Identification techniques parasitology

Question 1

Types of specimens which can be analysed?

Answer 1

Faeces e.g Entamoeba histolytica cysts
Blood e.g Plasmodium spp. trophozoites
Urine e.g. Schistosoma haematobium eggs
Cerebrospinal fluid (CSF) e.g. Trypansoma brucei spp.
Other body fluids e.g. vaginal discharge for Trichomonas vaginalis
Biopsy tissue e.g ‘skin snips’ for microfilariae

Question 2

Laboratory identification techniques?

Answer 2

Macroscopic observation – e.g. look for tapeworm segments, note characteristic smell in giardiasis
Microscopy – light microscope ( e.g. blood slide for malaria), electron microscope (e.g. faecal sample for microsporidia)
Immunological based assays (e.g. immunofluorescence for parasite antigens in faeces)
‘Point of care’ test ( e.g. dipstick for Leishmania spp parasites in blood)
Molecular techniques – (e.g.PCR to detect and determine species of Entamoeba spp in faeces)
Antibody detection techniques (e.g. serological tests for Toxoplasma gondii)

Question 3

Diagnosis of faecal parasites?

Answer 3

Collect faecal specimen and take to laboratory
In the laboratory :
Have a good look at it and record its colour, consistency and smell!
Look out for whole worms or segments (tapeworm proglottids)

Question 4

Techniques for detection of faecal parasites?

Answer 4

Light Microscopy - ‘classical’ method for most protozoan cysts and helminth ova
Electron Microscopy - for very small structures e.g. microsporidia
Immunofluorescence - developed for a few protozoan cysts
Enzyme linked immunoassay - several tests available for antigen in faeces and /or antibody in serum

Question 5

Light Microscopy- preparation?

Answer 5

Preparation of faecal sample
1.Direct wet smear
2. Concentration of sample before making smear :
Saturated salt/sugar solution (SSS) concentration
Formol-ether concentration

Question 6

Direct faecal smear- how is it prepared and what stain is used?

Answer 6

Use saline smear to look for large structures with their own colour, such as helminth eggs
Use iodine smear to highlight details inside smaller, transparent structures, such as protozoan cysts

Question 7

Outline the Saturated salt (or sugar) solution concentration

Answer 7

dipped and brought out after 45 mins

Question 8

Outline the Formol-ether technique

Answer 8

Formol water = 10% solution of formaldehyde
1g faeces + 7 ml formol water
MIX by shaking!
Sieve mixture into strong test tube
Add 3-4 ml diethyl ether
Vortex to mix thoroughly, then centrifuge
Examin -Sediment, containing eggs and cysts (if there were any in original sample)

Question 9

Stained faecal smear- how is it prepared and what stain is used?

Answer 9

Prepare saline smear but do not place coverslip on top
Leave to dry in air, then fix in alcohol
Stain with modified Ziehl-Neelsen stain (Cryptosporidium parvum)
Or specific fluorescent-labelled antibody (Cryptosporidium parvum, Giardia duoedenalis, Entamoeba histolytica)

Question 10

Electron microscopy- when is it used?

Answer 10

Not usually required in routine parasitological diagnosis
Used in diagnosis of microsporidia which produce spores (not cysts) - and are in fact probably fungi!
The spores are difficult to see by light microscopy (~1.5 mm)

Question 11

Electron Micrograph of Enterocytozoon bieneusi spore

Answer 11

Double rows of polar tubule coils - diagnostic of E.bieneusi

Question 12

Detection of blood-borne parasites

Answer 12

Protozoa at various life cycle stages
Collect blood at any time whether patient symptomatic or not
Filarial worms in larval stage (‘microfilariae’)
Some species only appear in peripheral blood every 12 or 24 hours, so have to time blood collection carefully!

Question 13

Techniques for Detection of blood-borne parasites?

Answer 13

Light Microscopy of stained thick and thin blood films and stained tissue smears
‘Dipstick’ tests for parasite antigens (Plasmodium spp.)
Quantitative Buffy Coat (Plasmodium spp. Trypanosoma spp.)
ELISA for anti-parasite serum antibodies

Question 14

Difference and similarity between a thick and thin blood film

Answer 14

Thick film - no spreading or alcohol fix

Use Giemsa stain

Question 15

OptiMAL® test

Answer 15

Antigen capture test method
Exploits the fact that Plasmodium spp. produce a Lactate Dehydrogenase enzyme (pLDH) which has no antigenic cross reaction with human LDH
Also Plasmodium falciparum produces antigenically distinct pLDH

Question 16

Quantitative Buffy Coat

Answer 16

1- Float
Acridine Orange (DNA intercalating agent - fluorescent)
Potassium oxalate (ant-coagulant)
2- Add patient’s blood and centrifuge the tube
3- Examine under ultra violet light
Any parasites inside RBCs will fluoresce because of the parasite DNA (uninfected RBCs have no DNA)
e.g. Trypanosoma spp., Plasmodium spp.

Question 17

The ability to detect a parasite via its DNA can be useful when?

Answer 17

The parasite is only present in tissues and/or organs

The parasite only occurs in the body in very small numbers an is difficult to culture in vitro

Question 18

Leishmania spp- how do you find it in a clinical specimen

Answer 18

Depending on species, take tissue biopsies or aspirates from bone marrow + spleen or buffy coat
Make smear on slide and stain with Giemsa stain
Leishmania parasites in stained smears are very small and hard to detect
Many species and sub-species of Leishmania, most of which were distinguished by DNA sequence - PCR using carefully designed primers

Question 19

Which species can be detected with PCR?

Answer 19

Many species and sub-species of Leishmania, most of which were distinguished by DNA sequence - PCR using carefully designed primers

Question 20

Molecular techniques can also be very useful in diagnosis of parasites.
Name an example?

Answer 20

For example in E. histolytica infection, some patients have mild diarrhoea, some have dysentery and a few have invasive amoebic disease
One causes the severe symptoms now called E.histolytica
The other is associated with mild or no symptoms, now called E.dispar

Question 21

Entamoeba dispar cysts and amoebae look exactly the same as Entamoeba histolytica cysts and amoebae,
How is it diagnosed?

Answer 21

Diagnosis by Polymerase Chain Reaction using species specific primers