Infection case 12
I2 許師偉
報告日期：2005/10/31
General Data
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M/28
HIV-seropositive
Low CD4 count (50 cells/mm3) for
approximately 1 year
Chief Complant
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Diarrhea for 10 weeks and a recent
weight loss of 20 lb
Subjective Finding
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Watery diarrhea and profuse but
nonbloody
Antidiarrheal medications (e.g.,
loperamide HC1 [Imodium]) were
not successful in alleviating his
condition.
Objective Finding
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Signs of dehydration on examination
A stool specimen was collected for
routine culture for bacterial
pathogens.(Negative finding)
Three stool specimens, collected on
alternate days, were examined for
ova and parasites.(Positive finding)
Positive stool exam finding
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Pale oval structures
The permanent stained
trichrome smear
showed evidence of a
protozoan parasite
A modified acid-fast
procedure(a special
stain for coccidia )
revealed elliptical pink
structures measuring 25
to 30 μm. Each
structure was
surrounded by a clear,
double-layer wall.
QUESTIONS 1
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Which protozoan parasite do you
think is causing this patient's illness?
What are the structures seen in the
patient's stool specimen?
Ans：(1)Isospora belli
(2) An oocyst of Isospora belli
showing 1 sporocyst
Adapt from UKNEQAS Parasitology

1.
2.
3.
4.
Routine procedure for the microscopic examination of
faecal samples for parasites
Direct microscopy should be done on all unformed and liquid
samples by mixing a small amount of the specimen in 0.9%
sodium chloride solution. This permits detection of trophozoites of
Entamoeba histolytica and Giardia lamblia. It can also provide
information on the content of the stool ie the presence of
leucocytes and red blood cells.
A formol-ether concentrate should be done on all faecal samples
examined for parasites. This reveals the presence of most
protozoan cysts, eggs of nematodes, cestodes and trematodes
and also the larval stages of some nematodes.
A permanently stained direct faecal smear should be used for all
bloody, liquid or semi-formed stools. The smear can reveal the
presence of intestinal parasites which can be either destroyed or
missed by the formol-ether concentration method eg.
Dientamoeba fragilis.
Specimens from patients with HIV should be left in 10% formalin
for hour before proceeding with parasite examination.
Adapt from UKNEQAS Parasitology
Faecal smears are made for the following reasons:
Provide information on the exudate present.
(Romanowsky stains)
Helpful in accurately identifying flagellates.
(Romanowsky stains, Iron haematoxylin)
When parasites cannot be detected in either the
direct wet preparation or concentrated deposit, a
permanent stain of a fresh faecal smear can
reveal the presence of intestinal parasites.
(Romanowsky stains, Trichrome stain, modified
Ziehl-Neelsen)
Useful in demonstrating the nuclear patterns of
cysts thus facilitating identification. (Iron
haematoxylin, Trichrome stain)

Adapt from UKNEQAS Parasitology
Trichrome Stain
The trichrome method for staining
protozoa is especially recommended
for identifying features of amoebic
cysts and trophozoites
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Adapt from UKNEQAS Parasitology
Modified Ziehl-Neelsen
Use of the modified Ziehl-Neelsen stain
for faecal smears has already been
established for coccidian protozoa, in
particular, oocysts of Cryptosporidium
species, but it is also useful to
confirm the presence of oocysts of
Isospora belli and Cyclospora
cayetanensis.
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J Antimicrob Chemother. 1996 May;37 Suppl B:61-70.
Recently recognised microbial enteropathies and HIV
infection.
Farthing MJ, Kelly MP, Veitch AM
Digestive Diseases Research Centre, Medical College of St
Bartholomew's Hospital, London, UK.
At least 80% of cases of persistent diarrhoea in patients
with HIV/AIDS can be attributed to a specific
enteropathogen. The coccidian parasites Cryptosporidium
parvum, Isospora belli and Cyclospora and the
Microsporidia account for at least 50% of cases of
persistent diarrhoea in the industrialised and developing
world
Adapt from UKNEQAS Parasitology

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Oocysts are thin walled,
transparent and ovoid in shape.
They can be demonstrated in
faeces after formal ether
concentration where they appear
as translucent, oval structures
measuring 20-33μm by 10-19μm.
Alternatively, oocysts can be seen
in a faecal smear stained by a
modified Ziehl-Neelsen method ,
where they stain a granular red
colour against a green
background, or by phenolauramine.
QUESTIONS 2

Why was the modified acid-fast
procedure needed to definitively
identify the parasite?
Adapt from UKNEQAS Parasitology

Modified Ziehl-Neelsen
Use of the modified Ziehl-Neelsen stain for faecal smears has already been
established for coccidian protozoa, in particular, oocysts of Cryptosporidium
species, but it is also useful to confirm the presence of oocysts of Isospora
belli and Cyclospora cayetanensis.
Method
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a. Faecal smears are made either directly from the stool
sample or from the concentration deposit.
b. Allow to air dry.
c. Fix in methanol for 3 minutes.
d. Stain with strong carbol fuchsin for 15-20 minutes.
e. Rinse thoroughly in tap water.
f. Decolourise in acid alcohol (1% HCl in methanol) for 1520 seconds.
g. Rinse thoroughly in tap water.
h. Counterstain with 0.4% malachite green (or methylene
blue) for 30-60 seconds.
i. Rinse thoroughly and air dry.
j. Examine using x40 and x100 objectives.
QUESTIONS 3
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Which other two coccidian parasites
give a similar reaction when stained
by the modified acid-fast procedure?
Adapt from UKNEQAS Parasitology
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Cryptosporidium parvum
Cyclospora cayetanensis
Adapt from UKNEQAS Parasitology

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Cryptosporidium
parvum
Definitive diagnosis of
cryptosporidiosis is by
finding the characteristic
spherical oocysts in faecal
samples.
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Sporulated oocysts measuring
4-6 m in diameter and
containing up to 4 sporozoites
are passed into the faeces.
Adapt from UKNEQAS Parasitology
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Cyclospora
cayetanensis
The oocysts of C.
cayetanensis are
spherical, measuring 810 in diameter
QUESTIONS 4
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How would you distinguish these
three parasites?
Adapt from UKNEQAS Parasitology
Microscopic
Characterists
cs
Cryptosporidiu
m parvum
Isospora
belli
Cyclospora
cayetanensi
s
Size
4-6
20-33 &
10 - 19
8 - 10
Identified in
formol-ether
concentrate by
light microscopy
No
Yes
Yes
Identified by
modified Ziehl-
Yes
Yes
Yes
Shape of the
oocyst
spherical
oval
spherical
Identified by
Phenol Auramine
Yes
Variable
No
Neelsen
stain
QUESTIONS 5

Describe the life cycle of the parasite
causing this patient's infection
Adapt from UKNEQAS Parasitology

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The life cycle of Isospora belli involves an asexual
(schizogonic stage) and a sexual (sporogonic stage)
Infection with I. belli occurs in both immunocompetent and
immunocompromised patients and begins when the mature
oocyst is ingested in water or food.The mature oocyst
contains 2 sporocysts, each containing 4 sporozoites. The
sporulated oocyst is the infective stage of the parasite and
they excyst in the small intestine releasing sporozoites
which penetrate the epithelial cells, thus initiating the
asexual stage of the lifecycle. The sporozoite develops in
the epithelial cell to form a schizont which ruptures the
epithelial cell containing it, liberating merozoites into the
lumen. These merozoites will then infect new epithelial
cells and the process of asexual reproduction in the
intestine proceeds. Some of the merozoites form
macrogametes and microgametes (sexual stages) which
fuse to form a zygote which matures to form an oocyst.
QUESTIONS 6

Why would this patient pose less
risk to laboratory personnel than
patients infected with other
coccidian parasites?
Cryptosporidium parvum
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Thick wall sporulated oocysts, containing
4 sporozoites, are excreted by the infected
host through feces and possibly other
routes such as respiratory secretions .
Transmission of Cryptosporidium parvum
occurs mainly through contact with
contaminated water (e.g., drinking or
recreational water). Occasionally food
sources, such as chicken salad, may serve
as vehicles for transmission.
Cyclospora cayetanensis
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When freshly passed in stools, the oocyst is not
infective (thus, direct fecal-oral transmission
cannot occur; this differentiates Cyclospora from
another important coccidian parasite, Cryptosporidium).
In the environment , sporulation occurs after
days or weeks at temperatures between 22°C to
32°C, resulting in division of the sporont into two
sporocysts, each containing two elongate
sporozoites. Fresh produce and water can serve
as vehicles for transmission and the sporulated
oocysts are ingested (in contaminated food or
water) .
Isospora belli
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At time of excretion, the immature oocyst
contains usually one sporoblast (more
rarely two) . In further maturation after
excretion, the sporoblast divides in two
(the oocyst now contains two sporoblasts);
the sporoblasts secrete a cyst wall, thus
becoming sporocysts; and the sporocysts
divide twice to produce four sporozoites
each . Infection occurs by ingestion of
sporocysts-containing oocysts
QUESTIONS 7
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How is infection with this parasite
transmitted?
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Infection is a result of the ingestion
of a infective of sporulated oocyst.
QUESTIONS 8
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How should this patient be treated?
NEJM Volume 320:1044-1047 April 20, 1989 Number 16
Treatment and prophylaxis of Isospora belli infection in patients with the acquired immunodeficiency
syndrome
JW Pape, RI Verdier, and WD Johnson
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I. belli responds well to treatment with
trimethoprim-sulfamethoxazole, but there
is a high rate of recurrence. We conclude
that isosporiasis in patients with AIDS can
be treated effectively with a 10-day course
of trimethoprim(160 mg) sulfamethoxazole(800 mg) four times a
day , and that recurrent disease can
subsequently be prevented by ongoing
prophylaxis with either trimethoprimsulfamethoxazole or sulfadoxinepyrimethamine.