Infection case 12

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Infection case 12

• I2 ???
• ????2005/10/31

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General Data

• M/28
• HIV-seropositive
• Low CD4 count (50 cells/mm3) for approximately 1
  year

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Chief Complant

• Diarrhea for 10 weeks and a recent weight loss of
  20 lb

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Subjective Finding

• Watery diarrhea and profuse but nonbloody
• Antidiarrheal medications (e.g., loperamide HC1
  Imodium) were not successful in alleviating his
  condition.

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Objective Finding

• 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)

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Positive stool exam finding

• 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.

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QUESTIONS 1

• 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

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Adapt from UKNEQAS Parasitology

• Routine procedure for the microscopic examination
  of faecal samples for parasites
• 1. 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.
• 2. 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.
• 3. 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.
• 4. Specimens from patients with HIV should be
  left in 10 formalin for hour before proceeding
  with parasite examination.

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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)

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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 May37 Suppl
  B61-70.
• 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

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Adapt from UKNEQAS Parasitology

• 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 phenol-auramine.

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QUESTIONS 2

• Why was the modified acid-fast procedure needed
  to definitively identify the parasite?

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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.
• Method
• 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 15-20 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.

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QUESTIONS 3

• Which other two coccidian parasites give a
  similar reaction when stained by the modified
  acid-fast procedure?

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Adapt from UKNEQAS Parasitology

• Cryptosporidium parvum
• Cyclospora cayetanensis

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Adapt from UKNEQAS Parasitology

• Cryptosporidium parvum
• Definitive diagnosis of cryptosporidiosis is by
  finding the characteristic spherical oocysts in
  faecal samples. 
• Sporulated oocysts measuring 4-6 m in diameter
  and containing up to 4 sporozoites are passed
  into the faeces.

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Adapt from UKNEQAS Parasitology

• Cyclospora cayetanensis
• The oocysts of C. cayetanensis are spherical,
  measuring 8-10 in diameter

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QUESTIONS 4

• How would you distinguish these three parasites?

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Adapt from UKNEQAS Parasitology
Microscopic Characteristscs Cryptosporidium parvum Isospora belli Cyclospora cayetanensis
Size 4 - 6 20-33 10 - 19 8 - 10
Identified in formol-ether concentrate by light microscopy No Yes Yes
Identified by modified Ziehl-Neelsen Yes Yes Yes
Shape of the oocyst spherical oval spherical
Identified by Phenol Auramine stain Yes Variable No
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QUESTIONS 5

• Describe the life cycle of the parasite causing
  this patient's infection

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Adapt from UKNEQAS Parasitology

• 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.

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QUESTIONS 6

• Why would this patient pose less risk to
  laboratory personnel than patients infected with
  other coccidian parasites?

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Cryptosporidium parvum

• 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.

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Cyclospora cayetanensis

• 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 22C to 32C, 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) .

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Isospora belli

• 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

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QUESTIONS 7

• How is infection with this parasite transmitted?

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• Infection is a result of the ingestion of a
  infective of sporulated oocyst.

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QUESTIONS 8

• How should this patient be treated?

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NEJM Volume 3201044-1047 April 20, 1989
Number 16Treatment and prophylaxis of Isospora
belli infection in patients with the acquired
immunodeficiency syndrome JW Pape, RI Verdier,
and WD Johnson

• 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 trimethoprim-sulfamethoxaz
  ole or sulfadoxine-pyrimethamine.