Transcript ppt

Toxoplasma and toxoplasmosis
Medical Parasitology 2012
Silvia N.J. Moreno
Alveolata
Dinoflagellata:
Many are photosynthetic.
transverse flagellum that encircles
the body (often in a groove known
as the cingulum) and a longitudinal
flagellum oriented perpendicular
Ciliata: some or all of
the surface is covered with
short, dense hairlike
structures (cilia) which
beat to propel the ciliate
through the water and/or to
draw in food particles.
Apicomplexa:
Diverse group. All parasitic.
Elements of the Apical
complex
Alveolata
• Cortical alveoli or inner
membrane complex (flattened
membranous cisternae
underlying the plasma
membrane)
• Subpellicular microtubuli
• Mitochondria with tubular
cristae
• Molecular phylogeny on rRNA,
tubulin and several other
genes supports this grouping
PM
IMC
MT
Balantidium coli
• Balantidium coli is the only
human pathogen from
this phylum
• Ciliates have 2 nuclei (one
macronucleus and one
micronucleus) and
reproduce by transverse
binary fission, conjugation,
autogamy, and cytogamy.
• Typical ciliate organization
(cilia, digestive vacuole,
pulsating vacuole etc.)
Balantidium coli
• Cosmopolitan distribution.
Relatively rare in humans (most
reported cases are from
Philippines). Association with
pig reservoir
• Balantidium coli has 2
developmental stages: a
trophozoite stage and a cyst
stage.
• Causes diarrhea and dysentery
in both swine and humans.
• Trophozoites can invade the
mucosa forming ulcers very
much like E. histolytica. Disease
can be fatal.
APICOMPLEXA
 Apical complex: an
assemblage of
cytoskeletal elements
and secretory organelles
 No flagella or cilia
except for the
microgamete (sperm)
 All members of the
phylum are parasitic
 Complex life cycle:
asexual and sexual
forms the development
of these stages are
tissue specific.
APICOMPLEXA
• Gregarines: parasites of invertebrates,
some quite big (used as early research
models), Cryptosporidium might belong into
this category
• Coccidians: tissue parasites of vertebrates
and invertebrates (can have single (e.g.
Eimeria) or two host (e.g. Toxoplasma).
Many parasites of medical and veterinary
importance. Sex produces a sporelike
oocyst
• Haemosporidians (Plasmodium) and
Piroplasms (Babesia & Theileria): small
parasites of blood cells which are
transmitted by arthropods
Plasmodium falciparum
bursting from red blood cells
Credit: James Marden
Gregarine parasites that causes metabolic disorders in dragonflies,
as it appears under an electron microscope at 100-micron scale.
Eimeria Tenella labelled with
antibodies against
microneme proteins
SPECIES OF MEDICAL IMPORTANCE
Intestinal parasites: Eimeria, Cyclospora and cryptosporidium.
Extraintestinal or tissue parasites: Toxoplasma, sarcocystis,
neospora.
Blood parasites: plasmodium, babesia and theileria
Toxoplasma gondii
• Initially found in 1908 as a tissue parasite of gondi (an african rodent)
• T. gondii is distributed worldwide. High prevalence in Europe. In the
US approx. 20-30% of the population is infected
• Broad host and tissue specificity: infects almost all nucleated cells.
• Transmission by tissue cysts (containing bradyzoites) or the oocyst
(containing sporozoites)
• Infection through ingestion of undercooked or raw
meat or contaminated water
• Clinical significance:
Asymptomatic in healthy individuals
CNS involvement in immunocompromised patients
Congenital disease
PARASITE FORMS
• TACHYZOITES: rapidly growing meront or zoite observed in the early
stage of of infection
• BRADYZOITES: Slow-growing zoite or meront inside the tissue cysts
• TISSUE CYSTS: Found in the tissues contain bradyzoites. Infective
form
This scanning electron micrograph shows a tissue
cyst with its many bradyzoites within the brain of
an infected mouse.Photo courtesy of David
Ferguson, Oxford University.
http://cmgm.stanford.edu/micro/boothroyd/boothroydlab
desc.html
• OOCYSTS: Zygote form.
Highly resistant form.
Infective form
• Sporocyst
• Sporozoites
Oocyst from a fecal sample of a cat
Life cycle of Toxoplasma gondii
From Chidoni, Moody & Manser, 2001
DEFINITIVE HOST
• Ingestion of TC by the cat initiates
the intestinal stage
• Tissue cysts are also found in the
cat
• Oocysts are only produced in the definitive
host (DH), members of the family Felidae
• Sexual cycle produces the
oocyst which is excreted in the
feces
•As the host
develops immunity
replication rate slows
down and parasite
form tissue cysts
(TC) containing
bradyzoites
• Sporulation results
in the formation of
sporozoites
• Sporulated oocyst
remain infective for
months
INTERMEDIATE HOST
• IH gets infected by ingesting sporulated oocysts which release sporozoites which penetrate the
intestinal epithelium, and invade macrophages and other types of cells.
• Parasites multiply by endodiogeny
• Tachyzoites are released which will invade new host cells
The intestinal phase in the cat
Cat ingests tissue cysts containing bradyzoites
Number of merogonous cycles is variable
Gametocytes develop in the small intestine but are more
common in the ileum
2-4% of gametocytes are male, each produces around 12
microgametes
Oocysts appear in the cat’s feces 3-5 days after infection by
cysts with peak production around 5 and 8
Oocysts require oxygen and they sporulate in 1-5 days
TOXOPLASMA TRANSMISSION
DEFINITIVE HOST
Sources of
infection:
Contaminated water or
food
Undercooked meat
Mother to fetus
Organ transplant (rare)
Blood transfusion (rare)
Latent bradyzoite cysts confer lifelong infection
• The acute infection is carried by
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tachyzoite stage
Tachyzoites divide rapidly and
aggresively destroy tissues, all
pathology is associated with
tachyzoites
Tachys can cross the blood-brain
barrier and the placenta
When the IR slows down tachy
replication, they differentiate in
bradys and form tissue cysts
Cysts form in brain and skeletal
muscle
Bradyzoite cyst persist in the
immune host for the rest of its life
Bradyzoites are resistant to all
currently available drugs
Bradyzoite cysts are highly
infective if ingested
• Bradyzoites marks the beginning
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of the chronic phase of infection
Tissue cysts show very little
evidence of inflammation or
immune cell infiltrates.
• Bradyzoites (not tachyzoites) are
resistant to low pH and
digestive enzymes during
stomach passage
• Protective cyst wall is finally
dissolved and bradyzoites infect
tissue and transform into tachys
• Tachyzoites: pathogenesis
Bradyzoites: epidemiology
Toxoplasma is an opportunistic
pathogen
• 15-70% of the population is chronically infected
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(current seroprevalence in the US is 21.5%)
Most people show no or only benign symptoms
(headache, sore throat, lymphadenitis, fever
Three situations can lead to severe disease:
1) congenital toxoplasmosis
2) occular toxoplasmosis in immunocompetent adults
3) loss of a functional immune system
PATHOGENESIS
• Congenital disease:
acute infection of the expectant mother.
Severity depends on the stage of pregnancy.
Spontaneous abortions or neurological disorders such as
blindness and mental retardation can result.
• Immunocompromised
individuals: toxoplasmic encephalitis
Reactivation of a latent infection (>95% cases)
Recently acquired infection
• Ocular toxoplasmosis
Congenital toxoplasmosis
• If a women is infected for the first
time during pregnancy the parasite
can transverse the placenta and
cause fetal disease
• Both the probability and severity of
the disease depend on when the
infection takes place during
pregnancy (early: low
transmission, but severe disease,
late: high transmission, more
benign symptoms)
• Several countries screen all
pregnant women for acute infection
but the efficacy of in utero
treatment is hotly debated
Congenital toxoplasmosis
Girl with hydrocephalus due to congenital toxoplasmosis.
• Children who are asymptomatic at birth often develop disease
later on, long term treatment of these kids is quite successful
• This include antiparasitic treatment as well as symptomatic
treatment against e.g. hydrocephalus
• Despite calcification throughout the brain this 10 month old child
developed completely normal
Toxoplasmic encephalitis (TE)
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Only 25-30% of seropositive patients who develop AIDS develop TE
Involvement of the CNS.
Symptoms: lethargy, loss of memory to severe dementia, focal to major motor
seizures
MRI result of an
homosexual male
diagnosed with HIV
admitted to a local
hospital complaining
of intermittent
headache, lethargy,
personality change,
abdominal pain and
vomiting.
T. gondii is a major pathogen in
late stages of AIDS
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In the majority of cases this is due
to reactivation of the chronic
infection rather than new infection
Usually a multifocal process.
Multiple brain abscesses: necrotic
central zone surrounded by
hyperemic region containing
parasites and infiltrating
mononuclear cells. Sometimes
disseminated TE
TE can be treated with
pyrimethamine and sulfa but not all
patients tolerate side effects
As for all other opportunistic
infections the incidence is much
lower in patient on HAART (highly
active antiretroviral therapy)
Ocular toxoplasmosis
• Ocular toxoplasmosis can be a
sequel of congenital infection
(neonatal or late forms)
• However recent epidemiological
studies show that it also occurs
after infection of seemingly fully
immunocompetent adults
• Severe retinochoroiditis
(cleared yellowish zone), after
treatment scaring occurs
• The acute phase responds well
to treatment, but
• 80% probability of relapse and
relatively poor prognosis of
eyesight in the long term
DIAGNOSIS OF TOXOPLASMOSIS
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Parasites can be detected in biopsied specimens or cerebral spinal fluid.
Not very reliable.
Numerous serologic procedures available: Sabin-Feldman dye test,
indirect haemagglutination assays, IFA, direct agglutination tests, etc.
Serologic results may be complicated because of the high prevalence of
seropositive individuals. High titer is not evidence of an acute infection.
Best to collect two samples 2-4 weeks apart. If there is increase in the titer
in the second sample may indicate acute infection.
Of particular interest is determining acute infection in pregnant women,
due to the risk of congenital toxoplasmosis.
This is complicated by the fact that many
women have existing IgG and IgM
antibodies to T. gondii.
Fixed tachyzoites are recognized by the patient's antibody (IgG), and a
fluorescent-labeled anti-human antibody (IgG) is added next to identify the
antibody by fluorescent microscopy. If no antibody against the T gondii tachyzoite
is seen, no fluorescence is present. (Photomicrographs and IFA preparation by Parkland
Memorial Health and Hospital System Humoral Immunology Laboratory.)
CHEMOTHERAPY OF TOXOPLASMOSIS
• Inhibitors of dihydrofolate reductase
Pyrimethamine in combination with sulfonamides
• Sulfonamides
Sulfadiazine, sulfamethazine, sulfamerzaine.
• Clindamycin
• Macrolides and azalides
Roxithromycin, azythromycin, clarithromycin, spiramycin
• Tetracyclines
Doxycline, minocycline
• Hydroxynaphthoquinones
Atovaquone.
• Others
Eimycine, aprinocid, dapsone, quinghasou, pentamidine
•Combination therapy
pyrimethamine-dapsone, clarithromycin-minocycline,
azithromycin-pyrimethamine, azithromycin-sulfadiazine, etc
• Biological response modifiers
Cytokines (IFN- , IL-2, TNF, IL-1, IFN- ) in combination with chemotherapy
Pyrimethamine and sulfadoxine
Sulfadoxine: The sulfonamides are structural analogues of para-aminobenzoic acid
(PABA), and competitive inhibitors of dihydropteroate synthetase.
Pyrimethamine: An antifolate, a highly selective and powerful competitive inhibitor of
dihydrofolate reductase.
TOXOPLASMA
Folate metabolism
HUMAN
Pteridine + PABA
Dihydropteroate
synthetase
sulfadoxine
Dihydropteroic acid
GLUTAMATE
Dihydrofolate
NADPH
Dihydrofolate
reductase
pyrimethamine
Preformed dietary
folates
NADPH
Dihydrofolate
reductase
NADP
Tetrahydrofolate
NADP
Tetrahydrofolate
FAH4 cofactors
Thymidine
DNA
Purines
DNA
RNA
FAH4 cofactors
Methionine
Glycine
f-met-tRNA
Thymidine
PROTEINS
DNA
Purines
DNA
RNA
Methionine
Glycine
f-met-tRNA
PROTEINS
Parasites synthesize their own folic acid; unlike man, they cannot import preformed folic acid. Tetrahydrofolic acid
is an essential cofactor for the synthesis of nucleic acid precursors, and some amino acids.
THE TACHYZOITE
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Crescent shaped
2 by 6 µm
Asexual form
Multiplies by endodyogeny
It can infect phagocytic and nonphagocytic, nucleated cells.
Organelles: pellicle, apical rings,
polar rings, conoid, rhoptries,
micronemes, dense granules,
apicoplast , acidocalcisome,
micropore, subpellicular microtubules,
mitochondrion, endoplasmic reticulum,
Golgi complex, ribosomes, rough and
smooth endoplasmic reticulum,
nucleus.
M. Black and J. C. Boothroyd (2000).
The lytic cycle of Toxoplasma gondii.
Microbiology and Molecular Biology Reviews, 64 (3) p. 607
THE TACHYZOITE
Carey et al, (2004) PNAS 101: 7433
Am, amylopectin
granule; Co, conoid;
Dg, electron-dense
granule; Go, Golgi
complex; Mn,
microneme; No,
nucleolus, Nu,
nucleus; Pv,
parasitophorous
vacuole; Rh, rhoptry.
Dubey, et al (1998).
Clinical Microbiology
Reviews, 11: 267-299.
THE CYTOSKELETON OF TOXOPLASMA
The anterior end of the parasite
is enlarged in the box to the
right so as to illustrate the
preconoidal rings, the conoid,
the two apical microtubules,
and the polar ring from which
22 subpellicular microtubules
emanate. The IMC is located
just beneath the plasma
membrane from the anterior to
the posterior poles and is
interrupted only by the
micropore (MP) located in the
middle of the parasite body.
Micrograph by J. Boothroyd and D. Ferguson
.
Diagram of endodyogeny
As a parasite begins to divide, two
IMCs begin to develop in the
middle of the cell. As the IMC
extends from these structures, the
nucleus (N) and mitochondrion
(Mitoch.) divide into these
membranous outlines. Nascent
apical organelles (NO) develop
within the anterior poles as the
daughter cells grow. Eventually,
the entire cytoplasm is divided
between the daughters and the
IMC of the mother dissociates. A
cleavage furrow divides the cells
from the anterior pole. This
division continues down the length
of the cells until it reaches the
posterior end, where it can leave a
residual body connecting the two
daughters.
M. Black and J. C. Boothroyd (2000). The lytic
cycle of Toxoplasma gondii. Microbiology and
Molecular Biology Reviews, 64 (3) p. 607
Transmission electron micrograph of a tachyzoite undergoing
endodyogeny within a mouse peritoneal macrophage
Cd, conoid of developing
tachyzoite; Co, conoid of
mother tachyzoite; Dg, dense
granule; Ga, Golgi adjunct;
Hm, host cell mitochondrion;
Hn, host cell nucleus; Id, inner
membrane complex of
developing tachyzoite; Im, inner
membrane complex of mother
tachyzoite; Mi, mitochondrion;
Mn, microneme; Nu, nuclei of
daughter tachyzoites; Pl,
plasmalemma of mother
tachyzoite; Pm,
parasitophorous vacuolar
membrane; Pv,
parasitophorous vacuole; Rh,
rhoptries of daughter
tachyzoites.
Dubey, J.P., et al (1998). Structures of Toxoplasma gondii tachyzoites, bradyzoites, and sporozoites and
biology and development of Tissue Cysts. Clinical Microbiology Reviews, 11: 267-299.
BRADYZOITE
• Crescent-shaped cell
• 7 x 1.5 m
• Morphologicaly similar to tachyzoites
except that it has higher content of
micronemes and amylopectin granules
• Only stage that can initiate the sexual
phase. When they invade the feline
enterocytes they differentiate into microand macrogamete.
TISSUE CYSTS
• Intracellular and bradyzoites divide by
endodyogeny.
• Intact tissue cysts do not cause any
harm and do not induce an
inflammatory response.
• The intracellular location protects
them from the immune system.
• More abundant in myocardium,
striated muscle and CNS.
• Some bradyzoites may escape from
the cyst and form daughter cysts.
• The cysts are resistant to HCl and
digestive enzymes.
• Vary in size; young cysts as small as
5 µm in diameter with only two
bradyzoites, while older ones may
contain hundreds
TISSUE CYST
A highly stretched
tissue cyst with
more than 1,000
bradyzoites in
an impression
smear of brain
homogenate from a
rat 14 months after
infection with the
VEG strain of T.
gondii. The cyst
wall (arrow) is
barely visible.
Dubey, J.P., Lindsay, D.S. and Speer, C.A.
(1998). Structures of Toxoplasma gondii
tachyzoites, bradyzoites, and sporozoites and
biology and development of Tissue Cysts.
Clinical Microbiology Reviews, 11: 267-299.
THE OOCYST
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The oocyst is noninfectious before sporulation.
Unsporulated oocysts are subspherical to spherical and are 10 by 12
m in diameter.
Sporulated oocysts are subspherical to ellipsoidal and are 11 by 13 m
in diameter.
Each oocyst has two ellipsoidal sporocysts.
Sporocysts measure 6 by 8 m.
Each sporocyst contains four sporozoites.
Shedding occurs 3-5 days after
ingestion of tissue cysts or 20-34
days after ingestion of oocysts.
Toxoplasmosis prevention
• Avoid raw or undercooked meat. (Cooking kills Toxoplasma)
• Wash raw fruits and vegetables well before eating.
• Remove feces from the litter box every day, to eliminate any parasites before oocysts sporulate
• Keep cats indoors to prevent hunting. Cats can become infected by eating infected rodents or birds.
• Feed cats only commercial food or well-cooked meat.
• Do not feed cats raw or undercooked meat.
• Do not adopt cats who have lived outdoors or who have been fed raw meat.
• Do not handle stray cats.
• Wash hands well with soap and warm water after:
• Gardening
• Yard Work
• Any Other Outdoor Activity Involving Contact With Soil that could be
contaminated with cat feces
• There is no treatment available to prevent Toxoplasma in cats, or to
prevent an infected cat from shedding the parasite in its stool.
Source: U.S. Centers for Disease Control and Prevention