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Infection disease
I2 洪俊斌
CASE11
A 32-year-old woman with pain in her right eye was seen
by her ophthalmologist, who diagnosed a corneal ulcer in
the affected eye. Her physical examination was
unremarkable otherwise; she had no history of eye
problems and had recently been examined by her primarycare physician, who declared her to be in good health.
Bacterial cultures taken from a discharge from her eye
were negative. Viral cultures were also negative. Specimens
were collected for culture for free-living amebae. No amebic
parasite was recovered in culture. Figure 11.1 shows the
parasite in a smear stained by the chromotrope Gram stain
method. The Warthin-Starry silver stain was used to
confirm the diagnosis.
Picture
Chromotrope Gram stain method
In this method, the samples are stained
in heated (50 degrees C to 55 degrees C)
solutions of crystal violet and iodine used
in Gram's stain, followed by a modified
chromotrope solution (heated to 50
degrees C to 55 degrees C). The modified
stain is composed of chromotrope 2R
(1%), fast green (0.15%), and
phosphotungstic acid (0.25%).
Gram-chromotrope" staining technique
can be used to detect microsporidian
spores in clinical specimens(stool).
How to think
Right eye corneal ulcer
Immunocompetent patient
Chromotrope Gram stain(+)
The Warthin-Starry silver stain(+)
In microscopic view: Many spores
like materials was seen in this
specien: blue or black
Microsporidian spores was highly
suspected
QUESTIONS
Which group of obligate intracellular
protozoan parasites fits the
description given and might be
causing this infection in this
immunocompetent woman?
ANS
Microsporidia are obligately intracellular,
spore-forming parasites belonging to the
phylum Microspora and the order
Microsporida. They are eukaryotic
organisms containing 70S ribosomes but
lacking mitochondria, peroxisomes, Golgi
membranes, and other typically eukaryotic
organelles. The phylum Microspora
contains over 1000 species. The host
range is extensive and includes honeybees,
fish, mosquitoes, fleas, grasshoppers,
rodents, rabbits, and other fur-bearing
mammals.
QUESTIONS
List several members of this group
that may be causing this patient's
infection
ANS
seven genera
Nosema corneae
Vittaforma ocularum
Enterocytozoon
Encephalitozoon
Pleistophora
Trachipleistophora
Brachiola
QUESTIONS
Which laboratory procedure may be
used to identify the parasite to
genus level?
ANS
Polymerase chain reaction is
available in some research
laboratories and can be used to
diagnose the following 3
Encephalitozoon species: (1) E
bieneusi, (2) V corneae, and (3)
Nosema species
QUESTIONS
What are the structures seen in the
smear?
ANS
The spore wall should stain
pinkish to red, with the
interior of the spore being
clear or perhaps showing a
horizontal or diagonal
stripe, which represents
the polar tube. The
background will appear
green or blue, depending
on the method
Arrow shows coiled polar
filament.
Silver stain
Nosema connori
QUESTIONS
Which other laboratory
procedures are available to
diagnose infections with this
group of protozoans?
ANS
Body fluid specimens
– Microscopic examination of stained stool samples
– Examine stools for other parasites (ova and parasite
exam) and bacteria.
– The modified trichrome stain (chromotrope 2R)
commonly is used to detect microsporidia in urine, stool,
or mucus.
– The rapid Gram chromotrope method can be performed
more quickly (about 11 min) and combines the
chromotrope method with a Gram-staining step. The
spores stain dark violet, and the equatorial stripe is
enhanced.
– Cytologic and histologic examinations are quite useful in
diagnosis. A conjunctival scraping or swab frequently
can reveal the organism after a Gram stain (organisms
usually stain gram-positive) or chromotrope stain.
– Stains that are used to detect microsporidia
include the Brown Brenn Gram stain, WarthinStarry silver stain, Giemsa, and trichrome blue.
– Fluorochrome stains, including calcofluor white
and uvitex 2B, have a high affinity for chitin.
– Microsporidia stain poorly with hematoxylineosin.
Immunofluorescence assays using monoclonal or
polyclonal antibodies are available in some
research laboratories and can be performed on
most specimens,
QUESTIONS
Describe the pathogenesis of this
infection
Humans acquire infection through ingestion or
inhalation of spores. Studies isolating
Encephalitozoon species in the urinary tract in
those with disseminated infections suggest that
sexual transmission is possible. The spore is the
infective form. It is environmentally resistant and
can survive for several months. The spore
extrudes its polar tubule and injects the infective
sporoplasm into the host cell. Once inside the cell,
it multiplies by binary fission or schizogony.
Development can occur directly inside the host
cell cytoplasm or inside parasitophorous vacuoles.
As mature spores develop and accumulate, the
cell expands and eventually ruptures, releasing
the spores.
QUESTIONS
How does this infection differ in
immunocompromised (AIDS)
patients?
ANS
– Immunocompetent patients with ocular involvement due
to Encephalitozoon species can have conjunctival
hyperemia. A slit lamp examination can reveal
keratoconjunctivitis, which is characterized by diffuse,
superficial, punctate keratopathy. Infection frequently is
bilateral.
– Corneal ulceration is rare in patients with AIDS. Retinal
involvement is not reported. But chronic diarrhea and
wasting can be found in some immunocompromised
patients.
– The symptom of corneal ulcer in immunocompromised
patients is mild and the destruction of corneal is less
serious than immunocompetent pateints due the low
response of immunity,so the ocular symptoms are
always neglected in some HIV infected patients.
Thanks for your attention !!