Parasitic Infection

Laboratory diagnosis of parasitic diseases

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⮚Protozoa: unicellular organisms, e.g. Plasmodium (malaria)
⮚Metazoa: multicellular organisms, e.g. helminths (worms)
and arthropods (ticks, lice)

⮚An endoparasite: “a parasite that lives within

another living organism” e.g. malaria, Giardia
⮚An ectoparasite: “a parasite that lives on the
external surface of another living organism” e.g.
lice, ticks

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 Why study Parasitology?
⚫ Many of these parasites are causative agents of major public health
problems of the world.

⚫Recent estimates of prevalence of parasites in the world are:

Ascaris 1.5 billion
Hookworms 1.3 billion
Whipworms 1 billion
Filarial worms 657 million
Malaria 500 million
Schistosomes 210 million
Amebiasis 50 million
Taenia tapeworms 50 million
Clonorchis 20 million
Chagas’ Disease 15 million  

⚫These parasites cause varying morbidities and even mortalities

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 The burden of some major parasitic infections
Parasite Diseases No. people infected
Plasmodium malaria 273 million

Soil transmitted helminths: 2 billion

•Roundworm (Ascaris) Pnemonitis, intestinal obstruction

•Whipworm (Trichuris) Bloody diarrhoea, rectal prolapse

•Hookworm (Ancylostoma and Coughing, wheezing, abdominal pain and

Necator) anaemia

Schistosoma Renal tract and intestinal disease 200 million

Filariae Lymphatic filariasis and elephantiasis 120 million

Trypanasoma cruzi Chagas disease (cardiovascular) 13 million

African trypanosomes African sleeping sickness 0.3 – 0.5 million

Leishamania
5 Cutaneous, mucocutaneous and visceral 12 million; 2 million new
leishmaniasis cases/yr
 Current disease portfolio from WHO report, 2001

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 Examples of important intestinal protozoa
Transmitted by the faecal-oral route and cause diarrhoea

Giardia lamblia: world-wide distribution, lives in the small

intestine and results in malabsorption

Entamoeba histolytica: may invade the colon and cause bloody

diarrhoea – amoebic dysentery. Also causes amoebic liver
abscess.

Cryptosporidium parvum: more prevalent in the

immunocompromised

Cyclospora cyatenensis : parasitizes the small intestinal mucosa

and may cause diarrhoea for several weeks

Balantidium coli: a large motile ciliated parasite that lives in the

colon of pigs, humans and rodents and can lead to colonic
ulceration
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Cyst of E. histolytica stained with
trichrome. Note the chromatoid body Trophozoites of E. histolytica with
with blunt ends (red arrow) ingested erythrocytes stained with
trichrome.

G. duodenalis trophozoite
Cryptosporidium sp. oocysts stained with trichrome
stained with modified acid-fast.
G. duodenalis cyst stained with
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trichrome.
Examples of important systemic protozoa
Detected in the blood
⚫ Plasmodium: the cause of malaria. There are 4 species
that infect man: P. falciparum, P. vivax, P. ovale and P.
malariae

⚫ Toxoplasma gondi: transmitted by the ingestion of

oocysts from cat faeces. Infection can lead to ocular
problems and is also a cause of neonatal toxoplasmosis
Typical lesion
⚫ Leishmania: transmitted by sand flies, can lead to of cutaneous
leishmaniasis
visceral, cutaneous and mucocutaneous leishmaniasis

⚫ Trypanosoma: haemoflagellates cause

⚫In Africa - sleeping sickness (transmitted by the Tsetse fly)

⚫In South America - Chagas disease (transmitted by the Reduviid

bug)

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Trypanosoma brucei ssp. in a thin blood T. cruzi trypomastigote in a thin blood smear
smear stained with Giemsa. stained with Giemsa.

Leishmania sp. amastigotes in a Giemsa-stained tissue scraping

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 Examples of important intestinal nematodes
⚫ Ascaris (roundworm)
⚫ Trichuris (whipworm)
⚫A soil transmitted helminth ⚫Found world-wide in conditions
⚫prevalent in warm, humid conditions of poor hygiene, transmitted by
⚫Can cause diarrhoea, rectal prolapse and the faecal- oral route
anaemia in heavily-infected people ⚫Adult worms lives in the small
intestine
⚫ Ancylostoma and Necator (hookworms)
⚫Causes eosinophilia
⚫A major cause of anaemia in the tropics

⚫ Strongyloides ⚫ Enterobius (pinworm or

⚫inhabits the small bowel threadworm)
⚫prevalent in cold and temperate
⚫infection more severe in
climates but rare in the tropics
immunospressed people (e.g.
HIV/AIDS, malnutrition, intercurrent ⚫found mainly in children
disease)

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Adult female A. lumbricoides. Unfertilized egg of A. lumbricoides Fertilized egg

Trichinella larva in tongue muscle of a rat,

Hookworm egg in an unstained wet mount
stained with hematoxylin and eosin
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 Examples of important systemic nematodes
⚫ Toxocara
Filaria worms including:
⚫A world-wide infection of
⚫ Onchocerca volvulus :
dogs and cats
Transmitted by the simulium black fly, ⚫Human infection occurs
this microfilarial parasite can cause
when embryonated eggs
visual impairment, blindness and severe
are ingested from dog or
itching of the skin in those infected
cat faeces
⚫It is common in children
⚫ Wuchereria bancrofti :
and can cause visceral
The major causative agent of lymphatic larva migrans (VLM)
filariasis

⚫ Brugia malayi :
Another microfilarial parasite that causes
lymphatic filariasis
Microfilaria of W. bancrofti in a thick blood
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smear stained with Giemsa
 Examples of important flatworms : cestodes
Intestinal :(“tapeworms”) Systemic
Taenia saginata
⚫ worldwide Echinococcus granulosus (dog
⚫ acquired by ingestion of tapeworm) and Echinicoccus
contaminated, uncooked beef multilocularis (rodent tapeworm)
⚫ a common infection but causes
minimal symptoms
Hydatid disease occurs when the
larval stages of these organisms
Taenia solium are ingested
⚫ worldwide
⚫ acquired by ingestion of
contaminated, uncooked pork The larvae may develop in the
that contains cystercerci human host and cause space-
⚫ Less common, but causes
cystercicosis – a systemic occupying lesions in several
disease where cysticerci organs, e.g. liver, brain
encyst in muscles and in the
brain – may lead to epilepsy

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Protoscoleces in a hydatid cyst removed
from lung tissue, stained with Taenia sp. egg in unstained wet mounts
hematoxylin and eosin (H&E).

Proglottid of T. saginata injected with India Ink

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 Examples of important trematodes (flukes)

Intestinal ⚫ Fasciola hepatica (liver fluke)- a

parasite of sheep, humans become
⚫ Fasciolopsis buski : A common parasite infected when ingest metacercariae
that have encysted on watercress.
of humans and pigs in South- east Asia. The adult trematode lives in the
This parasite is one of the largest intra-hepatic bile ducts of the liver.
trematodes to infect man (8cm in length) “Fascioliasis” can lead to severe
anaemia in humans
and lives in the upper intestine. Chronic
infection leads to inflammation,
⚫ Clonorchis sinensis (liver fluke):
ulceration and haemorrhage of the small Widespread in China, Japan, Korea
intestine and Taiwan, this parasite is
acquired by ingestion of infective
metacercariae in raw or pickled
⚫ Paragonimus westermani ( lung fluke)- fish
Widespread in the Far East and South east
Asia, the parasite is acquired by ingestion of
⚫ Schistosoma haematobium, S.
infective metacercariae in raw or pickled
crustaceans mansoni and S. japonicum
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 Egg of S. japonicum in an
Egg of S. haematobium Egg of S. mansoni in unstained wet mount of
in a wet mount of a urine unstained wet mounts. stool.
concentrate

Egg of F. hepatica in an unstained wet mount

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 Diagnosis of Parasitic Infections

1. Clinical

2. Laboratory

Purpose of laboratory diagnosis :

⚫ Confirmation of clinical suspicion

⚫ Identification of unsuspected infection
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 Specimens
❖Stool

❖Blood

❖Serum and plasma

❖Others (anal swab, duodenal aspirate, sputum, urine, urogenital

specimen)
❖Tissues and aspirates

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 Stool examination
Sample collection:

⚫Sample is collected in clean, dry container

⚫Handled carefully
⚫Sometimes use preservative (10% formalin)
⚫Samples in some cases fresh(amoeba, ciliates)
⚫Liquid and soft stool examined within 15 min
⚫Not mixed with urine or disinfectant (as they will kill trophozoites)
⚫Specimens obtained by enema or laxatives are often
positive for worm eggs or adult worm.

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 Examination of the stool sample:

Gross examination:
⚫Mucoid blood stained (acute amoebic dysentry), Parasites can
be detected (nematodes, cestodes)
Microscopic examination:
⚫Saline mount
⚫Iodine Mount
⚫Thick smears – not commonly used
⚫Permanent stained smears
⚫Iron hematoxylene
⚫Whearley’s trichrome stain
⚫Concentration methods
⚫Floatation techniques
⚫Sedimentation techniques

⚫Antigen detection
⚫Molecular diagnosis
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 Microscopic examination
Direct wet mount:

⚫ Thin emulsion of small amount of faeces

⚫ Few drops of saline

⚫ Sometimes add lugol’s iodine (nuclear details, glycogen vacuole in cyst)

⚫ Protozoa (trophozoite), cyst, eggs and larva of helminths, crystals (charcot

leyden)

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 Concentration methods
• Scanty parasites in the sample

• Floatation (eggs and cyst float , solution of high specific gravity)

1. saturated sodium chloride (ascaris, hookworms)

2. Zinc sulphate centrifugation floatation (cyst, nematodes).

• Sedimentation (solution of low specific gravity):

formol ether
Egg count in 1 gram

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 Stoll’s technique for counting helminth egg

3 gm stool and 42 ml water

0.15 ml on slid
Multiply result in 100
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Number in 1 gm
 Immunodiagnostic (antigen detection)

⚫ Fresh or preserved stool samples are the appropriate specimens

Amebiasis
⚫ EIA(enzyme immune assay) kits are commercially available for
detection of fecal antigens for the diagnosis of intestinal amebiasis.
⚫ These assays use monoclonal antibodies that detect the galactose-
inhibitable adherence protein in the pathogenic E. histolytica.
⚫ Several EIA kits for antigen detection of the E. histolytica/E. Dispar
(non-pathogenic amoeba) group are available , but only the TechLab kit
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is specific for E. histolytica.
 Cryptosporidiasis

Several kits are combined tests for Cryptosporidium,

Giardia, and E. Histolytica.

DFA test identifies oocysts in concentrated or unconcentrated

fecal samples by using a fluorescein isothiocyanate
(FITC)-labeled monoclonal antibody is the most sensitive.

Giardiasis
DFA assays may be purchased that employ FITC-labeled monoclonal
antibody for detection of Giardia cysts.

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 Molecular diagnosis
(using stool sample)

If an unequivocal identification of the parasite can not be made, the stool

specimen can be analyzed using molecular techniques such as polymerase

chain reaction (PCR). PCR amplified fragments can be analyzed by using

restriction fragment length polymorphisms (RFLP) or DNA sequencing if

further characterization is needed.

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⚫ Sample :
Fresh stool should be kept cold or frozen till DNA extraction.
Samples collected in a preservative should be compatible with molecular
detection (TotalFix, Unifix, modified PVA (Zn- or Cu-based), and Ecofix)

⚫ DNA Extraction better using commercially available kits (Qiagen)

⚫ PCR analysis:

Conventional PCR:
DNA is tested by PCR with diagnostic primers. Amplified DNA fragments are
electrophoretically resolved on an agarose gel for analysis of results.

Real-Time PCR
The DNA amplification is monitored by measuring the fluorescence signal
generated in the reaction vessel. The fluorescence signal is measured every
cycle and is proportional to the amount of accumulated PCR product.
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 Blood examination
⚫Fresh capillary blood of finger or ear lobe
⚫Venous blood collected in EDTA (anticoagulant)

Blood sample will be used for :

⚫Microscopic examination(Thin Smear, Thick smear, Wet mount for microfilaria).

⚫Molecular diagnosis
⚫Detection of parasite antigen
⚫Isolation of organisms
⚫Special tests

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Blood sample preparation for microscopic examination

Thin blood film

Thin blood film

Thick blood film

Thick blood film
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Thick blood film Thin blood film
In malaria Parasitized red blood cells and
⚫Screen large amount of parasites
blood (light infection) Definite species identification
⚫Can be stained latter

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 Microfilaria
⚫Sample collection according to periodicity of
microfilaria
⚫Concentration by sedimentation or membrane
filtration (examine the filter)
⚫DEC provacation method

Microfilaria of Wuchereria bancrofti

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 Molecular diagnosis
(using blood sample)

⚫Collect a 1-5 ml blood sample in tube with EDTA.

⚫Blood can be collected on filter papers (e.g Whatman)
⚫DNA is extracted using DNA extraction kits

Species-specific diagnosis of malaria

Detection and speciation of Plasmodium is done with a two

step nested PCR using the primers of Snounou et al 1993.

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 Detection of parasite antigens
(in blood sample)

⚫ Rapid diagnostic tests for malaria employing immunochromatographic

methods based on the detection of malarial antigens present in
peripheral blood.
⚫ only diagnose only P. falciparum malaria.

⚫ Currently, the only available RDT for malaria in the United States is
the BinaxNOW® Malaria Test.

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 Isolation of Organisms
(from blood)

❑The diagnosis of Leishmania spp. is made by microscopic identification of

the nonmotile, intracellular form (amastigote) in stained sections from lesions,
and by culture of the motile, extracellular form (promastigote) on suitable media.

❑ Slides should be fixed and stained before they are sent unless reagents are not
available.

❑Serologic tests are also available to detect for anti-leishmanial antibodies;

however, these tests are often not sensitive, particularly for diagnosing cutaneous
leishmaniasis.
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 Special Tests: MQ Testing
⮚Mefloquine is recommended by CDC as a prophylactic against malaria.
⮚ When individuals who are on mefloquine prophylaxis exhibit signs of malaria,
blood samples are collected and analyzed for the presence of the drug.

⮚The drug is extracted from the blood and the concentration is determined by
high-performance liquid chromatographic (HPLC) methods.
⮚Determining the level of mefloquine in the blood helps assess if the individual
was adherent with his/her medication.

⮚This procedure is also useful to determine treatment failure due to a

mefloquine resistant form of malaria. 42
 Serum, plasma and others

Specimen Requirements
oSerum/plasma is required for all parasitic disease immunodiagnostic tests.
oA single sample is sufficient; acute and convalescent specimens are not
necessary.
oCSF and eye fluids (vitreous or aqueous) are acceptable for selected diseases)
but MUST be accompanied by a serum specimen.

Serum for all tests: 0.5 ml serum/plasma separated from RBCs .

CSF: 0.5 ml. Acceptable only for cysticercosis and baylisascariasis testing.

Eye fluids: 0.1 ml neat fluid (no washings). Acceptable only for toxocariasis
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 ⚫Serology – All tests available
⚫IHA
⚫ELISA
⚫CIEP ⚫Skin Tests
⚫IF
⚫CFT Specificity low, cross reactions common
⚫More useful in
Examples:
⚫Amoebiasis
⚫Leishmaniasis ⚫Casoni’s test
⚫Malaria
⚫Toxoplasmosis ⚫Leishmanin test
⚫Trichinosis
⚫Filariasis
⚫Echinococcosis
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 Cultivation of parasites
Culture methods are used for :

⚫Amoeba

⚫Leishmania

⚫ Trypanosoma

⚫Malarial parasite

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 Animal inoculation

⚫Leishmania (young hamester)

⚫Trypanosomes (rat, mouse)

⚫Toxoplasma (all lab animals)

Xenodiagnosis:

In chagas’ disease

Vector infected experimentally

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 Sputum examination
Microscopic examination of sputum can identify:

⮚ Paragonimus westermani eggs

⮚ Strongyloides stercoralis larva
⮚ Ascaris lumbricoides larvae
⮚ hookworm larvae, and rarely Entamoeba histolytica.

❑ Sputum should be obtained from the lower respiratory passages not

saliva.
❑Sputum specimens should be collected first thing in the morning.

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 A sputum sample can be examined in several ways:

❑The unfixed specimen may be centrifuged and then the sediment examined as
a direct wet mount.

❑If the sputum is too viscous, an equal volume of 3% sodium hydroxide may be
added, then centrifuge, and examine the sediment.

❑The specimen can be preserved in 10% formalin and a formalin-ethyl acetate

❑ The specimen can be preserved in PVA if protozoa are suspected and stained
with trichrome stain.
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 Vaginal swabs
▪Demonstration of Trichomonas vaginalis trophozoites is usually done
by preparing wet mounts made from vaginal swabs or scrapings.

▪If the specimen cannot be examined immediately, it should be preserved in

PVA and stained smears examined later.

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 Tissue Specimens for Free-living Amebae (FLAs)

Tissue specimens, including biopsy, surgical or necropsy specimens, may be

collected for the detection of free-living amebae (Naegleria, Balamuthia, and
Acanthamoeba).

The desired specimens include:

•Tissue slides stained with hematoxylin and eosin (H&E).

•Unstained slides (for indirect Immunofluorescence, or IIF). Acanthamoeba cyst

•Unfixed brain tissue or CSF for PCR.

•Unfixed corneal scrapings (for Acanthamoeba).
•Paraffin-embedded tissue block.
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 Cellulose Tape or Swube Tube Procedure for Demonstration of
Pinworm Eggs

•The most reliable and widely used technique for demonstrating pinworm eggs
(Enterobius vermicularis) is the cellulose tape or swube tube procedure.

•The adhesive part of the swube tube or tape is applied to the perianal area first
thing in the morning.

•Specimens should be collected on three consecutive mornings prior to bathing.

• If an infection is present, eggs and sometimes adult worms

of Enterobius vermicularis will be present
on the tape and can be seen under the microscope
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 Urine Specimens
Urinary schistosomiasis

• Presence of S. haematobium eggs in urine is diagnostic for

•Eggs usually shed in the urine around midday, so an optimum urine specimen
for diagnosis should be collected at noon.

• The specimen should be immediately centrifuged at 400 × g and the sediment

examined by wet mount.

Trichomonas vaginalis
❑Motile trophozoites may also be found in the urine, especially in infected
male patients.

❑Urine specimen should be centrifuged at 400 × g, the sediment mixed with a

drop or two of saline, and examined by wet mount.

❑Temporary stains, such as methylene blue is helpful to see T. Vaginalis 53

 Artifacts
•Cysts and trophozoites must be examined carefully in different fields of view
and measurement is often essential. Objects such as epithelial cells and
macrophages are around the same size as amoebic trophozoites: the latter may
also move and contain red blood cells.

•White blood cells, plant and vegetable cells, fat globules, muscle fibers,
pollen grains, yeasts cells and air bubbles may be confused with cysts or eggs.

• Air bubbles trapped under adhesive tape often resemble Enterobius eggs.
•Plant hairs and fibers are easily confused with larvae
•Earthworms may resemble roundworms. 54
•Eggs of Heterodera, a parasitic nematode of root vegetables, may resemble
hookworm eggs.
•Eggs originating from harmless mites in cereals or flour could be confused
with hookworm ova
•A patient complaining of hematuria: we suspected Schistosoma haematobium
but, on closer analysis, the eggs contained unidentified insects

Here we show examples of artifacts that may be confused for parasitic life
stages.
Artifacts should be considered on the basis of size, shape, lack of organelles
and defining feature, and variable reactivity with common
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 Artifacts

White blood cells can be mistaken Image showing fat cells, which
for protozoan cyst (picture can be mistaken with
showing white blood cells) protozoan cyst

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 Platelets in thin blood smears. The nature of the platelets gives
them the appearance of trypomastigotes of Trypanosoma cruzi.

nucleated red blood cell in a thin blood smear, stained with Giemsa.
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may be confused for schizonts of Plasmodium spp.
 Fungal spore of Helicosporium (or related). Such objects are air-borne contaminants in
laboratories and may be mistaken for microfilariae in stained blood smears.

Howell-Jolly bodies in a thin blood smear, stained with Giemsa. They may be seen in
splenectomized patients or patients with an otherwise non-functioning or atrophic spleen, and
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in patients with severe anemia or leukemia.
Trichrome-stained leukocytes Image showing macrophage stained
can be mistaken with amoeba with trichrome, which can be easily
mistaken with amoeba

Image showing epithelial cell stained with trichrome, which

can be easily mistaken with amoeba
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 Pollen grain in a trichrome-stained
stool specimen. In this focal plane, Pollen grain in a concentrated wet mount of
the grain looks like the striated egg stool. This grain looks very similar to the fertile
of Taenia sp. However, notice the egg of Ascaris lumbricoides
lack of refractile hooks

Possible pollen grain or algal or fungal spore in

a concentrated wet mount of stool. Grains like
this one resemble the operculated eggs of
Clonorchis, Metagonimus. These grains are
usually
60 smaller than the trematode eggs
 Spore of a morel mushroom. Such spores may
Yeast in an iodine-stained concentrated wet be confused for helminth eggs, especially
mount of stool. Yeast in wet mounts may be hookworm.
confused for Giardia.

Fungal spore in concentrated Fungal spore in a wet

Yeast cells can be confused
wet mounts of stool. Such mount of stool. Such
with cryptosporidium
spores may be confused for spores may be confused
oocysts
protozoa such as Giardia for the cysts of Entamoeba
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 Charcot-Leyden crystal in the
picture can be mistaken with hair Diatoms do not specifically resemble any parasites
of humans, but their size and shape and apparent
structure are striking

Mite egg in a formalin-concentrated stool specimen. Mite eggs are similar to hookworm
eggs but are usually larger (but not always). In this specimen, leg buds can be seen in the
lower right area of the egg 62
 Image shown plant cell, which can be mistaken
Plant cell in stool. Such material can be common in with paragonimus egg
stool and may be confused for helminth eggs,
although they are larger than the eggs.

Plant hairs can be common in stool and may be confused for the larvae of hookworm or
Strongyloides stercoralis. However, they are often broken at one end, have a refractile
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center and lack the strictures seem in helminth larvae (esophagus, genital primordium)
Horsehair worms are parasites of insects and may be found in households and end up in
toilets. As a result, they are often sent to the public health laboratories for identification.

Earthworms (Lumbricus and related) are commonly sent to the public health laboratories for
identification. The presence of setae, segmentation, and a clitellum (red arrow) should
distinguish them from parasitic helminths
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 Aquatic larvae of flies. The free-living aquatic larvae of various flies breed in standing
water, including toilets, leading to the misconception they came from stool or urine. The
presence of prolegs, a head capsule, breathing tubes (arrow), segmentation and/or setae
will usually distinguish them from true parasitic worms.

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 Thank you
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