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 Introduction
Clinical association and diagnosis
Identification of parasites
Protozoa in wet mounts
References
Appendices
Introduction
Opportunistic parasitic
infections can cause severe morbidity and mortality. Since many of these
infections are treatable, an early and accurate diagnosis is important. This can
be accomplished by a variety of methods such as direct demonstration of the
parasite and by serological tests to detect antigen and/or specific
antibodies. Adherence to conventional procedure may not be appropriate in
patients with AIDS. For example, antibody response may be poor in these
patients and therefore immunodiagnostic tests have to be interpreted with
caution.
Pneumocystis carinii,
Cryptosporidium, Toxoplasma gondii, Microsporidia and Strongyloides
stercoralis are commonly detected parasites. Less common parasites are
Acanthamoeba, Cyclospora and Isospora belli. Detection of these parasites
will help in proper management and treatment of these patients since drugs
are available for most of these opportunistic infections.
Emphasis will be made on the
importance of these parasites and the methods of their diagnosis in HIV/AIDS
patients. 
Clinical
association and diagnosis
Pneumocystis carinii, a ubiquitous
extracellular protozoan is a common pulmonary pathogen in immunocompromised
hosts and is one of the AIDS defining events as part of the CDC’s case
definition of AIDS. In such subjects, the disease manifests as interstitial
pneumonia. Laboratory diagnosis is made by demonstrating cysts or
trophozoites by Giemsa, Toluidine blue or Methenamine silver nitrate stain in
lung biopsy, hypopharyngeal washings, transbronchial aspirations or
transthoracal needle aspiration. Circulating antigen demonstration by ELISA
or PCR is also confirmatory. For these tests, the samples will have to be
sent to the reference laboratory.
Cryptosporidiosis, a disease
commonly caused by Cryptosporidium parvum causes severe chronic and even
fatal diarrhoea with malabsorption and dehydration. Diarrhoea is watery
without blood and mucus, may be scanty or continuous as much as 12-17 litres
per day. Extraintestinal infections in liver and lungs are known in AIDS
patients. Laboratory diagnosis is made by demonstrating oocysts in the specimens
using modified acid fast stain or safranin methylene blue technique.
Toxoplasmosis, a disease caused
by an ubiquitous intracellular protozoan Toxoplasma
gondii, is a world wide zoonosis. The cat is the definitive host for the
sexual stage and produces oocysts that are eliminated in the stool. All other
infected animals and humans are intermediate hosts. In an immunocompromised
host the disease presents as toxoplasmic encephalitis. Diagnosis is made by
CT scan showing characteristic cerebral hypodense lesions with
ring-enhancement after IV contrast. For definitive diagnosis, brain biopsy is
done to demonstrate the organism. Toxoplasma antigen demonstration by ELISA
or PCR from blood or CSF is possible for which facilities may be available in
reference laboratories or centres of excellence and samples need to be sent
to these laboratories.
Microsporidiosis, a disease
commonly caused by Enterocytozoon bienneusii and Septata intestinalis, causes
intractable diarrhoea and weight loss in AIDS patients. Diagnosis is made by
demonstrating Microsporidia spores in stool specimens by modified Trichrome
stain or Gram Chromotrop stain and histologically from the intestinal biopsy.
Strongyloides stercoralis, an
intestinal nematode, is potentially lethal because of its potential to cause
an overwhelming autoinfection in immunocompromised hosts. Diarrhoea is the
major clinical manifestation. Eosinophilia and local or generalized rash may
also occur. Definitive diagnosis is made by demonstrating larvae in faeces or
duodenal fluid.
Cyclospora, another intestinal
coccidian parasite causes prolonged, often relapsing watery diarrhoea and
weight loss. Modified safranin staining technique is recommended to
demonstrate oocysts in the faecal smear.
Isosporiasis caused by Isospora
belli produces protracted and sometimes profuse diarrhoea. The oocysts are
demonstrated in faecal smears by modified acid fast stain.
Safety
considerations
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Specimen collection
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Universal precautions to be followed, avoid
contact with specimens with ungloved hands.
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Specimen transport and storage
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Sterile leak-proof container in a sealed
plastic bag.
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Specimen processing
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At least biosafety level II with good
laboratory practice.
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The
above guidelines should be supplemented with recommendations from the local
health hazard and risk assessment committee.
Specimen collection
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Optimum time of specimen collection
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Ideally as close to the onset of symptoms as
possible;
Before initiation of antiparasitic therapy;
First morning sample
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Correct specimen type
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Waxed cardboard box with an overlapping lid,
or a plastic cup or box with a tight-fitting lid and two applicator sticks
Three consecutive stool specimens at1-day
interval is ideal
For P. carinii, collect transbronchial
aspirate, 10 ml bronchial washing or biopsy in plain sterile vial
For antigen detection collect 3-5 ml blood
in plain vial or 1-2 ml CSF in a plain sterile vial
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Specimen
transport and storage
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Time between specimen collection and processing
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The specimen must reach the laboratory
within half an hour after passage. If this is not possible, specimens must
be treated with preservatives like10% formaline or PVA
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Storage
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Specimen should be stored in ice box or
refrigerator.
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Specimen
processing
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Proper documentation upon receipt of specimen
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Maintenance of laboratory records with lab
number. Laboratory request form should indicate the specific test for P.
carinii and T. gondii
Laboratory record and laboratory number
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Initial processing
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Depends upon the clinical criteria and
parasite suspected
Stool sample; examine for consistency,
blood, mucous or any adult parasite;
Blood sample; centrifuge at 1000-1500 rpm
for five minutes and separate serum and store at –200C for antigen
detection;
CSF and other body fluids to be centrifuged
at 2000 – 2500 rpm for 10 minutes, and supernatant collected and stored at
–200C for antigen detection;
Sediment to be used for smear and wet mount;
Blood specimen for PCR and antigen detection
to be sent to the reference laboratory, and
Impression smear or necropsy or biopsy
specimen for P. carinii and T. gondii.
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Microscopy
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Very important; can predict the nature of
the infection
Procedure employed depends upon specimen
Examine saline or iodine wet mount for
protozoa and larvae, directly from faecal material or concentrated specimen
Giemsa stain or toluidine blue stain for P.
carinii and T. gondii
Modified acid fast stain or Safranin
methylene blue for Cryptosporidium and Cyclospora
Gram chromotrope or modified trichrome stain
for Microspodia
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Histopathology
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Hematoxylin eosin stain, PAS stain and
methenamine silver nitrate stain for P. carinii
Hematoxylin eosin stain and PAS stain for T.
gondii
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Serology
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Supernatant from centrifuged blood or body
fluids to be used for antigen or antibody detection where applicable.
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Reporting procedure
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Reporting should include appearance,
consistency, presence of blood, mucus, pus, worms or worm fragments
The report should mention the presence of
RBCs, WBCs and parasite detected. The results should be reported immediately
by telephone personally if possible.
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Preservation of specimens
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Preserve stool samples in equal amount of
10% buffered formalin.
Screw the cap of the vial securely.
Wrap a piece of adhesive tape around the top
of vial to prevent leakage.
Pack the vial(s) carefully in the box for
sending to reference laboratory.
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Disposal of specimen
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Stool samples collected in paper boxes,
dispose of by burning the entire container. If collected in metal or glass
container, add enough 10% formalin to kill the parasite.
Used slides to be put in a pan of
disinfectant such as 1% sodium hypochlorite solution for at least one hour
before washing.
Push the coverslip off the slide into the
disinfectant pan with applicator stick.
All used glassware to be put into disinfectant
pan for at least one hour before washing.
Applicator sticks and gauge pieces should be
burnt.
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Collection
of faecal specimens
Because of the fragile nature
of many intestinal parasites, and the need to maintain their morphology for
accurate identification, reliable microscopic diagnosis cannot be made unless
the stool is collected properly.
Give the patient the following:
 Because of the fragile nature of many
intestinal parasites, and the need to maintain their morphology for accurate
identification, reliable microscopic diagnosis cannot be made unless the
stool is collected properly.
Give the patient the following:
If waxed boxes or plastic cups
are not available, tin boxes or glass jars can be used. Three specimens are
usually recommended, at one-day intervals, to detect all parasitic
infections. A variety of substances may interfere with the examination of
stool specimens for parasites (e.g. laxatives, antacids, ingested contrast
media and certain antibiotics).
The container with the
specimens should be labelled clearly with the following information:
Patient’s name or number
Date of collection
Time the patient passed the stool (ask the
patient when he/she passed the stool).
Tell the patient to pass the
stool specimen directly into the container, or to pass the stool on to a
piece of paper and use the applicator sticks to transfer it to the container.
If paper is not available, the faeces can be passed on to a large, clean
leaf. However, the stool must be transferred immediately to the specimen container.
It should not remain on the leaf or be brought to the laboratory on the
paper/leaf.
The stool specimen must be
large enough for satisfactory examination. The smallest quantity that should
be accepted is about the size of a pigeon’s egg. Urine and dirt should be
excluded. Urine will destroy any protozoan trophozoites and dirt will
interfere with the examination. If the specimen is too small, or if it is
mixed with urine or dirt, it should not be accepted. Ask the patient to pass
another specimen.
Specimen transport and storage
Some organisms, especially
amoebic trophozoites, will begin to disintegrate or change within a short
time after passage and become unrecognizable. Warm temperatures will hasten
these changes. Therefore, specimens must reach the laboratory very soon
(i.e., within half an hour) after passage. If this is not possible, the
specimen must be treated with preservatives.
Keep the carton containing the
specimen in a refrigerator, or if this is not possible in the coolest,
shadiest area in the laboratory. Do not keep the specimen artificially warm
and do not leave it in the sun.
Specimen
examination
Macroscopic examination of stool
 As soon as the specimen is received in the
laboratory, check the consistency (degree of moisture) and write one of the
following on the container: formed, soft, loose, or watery. The consistency,
or degree of moisture, will be a guide as to whether the trophozoite stage is
in the stool. The appropriate techniques to be used are shown in the Table.
If several specimens are received at the same
time, those containing blood and mucus should be examined first followed by
liquid specimens. These specimens are the most likely to contain amoebic
trophozoites (which die soon after being passed) and must be examined within
an hour after passage. Formed specimens may be examined at any time during
the first day, but should not be left overnight (cysts may disintegrate).
Microscopic examination of Wet Mounts
Wet mounting is the simple and
easiest technique for examination of faeces, and this method should be
performed in all laboratories at the peripheral level.
A wet mount can be prepared
directly from faecal material or from concentrated specimens. The basic types
of wet mount to be used for each faecal examination are saline and iodine.
The saline wet mount is used
for the initial microscopic examination of stools. It is employed primarily
to demonstrate worm eggs, larvae, protozoan trophozoites, and cysts. This
type of mount can also reveal the presence of red blood cells and white blood
cells.
Categories of Stool and
Appropriate Techniques to be used
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Consistency
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Protozoan stage
most likely to be found*
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Technique to be
used
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Saline
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Iodine
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Buffered
Methylene blue
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Formed
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Cysts
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+
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+
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+
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Soft
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Cysts
(occasionally trophozoites)
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+
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+
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+
+
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Loose
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Trophozoites
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+
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+
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*Worm eggs and larvae may be
found in stools of any consistency
The Iodine wet mount is used mainly to stain
glycogen and the nuclei of cysts, if present. Cysts can usually be
specifically identified in this mount.
Buffered Methylene Blue (BMB) wet mount should
be prepared every time amoebic trophozoites are seen in a saline wet mount or
when their presence is suspected. BMB stain does not stain amoebic cysts. It
is only appropriate for fresh unpreserved specimens.
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