear chromatin is evenly distributed
along the nuclear membrane as fine granules.
 karyosome is small and is often centrally located,
with fine fibrils, which generally are not visible,
attaching it to the nuclear membrane.

 The cytoplasm is finely granular, and in invasive
organisms, no inclusions or only erythrocyte
inclusions are seen.
 characteristic that is pathognomonic for E.
histolytica :
 phagocytosis of erythrocytes
 very rarely occurs with other species.

 Noninvasive organisms
 may contain ingested bacteria
 In degenerating organisms, the cytoplasm
may become vacuolated and nuclei may show
abnormal chromatin clumping.

 Cysts of E. Histolytica
 spherical and measure 10–20 µm (usually 12–15
µm) in diameter.

 The rounded precyst stage has a single
nucleus but does not have a refractile cyst
wall.
 As it matures, the cyst develops four nuclei,
each approximately one-sixth the diameter of
the cyst.

 The cyst cytoplasm may contain glycogen
vacuoles and chromatoid bodies with blunted
or rounded ends.
 important diagnostic criteria for identifying
cysts
 number and size of nuclei
 appearance of chromatoid bodies

1.2 NON-PATHOGENIC AMEBAE
 Identification of trophozoites is based on :
 size and nuclear and cytoplasmic characteristics
 identification of cysts is based on :
 size, number and characteristics of nuclei, and
presence and character of chromatoid bodies and
glycogen masses.

 E. hartmanni
 morphologic characteristics similar to those of E.
histolytica, except trophozoites have a maximum
diameter of 12 µm and cysts have a maximum
diameter of 10 µm, and cysts often have a single
nucleus.

 Entamoeba coli
 a common lumen-dwelling ameba
 difficult to differentiate from E. histolytica.
 cytoplasm stains somewhat more darkly than the
cytoplasm of E. histolytica and is more
vacuolated, containing numerous ingested
bacteria, yeasts, and other materials.

 Mature cysts of E. coli
 contain eight nuclei
 occasional cysts contain 16 or more.
 Immature cysts of E. coli
 not common
 four nuclei that are larger (one-fourth the
diameter of the cyst) than nuclei of E. histolytica
(one-sixth the diameter of the cyst) and may
contain glycogen.

 Chromatoid bodies in E. Coli
 when present, are irregular in shape with
splintered or pointed ends, rather than the
rounded ends seen in E. histolytica.

 Endolimax nana
 smallest ameba to infect humans.
 Trophozoites
 atypical nuclei that contain a triangular chromatin
mass, a band of chromatin across the nucleus, or
two discrete masses of chromatin on opposite sides
of the nuclear membrane (see Fig. 62-12).
 Cysts
 contain four nuclei, although smaller numbers may
be seen.

 E. Nana trophozoite also may also show:
 clear halo or karyolymph space surrounds the
karyosome and extends to the nuclear membrane.
 Atypical nuclear forms may be helpful in
differentiating E. nana from I. bütschlii, which is
similar in appearance but larger.

 Glycogen
 when present, occurs diffusely in the cytoplasm
rather than as a discrete mass.
 Cysts may be confused with Blastocystis
hominis organisms.
 The nuclei of B. Hominis lack the halos that are
typically seen with E. nana cysts.

 nuclei of I. bütschlii trophozoites and
cysts
 large, centrally located karyosome frequently
surrounded by achromatic granules that may
not be distinct but appear only as a muddy
karyolymph space or halo.
 Cysts contain a single nucleus, in which the
karyosome is often eccentric with a nearby
crescent of achromatic granules.

 cyst of I. bütschlii
 characterized by a prominent vacuole of glycogen
that stains reddish brown in iodine-stained wet
mounts, thus the name of the organism.
 Glycogen
 dissolved by aqueous fixatives  not be
demonstrable

1.3 BLASTOCYSTIS HOMINIS
 inhabits the large bowel and is frequently
found in stool specimens of asymptomatic
individuals.
 Blastocystis may assume one of three forms:
 vacuolated (seen most commonly)
 Ameboid
 granular.

1.3 BLASTOCYSTIS HOMINIS
 The vacuolated form
 also known as the central vacuolar
 spherical and variable in size (5–20 µm) and has a
central clear area and two to four peripheral
nuclei.
 The presence of Blastocystis should be
reported, especially when they are numerous
(five or more per 400× field).

2.1 DIENTAMOEBA FRAGILIS
 an ameboid pathogen that infects the colon
and has been associated with diarrheal
disease, especially in young children.
 similar in appearance to amebae but has been
reclassified as a flagellate on the basis of
ultrastructural details and antigenic
similarities.
 Also, no cyst stage has been described.

 Symptoms
 diarrhea
 abdominal distention
 Approximately 25% of persons infected with
this parasite have symptomatic disease.

 usually is not associated with other fecal
protozoa but does show a 10–20 times
greater than expected association with
enterobiasis
 This association and some experimental
evidence suggest that D. fragilis infection
may be spread by ingestion of pinworm eggs
infected with D. Fragilis.

 D. fragilis infection will be overlooked unless
permanently stained slides are examined.
 Multiple specimens may need to be
submitted because shedding varies from day
to day.
 When smears are prepared, the last portion
of the stool evacuated should be examined
because the number of parasites found there
tends to be greater.

 Two thirds to four fifths of the organisms contain
two nuclei that consist of a cluster of four to eight
karyosomal granules, which may appear as one large
irregular karyosome (see Fig. 62-12).

 Uninucleate D. fragilis may be confused with
trophozoites of E. nana or I. bütschlii.
 The cytoplasm is finely granular and often
contains ingested bacteria.
 Trophozoites
 delicate and may be easily overlooked, so stained
slides must be carefully examined.

2.2 GIARDIA LAMBLIA
 Infection may be asymptomatic or may cause
disease ranging from mild diarrhea with
vague abdominal complaints to a
malabsorption syndrome with diarrhea and
steatorrhea, similar to that of sprue.

2.2 GIARDIA LAMBLIA
 should be considered in any patient
presenting with diarrhea of longer than 10
days’ duration.
 Diagnosis
 demonstration of Giardia trophozoites or cysts, or
both, in fecal specimens.

2.2 GIARDIA LAMBLIA
 trophozoites
 multiply in the small bowel and attach to the
mucosa by a ventral concave sucking disk.
 predominate in diarrheic stool
 infectious cysts
 more likely to be found in formed stool.

2.2 GIARDIA LAMBLIA
 examination of multiple specimens, collected
on different days, may be necessary
 passage of organisms varies from day to day
 Direct wet mounts
 helpful for demonstrating trophozoites, with their
so-called falling leaf motility, in a diarrheic or
aspirate specimen.

2.2 GIARDIA LAMBLIA
 Cysts can be seen in direct wet mount and
concentration techniques
 both trophozoites and cysts may be
demonstrated on permanently stained
slides.

2.2 GIARDIA LAMBLIA
 In some cases, the organisms cannot be
demonstrated in fecal specimens, and small
bowel aspirates or so-called string test
specimens may be required.
 Giardia trophozoites
 pear-shaped with a tapered posterior end
 two nuclei that give the appearance of a smiling
face with prominent eyes

2.2 GIARDIA LAMBLIA
 viewed from the side:
 anterior end of the organism is thicker and tapers
posteriorly
 anterior half to three quarters consists of the
sucking disk on the ventral surface.
 The four lateral, two ventral, and two caudal
flagella usually are not evident in wet mounts
or in stained preparations.

2.2 GIARDIA LAMBLIA
 Cysts are oval and usually quadrinucleate.
 Below the nuclei are dark-staining median
bodies that cross longitudinal fibrils,
providing distinctive internal characteristics.
 The cytoplasm often is retracted from the
cyst wall.

2.3 CHILOMATIX MESNILI
 consistent location of the single nucleus at
one end of the organism and the tapering of
the end opposite the nucleus .
 If multiple organisms are examined, the
cytostome and the spiral groove are visible in
some.

2.3 CHILOMATIX MESNILI
 The three external flagella usually are not
visible in stained or formalin-fixed
preparations.
 The lemon-shaped cysts contain various
curved cytostomal fibers with a safety pin–
like appearance.

2.4 PENTATRICHOMONAS
HOMINIS
known previously as
Trichomonas
hominis (see Table
62-11 and Fig. 62-
13, B)

2.4 PENTATRICHOMONAS
HOMINIS
 Do not stain particularly well and often are
distorted in permanent smears.
 single Entamoeba-like nucleus, undulating
membrane and associated costa, and flagella.
 A prominent rod-like object, the axostyle, runs
through the organism and protrudes from the
posterior end.
 No cyst stage has been described.

2.5 TRICHOMONAS VAGINALIS
 common cause of vaginitis, characterized by
inflammation, itching, vaginal discharge, and,
occasionally, dysuria.
 spread by sexual intercourse, often by males
who have an asymptomatic infection.

Occasionally, males may have symptomatic
prostatitis or urethritis.
diagnosed in the physician’s office by direct wet
mount examination of vaginal fluid, prostatic
fluid, or sediments of freshly passed urine.

Morphologically,
resembles P. hominis
but is larger (up to 23
µm), and the undulating
membrane extends only
half the length of the
body.

 Direct wet mount
 may be insensitive
 use of culture or commercially available
immunoassay techniques is recommended when
infection is not readily diagnosed.
 Cultures, including use of a convenient
“pouch” system, have a sensitivity of about
90%, as do DFA and EIA techniques that use
monoclonal antibodies.

 Papanicolaou-stained gynecologic smears may reveal T.
vaginalis on occasion but have poor sensitivity and
specificity.

2.6 OTHER FLAGELLATES
 Enteromonas hominis
 Retortamonas intestinalis
 small, nonpathogenic, intestinal flagellates that
are seen infrequently but, when present, may
occur in large numbers.

 Trichomonas tenax
 trichomonad that occasionally is recovered
from the oral cavity but does not cause
disease.

3.1 BALANTIDIUM COLI
 may cause a dysentery-like syndrome with
colonic ulcerations similar to that of
amebiasis, but it does not produce liver
abscesses or other systemic lesions.

 Acquired from hogs
 the largest protozoan to infect humans.
 Trophozoites
 between 40 µm and more than 200 µm in
greatest dimension (most measure 50–100 µm)
 uniformly covered with cilia that are slightly
longer at the anterior end adjacent to the
cytostome.

 large macronucleus is readily seen in stained
preparations
 smaller micronucleus is infrequently visible.
 Numerous food vacuoles and contractile
vacuoles are present in the cytoplasm.

 Cysts
 Rounded
 50–70 µm in length.
 Cilia may be seen within younger cysts
 nuclear characteristics are similar to those of
trophozoites.

4. COCCIDIA
Genera infecting the intestine of humans:
Isospora
Sarcocystis
Cryptosporidium
Cyclospora

4.1 CYTOISOSPORA BELLI
 formerly known as
Isospora belli
 undergoes both
asexual and sexual
development in
the cytoplasm of
small intestine
epithelial cells
(Fig. 62-15, A).

 Sexual development
 results in the production of oocysts, which are passed
in the stool and mature to the infective stage in the
environment.
 cause diarrhea and malabsorption -- generally
are self-limited.
 In patients with AIDS or other
immunosuppressive disorders, disease may
persist for months or years, and may contribute
to death

 Diagnosis
 finding the unsporulated oocysts measuring 12 ×
30 µm in fecal specimens, usually in direct wet
mounts or concentration preparations (Fig. 62-15,
B).
 If the unfixed specimen is left at room
temperature for 24–48 hours, sporulation
occurs.

 The infectious oocyst
contains two sporocysts,
each with four sporozoites
(see Fig. 62-13, A).
 These oocysts are similar to
those of Cryptosporidium in
that they stain acid-fast.

4.2 SARCOCYSTIS SPP.,
 two-host coccidia
 sexual phase develops in the intestinal
mucosa of carnivorous animals
 asexual, extraintestinal phase occurs in the
muscles and tissues of various intermediate
hosts.
 Humans may serve as definitive or
intermediate hosts, depending on the species
of Sarcocystis.

 Intestinal infection with Sarcocystis hominis
and Sarcocystis suihominis is acquired by
ingestion of raw or incompletely cooked beef
or pork, respectively, which contains tissue
cysts (sarcocysts).
 Infection usually is asymptomatic, but
occasional patients have transient diarrhea,
abdominal pain, or anorexia.

 Intestinal infection is self-limited because
asexual multiplication occurs in the
intermediate host and is not repeated in the
definitive host.
 Oocyst production is limited by the number
of organisms ingested in the form of
sarcocysts.

 diagnosis :
 detection of sporulated 25 × 33
µm sporocysts in the stools
 Each mature sporocyst
contains four sporozoites.
 The oocyst wall is thin and
often is not detectable, or has
already ruptured, releasing the
two sporocysts.

4.3 CRYPTOSPORIDIUM SPP.,
 use a single host in their life cycle but may infect
humans (predominantly C. hominis and C.
parvum) and a wide variety of animals, including
cattle and sheep.
 Parasites develop in the brush border of
epithelial cells of the small and large intestine
 occasionally spread to other sites such as the
gallbladder, the pancreas, and the respiratory
tract.

 Common cause of acute, self-limited diarrhea
in normal persons, especially in children who
attend day care.
 Oocysts are refractory to usual chlorination
levels of drinking water.
 In patients with AIDS, may cause chronic
secretory diarrhea that can last for months to
years and may contribute to death.

 incubation period
 about 8 days
 in previously healthy persons, illness lasts 9–
23 days.
 malaise, fever, anorexia, abdominal cramps,
and diarrhea

 Diagnosis
 stool examination.
 Various concentration methods,
including formalin–ethyl acetate
sedimentation and Sheather’s sugar
flotation, work well

 Smear is prepared from the sediment and
stained with an acid-fast stain or
immunofluorescent reagents.
 Several acid-fast staining methods, including
auramine-O, have been evaluated, but a
modified cold Kinyoun method is used most
widely.

 oocysts
 Spherical
 measure 4–6 µm in diameter
 when stained by the modified Kinyoun procedure,
appear a deep fuchsia
 Positive control slides must be used with
every run.

4.4 CYCLOSPORA CAYETANENSIS
 Causes a flu-like illness with nausea,
vomiting, weight loss, and explosive watery
diarrhea lasting 1–3 weeks.
 Oocysts
 passed unsporulated
 appear as nonrefractile spheres 8–10 µm in
diameter
 contain a cluster of refractile globules enclosed
within a membrane when viewed by light
microscopy.

 Oocysts autofluoresce bright
green to intense blue under
ultraviolet epifluorescence
 they stain acid-fast when
modified acidfast or auramine-O
staining techniques are used.
 must be differentiated from
oocysts of Cryptosporidium,
which stain in an identical
fashion but are smaller (4–6 µm)
(Fig. 62-15, E).

 A total of 1–2 weeks is required for
sporulation, after which the mature oocyst
contains two sporocysts, each with two
sporozoites.
 trichrome-stained smears
 oocysts appear as clear, round, and somewhat
wrinkled objects.

5. MICROSPORIDIA
 obligate intracellular, spore-forming
protozoan parasites in the phylum
Microspora that infect a variety of animals,
including humans
 serious pathogens in immunocompromised
hosts, especially those with AIDS - up to 30%
of otherwise unexplained diarrheal disease.

 Two species implicated most commonly in
human intestinal infection:
 Enterocytozoon bieneusi
 Encephalitozoon intestinalis
 cause protracted diarrhea and weight loss in AIDS
patients similar to that caused by
Cryptosporidium.
 E. intestinalis may also cause disseminated
disease.

 Organisms multiply intracellularly
(merogony) and form resistant spores
(sporogony) that eventually rupture the host
cell and infect adjacent cells or are passed out
of the body.
 Spore contains a coiled polar tubule, which is
forcefully extruded under appropriate
environmental stimuli and penetrates the
membrane of the recipient cell.

 The parasite’s sporoplasm is injected through
the tubule into the host cell cytoplasm, where
multiplication ensues.
 Reservoir hosts have not been identified.
 Occasionally, patients have been infected by
other genera of microsporidia:
 Encephalitozoon (hepatitis, ocular infection, CNS
disease)
 Nosema (disseminated infection)
 Pleistophora (myositis).

 Until recently, diagnosis required examination of
tissues submitted for routine light (Fig. 62-15, F
and G) and electron microscopy.

 Detection:
 Modified trichrome
staining method
 for examination of stool
specimens for spores.
 small (1.5–3 µm),
elliptical spores stain red
against a faint green
background, and some
display a characteristic
midbody cross-band
(Fig. 62-15, H).

 Fluorochrome stains such as Uvitex 2B and
Calcofluor white
 more sensitive in detecting spores and may be
useful in the initial screening of specimens.

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