Infection case 12Infection case 12

I2 I2 許師偉許師偉報告日期：報告日期： 2005/10/312005/10/31

General DataGeneral Data

M/28M/28 HIV-seropositive HIV-seropositive Low CD4 count (50 cells/mm3) for Low CD4 count (50 cells/mm3) for

approximately 1 year approximately 1 year

Chief ComplantChief Complant

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

Subjective FindingSubjective Finding

Watery diarrhea and profuse but Watery diarrhea and profuse but nonbloody nonbloody

Antidiarrheal medications (e.g., Antidiarrheal medications (e.g., loperamide HC1 [Imodium]) were not loperamide HC1 [Imodium]) were not successful in alleviating his condition. successful in alleviating his condition.

Objective Finding Objective Finding

Signs of dehydration on examinationSigns of dehydration on examination A stool specimen was collected for A stool specimen was collected for

routine culture for bacterial routine culture for bacterial pathogens.(pathogens.(Negative findingNegative finding))

Three stool specimens, collected on Three stool specimens, collected on alternate days, were examined for alternate days, were examined for ova and parasites.(ova and parasites.(Positive findingPositive finding) )

Positive stool exam findingPositive stool exam finding Pale oval structuresPale oval structures The permanent stained The permanent stained

trichrome smear showed trichrome smear showed evidence of a evidence of a protozoan protozoan parasiteparasite

A modified acid-fast A modified acid-fast procedure(procedure(a special a special stain for coccidiastain for coccidia ) ) revealed elliptical pink revealed elliptical pink structures measuring 25 structures measuring 25 to 30 μm. Each structure to 30 μm. Each structure was surrounded by a was surrounded by a clear, double-layer wall. clear, double-layer wall.

QUESTIONS 1QUESTIONS 1

Which protozoan parasite do you Which protozoan parasite do you think is causing this patient's think is causing this patient's illness? What are the structures illness? What are the structures seen in the patient's stool seen in the patient's stool specimen?specimen?

AnsAns ：： (1)(1)Isospora belliIsospora belli  

(2) An oocyst of (2) An oocyst of Isospora belliIsospora belli

showing 1 sporocystshowing 1 sporocyst

Adapt from UKNEQAS ParasitologyAdapt from UKNEQAS Parasitology

Routine procedure for the microscopic examination of Routine procedure for the microscopic examination of faecal samples for parasitesfaecal samples for parasites

1. 1. Direct microscopyDirect microscopy should be done on all unformed and liquid should be done on all unformed and liquid samples by mixing a small amount of the specimen in 0.9% samples by mixing a small amount of the specimen in 0.9% sodium chloride solution. This permits detection of sodium chloride solution. This permits detection of trophozoitestrophozoites of of Entamoeba histolyticaEntamoeba histolytica and and Giardia lamblia Giardia lamblia. It can also provide . It can also provide information on the content of the stool ie the presence of information on the content of the stool ie the presence of leucocytes and red blood cellsleucocytes and red blood cells. .

2. 2. A formol-ether concentrateA formol-ether concentrate should be done on all faecal samples should be done on all faecal samples examined for examined for parasitesparasites. This reveals the presence of most . This reveals the presence of most protozoan cysts, eggs of nematodes, cestodes and trematodes protozoan cysts, eggs of nematodes, cestodes and trematodes and also the larval stages of some nematodes. and also the larval stages of some nematodes.

3. 3. A permanently stained direct faecal smear should be used for all A permanently stained direct faecal smear should be used for all bloody, liquid or semi-formed stools. The smear can reveal the bloody, liquid or semi-formed stools. The smear can reveal the presence of intestinal parasites which can be either destroyed or presence of intestinal parasites which can be either destroyed or missed by the formol-ether concentration methodmissed by the formol-ether concentration method eg. eg. Dientamoeba fragilisDientamoeba fragilis. .

4. Specimens from patients with HIV should be left in 10% formalin 4. Specimens from patients with HIV should be left in 10% formalin for hour before proceeding with parasite examination. for hour before proceeding with parasite examination.

Adapt from UKNEQAS ParasitologyAdapt from UKNEQAS Parasitology

Faecal smears are made for the following reasonsFaecal smears are made for the following reasons: : Provide information on the exudate present. Provide information on the exudate present.

(Romanowsky stains) (Romanowsky stains) Helpful in accurately identifying flagellates. Helpful in accurately identifying flagellates.

(Romanowsky stains, Iron haematoxylin) (Romanowsky stains, Iron haematoxylin) When parasites cannot be detected in either the When parasites cannot be detected in either the

direct wet preparation or concentrated deposit, a direct wet preparation or concentrated deposit, a permanent permanent stainstain of a fresh faecal smear can of a fresh faecal smear can reveal the presence of intestinal parasites. reveal the presence of intestinal parasites. (Romanowsky stains, (Romanowsky stains, Trichrome Trichrome stainstain, , modified modified ZiehlZiehl--NeelsenNeelsen) )

Useful in demonstrating the nuclear patterns of Useful in demonstrating the nuclear patterns of cysts thus facilitating identification. (Iron cysts thus facilitating identification. (Iron haematoxylin, haematoxylin, Trichrome Trichrome stainstain) )

Adapt from UKNEQAS ParasitologyAdapt from UKNEQAS Parasitology

Trichrome StainTrichrome Stain

The trichrome method for staining The trichrome method for staining protozoa is especially recommended protozoa is especially recommended for identifying features of amoebic for identifying features of amoebic cysts and trophozoitescysts and trophozoites

Adapt from UKNEQAS ParasitologyAdapt from UKNEQAS Parasitology

Modified Ziehl-NeelsenModified Ziehl-Neelsen

Use of the modified Ziehl-Neelsen stain Use of the modified Ziehl-Neelsen stain for faecal smears has already been for faecal smears has already been established for coccidian protozoa, in established for coccidian protozoa, in particular, particular, oocysts of oocysts of CryptosporidiumCryptosporidium species species, but it is also , but it is also useful to confirm the presence of useful to confirm the presence of oocysts of oocysts of IsosporaIsospora bellibelli and and CyclosporaCyclospora cayetanensiscayetanensis..

J Antimicrob Chemother. 1996 May;37 Suppl B:61-70. J Antimicrob Chemother. 1996 May;37 Suppl B:61-70. Recently recognised microbial enteropathies and HIV Recently recognised microbial enteropathies and HIV

infection.infection.FFarthing MJarthing MJ, , Kelly MPKelly MP, , VeitchVeitch AM AM

Digestive Diseases Research Centre, Medical College of St Digestive Diseases Research Centre, Medical College of St Bartholomew's Hospital, London, UK.Bartholomew's Hospital, London, UK.

At least 80% of cases of persistent diarrhoea in patients At least 80% of cases of persistent diarrhoea in patients with HIV/AIDS can be attributed to a specific with HIV/AIDS can be attributed to a specific enteropathogen. enteropathogen. The coccidian parasitesThe coccidian parasites Cryptosporidium Cryptosporidium parvum, Isospora belli and Cyclospora and the parvum, Isospora belli and Cyclospora and the Microsporidia account for at least 50% of cases of Microsporidia account for at least 50% of cases of persistent diarrhoeapersistent diarrhoea in the industrialised and developing in the industrialised and developing worldworld

Adapt from UKNEQAS ParasitologyAdapt from UKNEQAS Parasitology

Oocysts are thin walled, Oocysts are thin walled, transparent and ovoid in shape. transparent and ovoid in shape. They can be demonstrated in They can be demonstrated in faeces after formal ether faeces after formal ether concentration where they appear concentration where they appear as translucent, as translucent, oval structuresoval structures measuring measuring 20-3320-33μμm by 10-19m by 10-19μμmm. .

Alternatively, oocysts can be seen Alternatively, oocysts can be seen in a faecal smear stained by a in a faecal smear stained by a modified Ziehl-Neelsen method , modified Ziehl-Neelsen method , where they  stain a granular red where they  stain a granular red colour against a green colour against a green background, or by phenol-background, or by phenol-auramine.auramine.

QUESTIONS 2QUESTIONS 2

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

Adapt from UKNEQAS ParasitologyAdapt from UKNEQAS Parasitology Modified Ziehl-NeelsenModified Ziehl-Neelsen Use of the modified Ziehl-Neelsen stain for faecal smears has already been Use of the modified Ziehl-Neelsen stain for faecal smears has already been

established for coccidian protozoa, in particular, oocysts of established for coccidian protozoa, in particular, oocysts of CryptosporidiumCryptosporidium species, but it is also useful to confirm the presence of species, but it is also useful to confirm the presence of oocysts of oocysts of IsosporaIsospora bellibelli and and CyclosporaCyclospora cayetanensiscayetanensis. .

Method Method a. Faecal smears are made either directly from the stool a. Faecal smears are made either directly from the stool

sample or from the concentration deposit. sample or from the concentration deposit. b. Allow to air dry. b. Allow to air dry. c. Fix in methanol for 3 minutes. c. Fix in methanol for 3 minutes. d. Stain with strong carbol fuchsin for 15-20 minutes. d. Stain with strong carbol fuchsin for 15-20 minutes. e. Rinse thoroughly in tap water. e. Rinse thoroughly in tap water. f. Decolourise in acid alcohol (1% HCl in methanol) for 15-f. Decolourise in acid alcohol (1% HCl in methanol) for 15-

20 seconds. 20 seconds. g. Rinse thoroughly in tap water. g. Rinse thoroughly in tap water. h. Counterstain with 0.4% malachite green (or methylene h. Counterstain with 0.4% malachite green (or methylene

blue) for 30-60 seconds. blue) for 30-60 seconds. i. Rinse thoroughly and air dry. i. Rinse thoroughly and air dry. j. Examine using x40 and x100 objectives.j. Examine using x40 and x100 objectives.

QUESTIONS 3QUESTIONS 3

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

Adapt from UKNEQAS ParasitologyAdapt from UKNEQAS Parasitology

Cryptosporidium parvumCryptosporidium parvum Cyclospora cayetanensisCyclospora cayetanensis

Adapt from UKNEQAS ParasitologyAdapt from UKNEQAS Parasitology

Cryptosporidium Cryptosporidium parvumparvum

Definitive diagnosis of Definitive diagnosis of cryptosporidiosis is by cryptosporidiosis is by finding the characteristic finding the characteristic spherical oocystsspherical oocysts in faecal in faecal

samples.samples.   Sporulated oocysts measuring Sporulated oocysts measuring

4-6 m in diameter and 4-6 m in diameter and containing up to 4 sporozoites containing up to 4 sporozoites

are passed into the faeces.are passed into the faeces.

Adapt from UKNEQAS ParasitologyAdapt from UKNEQAS Parasitology

Cyclospora Cyclospora cayetanensiscayetanensis

The oocysts of C. The oocysts of C. cayetanensis are cayetanensis are sphericalspherical, measuring 8-, measuring 8-

10 in diameter10 in diameter

QUESTIONS 4QUESTIONS 4

How would you distinguish these How would you distinguish these three parasites?three parasites?

Adapt from UKNEQAS ParasitologyAdapt from UKNEQAS ParasitologyMicroscopic Microscopic CharacteristsCharacteristscscs

CryptosporidiuCryptosporidium parvumm parvum

Isospora Isospora bellibelli

Cyclospora Cyclospora cayetanenscayetanensisis

SizeSize 4 - 64 - 6 20-33 & 20-33 & 10 - 1910 - 19

8 - 108 - 10

Identified in Identified in formol-ether formol-ether concentrate by concentrate by light microscopylight microscopy

NoNo YesYes YesYes

Identified by Identified by modified Ziehl-modified Ziehl-

NeelsenNeelsen YesYes YesYes YesYes

Shape of the Shape of the oocystoocyst

sphericalspherical ovaloval sphericalspherical

Identified by Identified by Phenol Auramine Phenol Auramine

stainstain YesYes VariableVariable NoNo

QUESTIONS 5QUESTIONS 5

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

Adapt from UKNEQAS ParasitologyAdapt from UKNEQAS Parasitology

The life cycle of The life cycle of Isospora belliIsospora belli involves an asexual involves an asexual (schizogonic stage) and a sexual (sporogonic stage) (schizogonic stage) and a sexual (sporogonic stage)

Infection with Infection with I. belliI. belli occurs in both immunocompetent and occurs in both immunocompetent and immunocompromised patients and begins when the mature immunocompromised patients and begins when the mature oocyst is ingested in water or food.The mature oocyst oocyst is ingested in water or food.The mature oocyst contains 2 sporocysts, each containing 4 sporozoites.  The contains 2 sporocysts, each containing 4 sporozoites.  The sporulated oocystsporulated oocyst is the infective stage of the parasite and is the infective stage of the parasite and they excyst in the small intestine releasing sporozoites they excyst in the small intestine releasing sporozoites which penetrate the epithelial cells, thus initiating the which penetrate the epithelial cells, thus initiating the asexual stage of the lifecycle.  The sporozoite develops in asexual stage of the lifecycle.  The sporozoite develops in the epithelial cell to form a schizont which ruptures the the epithelial cell to form a schizont which ruptures the epithelial cell containing it, liberating merozoites into the epithelial cell containing it, liberating merozoites into the lumen.  These merozoites will then infect new epithelial lumen.  These merozoites will then infect new epithelial cells and the process of asexual reproduction in the cells and the process of asexual reproduction in the intestine proceeds.  Some of the merozoites form intestine proceeds.  Some of the merozoites form macrogametes and microgametes (sexual stages) which macrogametes and microgametes (sexual stages) which fuse to form a zygote which matures to form an oocyst. fuse to form a zygote which matures to form an oocyst.

QUESTIONS 6QUESTIONS 6

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

Cryptosporidium parvumCryptosporidium parvum

Thick wallThick wall sporulated oocysts, containing sporulated oocysts, containing 4 sporozoites, are excreted by the infected 4 sporozoites, are excreted by the infected host through feces and possibly other host through feces and possibly other routes such as routes such as respiratory secretionsrespiratory secretions . . Transmission of Transmission of Cryptosporidium parvumCryptosporidium parvum occurs mainly through contact with occurs mainly through contact with contaminated water (e.g., drinking or contaminated water (e.g., drinking or recreational water).  Occasionally food recreational water).  Occasionally food sources, such as chicken salad, may serve sources, such as chicken salad, may serve as vehicles for transmission.as vehicles for transmission.

Cyclospora cayetanensisCyclospora cayetanensis When freshly passed in stools, the oocyst is When freshly passed in stools, the oocyst is not not

infectiveinfective (thus, (thus, direct fecal-oral transmission direct fecal-oral transmission cannot occurcannot occur; this differentiates; this differentiates Cyclospora Cyclospora from from another important coccidian parasite, another important coccidian parasite, CryptosporidiumCryptosporidium). In the environment , sporulation ). In the environment , sporulation occurs after days or weeks at temperatures occurs after days or weeks at temperatures between 22°C to 32°C, resulting in division of the between 22°C to 32°C, resulting in division of the sporont into two sporocysts, each containing two sporont into two sporocysts, each containing two elongate sporozoites. Fresh produce and water elongate sporozoites. Fresh produce and water can serve as vehicles for transmission and the can serve as vehicles for transmission and the sporulated oocysts are ingested (in contaminated sporulated oocysts are ingested (in contaminated food or water) . food or water) .

Isospora belliIsospora belli

At time of excretion, the immature oocyst At time of excretion, the immature oocyst contains usually one sporoblast (more contains usually one sporoblast (more rarely two) .  In further maturation after rarely two) .  In further maturation after excretion, the sporoblast divides in two excretion, the sporoblast divides in two (the oocyst now contains two sporoblasts); (the oocyst now contains two sporoblasts); the sporoblasts secrete a cyst wall, thus the sporoblasts secrete a cyst wall, thus becoming sporocysts; and the sporocysts becoming sporocysts; and the sporocysts divide twice to produce four sporozoites divide twice to produce four sporozoites each .  each .  Infection occurs by ingestion of Infection occurs by ingestion of sporocysts-containing oocystssporocysts-containing oocysts

QUESTIONS 7QUESTIONS 7

How is infection with this parasite How is infection with this parasite transmitted?transmitted?

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

QUESTIONS 8QUESTIONS 8

How should this patient be treated?How should this patient be treated?

NEJM Volume 320:1044-1047NEJM Volume 320:1044-1047 April 20, 1989April 20, 1989 Number 16Number 16Treatment and prophylaxis of Isospora belli infection in patients with the acquired immunodeficiency Treatment and prophylaxis of Isospora belli infection in patients with the acquired immunodeficiency

syndrome syndrome

JW Pape, RI Verdier, and WD JohnsonJW Pape, RI Verdier, and WD Johnson I. belli responds well to treatment with I. belli responds well to treatment with

trimethoprim-sulfamethoxazole, but there trimethoprim-sulfamethoxazole, but there is a high rate of recurrence.is a high rate of recurrence. We conclude We conclude that isosporiasis in patients with AIDS can that isosporiasis in patients with AIDS can be treated effectively with a 10-day course be treated effectively with a 10-day course of trimethoprim(160 mg) -of trimethoprim(160 mg) -sulfamethoxazole(800 mg) four times a sulfamethoxazole(800 mg) four times a day , and that recurrent disease can day , and that recurrent disease can subsequently be prevented by ongoing subsequently be prevented by ongoing prophylaxis with either trimethoprim-prophylaxis with either trimethoprim-sulfamethoxazole or sulfadoxine-sulfamethoxazole or sulfadoxine-pyrimethamine. pyrimethamine.