Diagnostic Parasitology

DIAGNOSTIC PARASITOLOGY

PROTOZOA / HELMITHES

 

          The following are the main ways in which parasitic infections are diagnosed in the laboratory.

 

Microscopic examination:  The majority of intestinal, urinary, and blood parasites can be detected microscopically in unstained or stained preparations, either directly or following concentration techniques.

 

Culture techniques: Only a minority of parasitic infection is diagnosed routinely by culture   techniques.

 

Immunodiagnosis: With the development of reagents, which are both more sensitive and specific, immunodiagnosis techniques are becoming increasingly used in diagnosis and in studies involving the epidemiology and control of parasitic diseases.

 

DIRECT EXAMINATION OF FECAL SPECIMENS:

 

Feces should be examined within a few hours of being passed. If amoebic dysentery is suspected, the specimen should be examined as soon as possible and kept in a warm environment until it is examined.

 

Gross examination:

Report the appearance of the specimen:

Mention: Color, Consistency, (i.e. whether it is formed, semiformed, unformed, or fluid), whether it contains blood, mucus, pus, whether it contains worms or worm segments.

 

Microscopic examination:

Place a drop of fresh physiological saline on one end of a slide and a drop of Lugol’ iodine on the other end. Using a piece of stick, mix a small amount of specimen (i.e. matchstick head size) in the saline and iodine. Make wet preparations.

 

Use of saline and Iodine:

In a saline preparation motile parasites such as amoeba, flagellates, larvae, and ciliates can be identified. Helminth eggs can be readily identified. Cysts can also be detected but they are much more easily seen in iodine prepration because it stain nuclei and glycogen mass.

 

Examine systematically the entire saline preparation using 10X objective.Use 40X objective to identify small parasites.

 

Normal structures found in feces.

Care must be taken not to report as parasites those structures, which can be normally found in feces such as muscle fibers, vegetable fibers, starch cells, pollen grains, fatty acid crystals, soaps, spores, yeasts, and hairs.

 

Concentration techniques for fecal parasites:

Concentration methods to detect parasites in feces may be necessary for the following reasons:

  • To detect parasites when they are not found in a direct examination but the symptoms of intestinal parasitic infection continue.
  • To detect the eggs of parasites, which are often, few in number such as those of Schistosoma or Taenia species.
  • To check whether treatment has been successful.
  • To investigate the prevalence and incidence of parasitic infection as part of an epidemiological survey.

 

 

The following techniques are used commonly:

 

Floatation Technique

  1. Formal ether technique in which parasites are sedimented by centrifugal force. Ether is used to dissolve fecal fat and to separate the fecal matter from the sedimented parasites.
  2. Formal detergent technique in which parasites are sedimented by gravity using a solution of low specific gravity with detergent added to clear the fecal mater.

 

Concentration Technique:

  1. Sodium chloride floatation technique in which parasites are floated in saturated sodium chloride.
  2. Zinc sulphate floatation technique in which parasites are float in 33% w/v zinc sulphate.

 

The recommended technique for hospital laboratories is the formal ether technique. It is rapid and gives good concentration of parasitic cysts, eggs, and larvae, in fresh or preserved feces. The formal detergent sedimentation technique is suitable replacement for the formal ether technique if a centrifuge is not available and results are not required urgently. Floatation techniques are not recommended as routine concentration techniques because they concentrate only a small range of fecal parasites.

 

Examination of Blood:

Examination of blood smear stained with Giemsa stain is the most common method of detecting Plasmodium spp., Babesia spp., Trypanosoma spp., and some species of Microfilariae. Although motile organisms such as Trypanosoma spp. And Microfilaria can be detected on a wet preparation of a fresh blood specimen under low and high power magnification, identification is made on the basis of characteristics seen on a permanently stained smear. Concentration methods using membrane filters can be used to detect Trypanosoma spp. or Microfilariae but are rarely performed in the clinical laboratory.

 

Collection and preparation of the blood specimen:

Blood taken directly from a finger stick should be used for a malarial smear because it tends to give best staining characteristics. Blood collected in EDTA gives adequate staining if processed within 1 hour. With Giemsa stain cytoplasm of the parasite stains bluish and the chromatin red to purple red. Giemsa staining gives best morphological details but it is a time consuming procedure.

 

Identification of the organism:

For the suspected case of blood parasites, both a thick and thin film should be made. Both preparations can be made on the same slide or on separate slides.

 

A thick film is best for detection of parasites, because organisms are concentrated in relatively small area. The thick film is made by pooling several drops of blood on the slide and then spreading it onto a 1.5 cm area. Thickness is optimal when newsprint is barely visible through the drop of blood before it dries. The blood should be allowed to dry for at least 6 hours before staining. It should not be fixed with methanol. Initial scanning of the stained smear at 10X detects microfilariae. At least 100 oil immersion fields should be examined before a negative result is reported.

 

Species identification should be made from a thin film, because the characteristics of the parasite and the RBC can be seen. The thin film is made in the same way as that for a differential count. It should be fixed in methanol for 1 minute and air dried before staining with Giemsa stain. The entire smear should be scanned at 10X for detection of the large parasites such as microfilariae; then at least 100 oil immersion fields must be examined for the presence of organisms such as Trypanosoma spp. or for intracellular organisms such as Plasmodium spp. or Babesia spp.

 

 

Immunodiagnosis of parasitic infection:

Immunodiagnosis is used to assist in the clinical diagnosis of parasitic infection and in the epidemiology and control of parasitic diseases.

 

Immunodiagnosis techniques are based on the detection of:

  • Antibody in a person’s serum, produced in response to a particular parasitic infection. The antibody may persist for a long period in the serum after an infection has ended and therefore antibody tests are unable to distinguish between a past or present infection.
  • Antigen, which is excreted by parasites and can be found in serum, urine, CSF, feces, or other specimens.

 

Immunodiagnostic techniques are required when:

  • Parasites live in the tissues of internal organs and cannot therefore be easily removed for examination.
  • Parasites can be found in specimens only in certain stages of an infection.
  • Parasites are present only intermittently or in to few numbers to be easily detected in specimens.
  • The techniques used to detect parasites are complex or time-consuming.

 

 

Those parasitic diseases for which Immunodiagnosis is of particular value include:

  • South American trypanosomiasis, chronic stage.
  • African trypanosomiasis, when parasitaemia is low.
  • Visceral leishmaniasis.
  • Cutaneous and mucocutaneous leishmaniasis.
  • Amoebic liver abscess.
  • Hydatid disease.
  • Filariasis, occult and chronic infection.
  • Schistosomiasis, chronic stage.

 

 


 

PARASITES FOUND IN DIFFERENT SPECIMENS

Specimen

Parasite

Form

Feces

E. histolytica

Cysts, amoeba

G. lamblia

Cysts, flagellate

B. coli

Cyst, ciliate

I. belli

Oocyst

Cryptosporidium

Oocyst

Taenia

Egg, segment

H. nana

Egg

D. latum

Egg

F. hepatica

Egg

F. buski

Egg

Schistosoma

Egg

A. lumbricoides

Egg, worm

E. vermicularis

Worm

S. stercoralis

Worm

T. trichiura

Egg

A. duodenale

Egg

N. americanus

Egg

Blood

Plasmodium

Trophozoite, schizont, gametocyte

Trypanosoma

Trypomastigote

W. bancrofti

Microfilaria

B. malayi

Microfilaria

L. loa

Microfilaria

Mansonella

Microfilaria

Urine

S. haematobium

Egg

S. mansoni

Egg

T. vaginalis

Flagellate

W. bancrofti

Microfilaria

O. volvulus

Microfilaria

Sputum

Paragonimus

Egg

CSF

Trypanosoma

Trypomastigote

Bone marrow

L. donovani

Amastigote

T. gondii

Toxoplasma

Lymph gland & aspirate

Trypanosoma

Trypomastigote

L. donovani

Amastigote

T. gondii

Toxoplasma

Liver aspirate

E. histolytica

Amoeba

L. donovani

Amastigote

T. gondii

Toxoplasma

Spleen aspirate

L. donovani

Amastigote

T. gondii

Toxoplasma

Skin

Leishmania

Amastigote

O. volvulus

Microfilaria

D. medinensis

Larva

E. vermicularis

Egg

Muscle

Trichinella

Larva

Duodenal aspirate

G. lamblia

Flagellate

F. hepatica

Egg

S. stercoralis

Larva

Bronchial biopsy aspirate

P. carinni

cyst


IMPORTANT MICROSCOPIC FEATURES OF COMMONLY FAECAL PARASITES.

 

Parasites Form Microscopic Features
E.histolytica Amoeba *Average size is about 25*20 µm.

*Active amoeboid movement (directional) in fresh warm

specimens.

*Contains ingested red cells.

Cyst *Round, measuring 10-15 µm.

*Contains 1, 2, or 4 nuclei.

*Chromatoid bars can be seen in immature cysts.

Giardia

lamblia

Flagellate *Pear-shaped, usually measuring 10-12*6 µm.

*Upper end has a concavity with a sucking disc.

*There are 8 flagella.

Cyst *Oval in shape.

*Small, measuring about 10*6 µm.

*Contains the remains of axonemes and parabasal bodies.

*Thread-like remains of flagellate may also be seen.

*4 nuclei.

B.coli Ciliate *Large, measuring 50-200*40-70 µm.

*Rapid revolving motility.

*Beating cilia can be seen, especially around the cytostome.

Cyst *Large, measuring 50-60 µm.

*Thick-walled.

*Cilia may be seen in younger cysts.

*Macronucleus visible.

I.belli Oocysts *Oval, measuring about 32*16 µm.

*Usually contain a central undivided mass of protoplasm.

Taenia Segment

T.saginata

*Appears white and opaque and measures about 20 mm long

by 6 mm wide when freshly passed.

*Uterus has a central stem which has more than 13 main side

branches on each side.

Segments

T.solium

*Appears gray-blue and translucent and measures about 13

mm long by 8 mm wide when freshly passed.

*Uterus has a central stem which has up to 13 main side

branches on each side.

Egg

T.saginata

T.solium

*It is round to oval, measuring 33-43 µm.

*A thick, brown, radially striated wall surrounds the embryo.

*Hooklets (3 pairs) are present in the embryo.

H.nana Egg *It is colorless, oval or round, measuring 30-45 µm.

*Hooklets are present in the embryo.

*At the each end of egg thread-like structures called polar

filaments are usually visible.

D.latum Egg *It is pale yellow and oval in shape, measuring about 70*45

µm.

*It has an operculum.

*Contains a mass of granulated yolk cells surrounding an

undeveloped ovum.

*A small projection is sometimes visible at the non-

operculated end of the egg.

F.buski

F.hepatica

Egg *It is pale yellow-brown, large and oval, measuring about

140*85 µm.

*Contains an unsegmented ovum surrounded by many yolk

cells.

*Has a small operculum.

S.mansoni Egg *It is pale yellow-brown, large, and oval, measuring about

150*60 µm.

*Has a characteristic side spine.

*Contain a fully developed miracidium.

*A viable egg shows flickering of the excretory flame cells.

*A non-viable egg is dark-colored and shows no structural

detail or flame cell movement.

A.lumbricoides Fertilized

egg

*It is yellow-brown and the shell is covered by an uneven

albuminous coat.

*Oval or round and measures 60*40 µm.

*Contains a central granular mass which is the unsegmented

fertilized ovum.

Unfertilized egg *It is darker in color and has a more granular albuminous

covering.

*More elongated, measuring about 90*45 µm.

*Contains a central mass of large refractile granules.

E.vermicularis Egg *It is colorless and has a clear shell.

*Oval in shape and usually flattened on one side. It measures

about 55*30 µm.

*Contains a larva.

S.stercoralis Larva *It is actively motile.

*It is large, measuring 200-300*15 µm and is unsheathed.

*Shows a typical rhabditiform bulbed esophagus.

*It can be distinguished from a hookworm larva by its

shorter buccal cavity.

T.trichiura Egg *It is yellow-brown and measures about 50*25 µm.

*Has a characteristic barrel shape with a colorless protruding

Mucoid plug at each end.

*Contains a central granular mass which is unsegmented

ovum.

A.duodenale

N.americanus

Egg *It is colorless with a thin shell which appears as a black line

around the ovum.

*Oval in shape, measuring about 65*40 µm.

*Contains an ovum, which usually appears segmented. If

specimen is more than 12 hours old, a larva may be seen

inside the egg. If the feces is more than 24 hours old than

larva may hatch and seen free in the feces.

 

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