MEDICAL HELMINTHOLOGY AND PROTOZOOLOGY

09/18/2022 (3rd week)

SPECIMEN COLLECTION HANDLING AND PROCESSING
PART 1
PURPOSE OF PARASITOLOGY LAB
 confirm a clinical impression that the condition has a
parasitic nature;
 rule out differential diagnoses;
 aid a clinician in the choice of proper medication; to
tab
 help in monitoring the effect of a treatment regimen
DIAGNOSIS OF PARASITIC INFECTION FECT – Formal Ether Concentration Techniques
• Demonstration of parasite components (definitive
diagnosis)
o Adults
o Eggs
o Larvae
o Oocyst
o Trophozoites
o Antigen
• Detection of host immune response
(presumptive/evidence of infection)
o Antibody
SPECIMENS FOR EXAMINATION
• Stool (most common) • CSF
• Urine • Tissue aspirate
• Blood • Tissue biopses
• Sputum • Orifice swab
EXAMINATION OF STOOL OR FECAL SMEAR
• Most common – helminths (nematodes, cestodes,
trematodes) or protozoan parasite
• Stool helps in demonstration of eggs, larvae, adults,
trophozoites, cysts or oocysts in stool
• Specimen should be submitted with the following
information
• Patient’s name
• sex
• date/time of collection
• requesting physician
• requested procedure
• presumptive diagnosis gi disorder amoebiasis
• prior infections
• travel history
Labels on the container: MICROSCOPIC EXAMINATION
• Name: • WET mount is the simplest and easiest technique:
• Date and time of collection: • Saline wet mount
o initial microscopic examination
o Demonstration of worm eggs, larvae, protozoan
cyst and trophozoites
o Reveals the presence of red blood cells
• Iodine wet mount
o Mainly to stain glycogen and nuclei of cysts
• Buffered methylene blue wet mount
o Should be prepared when are or
presence is suspected
MEDICAL HELMINTHOLOGY AND PROTOZOOLOGY
09/18/2022 (3rd week)
o Stains troph but not cyst

Kato Thick Smear or Kato Katz smear

MICROSCOPIC EXAMINATION
• White blood cell (PMN, eosinophil)
• Red blood cells
• Macrophages
• Charcot leyden crystals
• Epithelial cells
• Eggs of arthropods, plant nematodes and other
spurious parasites
• Fungal spores like candida, yeast
3. Concentration Techniques
• Elements of plant origin (pant cells, pollen grains,
• Concentration techniques can separate protozoan
starch granules, vegetable spiral)
cysts and helminth eggs from a larger amount of stool
• Plant and animal hair
(usually 1 g in amount) based on differences in specific
gravity to tab
TECHNIQUES USED FOR STOOL EXAMINATION
• In cases of light infections, or if there is a need to
1. Direct fecal smear
recover more parasites, stool concentration procedures
- routine method of stool examination primarily useful
are recommended
in the
A. Sedimentation Procedures
- detection of motile protozoantrophozoites(BMB
i. Acid Ether Concentration Technique (AECT)
stain)
- 40% HCI (dissolve albuminous material
- Protozoan cysts
- Ether (dissolve neutral fats in stool)
- Helminth eggs and larvae
- Recommended for the recovery of Trichuris, Capillaria,
- About 2 mg of stool (amount forming a low cone at the
and trematode eggs, especially Schistosoma
tip of an applicator stick) is comminuted thoroughly
- This is also the choice if stool material comes from
with a drop of 0.85% sodium chloride solution (NSS)
animals like cats and dogs.
and then covered with a cover slip.
- Drawbacks in the use of this technique include: loss of
parasite to the plug of debris and possible destruction of
protozoan cysts.
MEDICAL HELMINTHOLOGY AND PROTOZOOLOGY
09/18/2022 (3rd week)
- There is no need for centrifugation since helminths egg
rise to the surface of the solution.
- This technique is low cost and simple but helminth eggs
like hookworm and Schistosoma become badly
shrunken.
- This is for Operculated eggs like Clonorchis, Opistorchis,
and heterophyids because these do not float in brine
solution

ii. Formalin-ether/ethyl acetate concentration technique

(FECT)
- 10% formalin (fixative)
- Ether
- This is useful in the recovery of both helminth
eggs and protozoan cysts.
- FECT can also be done with formalin-preserved
and PVA-preserved stools.
- More parasites can be from formalin preserved
samples.
- Parasite morphology is also better preserved in
formalin than in PVA.
- Sediments from FECT can stored for a long
period of time

B. Floatation Procedures
i. Zinc sulfate floatation
- 33% Zinc sulfate (SG 1.18-1.20)
- If parasites are exposed to high specific gravity,
distortion and shrinkage of protozoan cysts and thin-
walled nematode eggs may occur
ii. Brine floatation
- saturated table salt solution
MEDICAL HELMINTHOLOGY AND PROTOZOOLOGY
09/18/2022 (3rd week)
MEDICAL HELMINTHOLOGY AND PROTOZOOLOGY
09/18/2022 (3rd week)
SPECIMEN COLLECTION HANDLING AND PROCESSING After airdrying, slides are fixed with
PART 2 methanol before staining.

METHODS OF ANALYSIS
A. Finger prick
1. Wet/Fresh preparation
Microfilariae and trypomastigotes are large
and motile in fresh blood preparations. Their
presence in the sample can therefore be
easily detected. Species identification,
however, is not possible with the wet mount.

BLOOD STAINS
2. Stained Smears  Giemsa – red cells stain pale red, white cell nuclei
a. Thick Film – prepare from two to three stain purple, eosinophils stain bright purple red, and
small drops of blood which are mixed neutrophils stain deep pink purple.
and spread with continuous movement
over an area which is about 2 cm in
diameter. Films are then thoroughly
dried and then dehemoglobinized prior
to staining.

 Wright’s Stain – already contains alcohol, so fixation

is not needed before staining. Stained smears show
light red erythrocytes, bright blue nuclei of
leukocytes, bright red eosinophilic granules, and pink
neutrophilic granules.
b. Thin Smear – prepared in such a way
that they are thick at one end, and thin
and feathery at the other end. Streaks
and holes should be avoided in the film.
Clean slides and spreaders are used.
MEDICAL HELMINTHOLOGY AND PROTOZOOLOGY
09/18/2022 (3rd week)
 Delafield hematoxylin stain – is mainly useful in
demonstrating the detailed structures of
microfilariae. In this method, thick films are
dehemoglobinized in 2% formalin with 1% acetic
acid. The main stain is a mixture of hematoxylin and
ammonium alum which enhances nuclear detail and
morphological features.
B. Venous blood
a. Knott’s concentration
In cases low microfilaremia, 1 ml of blood
can be mixed with 10 ml of 2% formalin and
then centrifuged. The supernate is discarded
and the sediment is studied. Part of the
sediment can be spread like a thin blood film
and stained.

b. Membrane Filtration
3. Capillary tube method
Microfilariae and trypomastigotes are large
and motile in fresh blood preparation. Their
presence in the sample can therefore be
easily detected. Species identification,
however, is not possible with the wet mount.
This technique makes use of syringe
attached to a Swinney filter holder. One ml
of fresh or anticoagulated blood is drawn up
into the syringe and lyzed by adding 10 ml of
distilled water. The lyzed blood is then
passed through the Swinney membrane
filter where microfilariae will be recovered.
The membrane filter can be examined like
wet smear preparation or may be dried
fixed, and then stained.

EXAMINATION OF SPUTUM
 Migrating larvae of Ascaris lumbricoides,
Strongyloides stercoralis, and hookworms
 Paragonimus ova
 Echinococcus granulosus hooklets from pulmonary
hydatid cysts
 Protozoa such as:
o Entamoeba histolytica trophozoites
from pulmonary amebic abscess
o Crypstosporidium parvum oocysts,
although very rare
MEDICAL HELMINTHOLOGY AND PROTOZOOLOGY
09/18/2022 (3rd week)
o Non-pathogenic Entamoeba gingivalis
and Trichomonas tenax

EXAMINATION OF URINE
• First morning specimen (best)
• Very good for diagnosis of Trichomonas vaginalis
• Wuchereria bancrofti microfilariae from chyluric
samples
• Schistosoma haematobium
MEDICAL HELMINTHOLOGY AND PROTOZOOLOGY
09/18/2022 (3rd week)

ADVANCES IN DIAGNOSTIC PARASITOLOGY –

IMMUNODIAGNOSIS
• Immunofluorescent assay (IFA)
• Enzyme-linked immunosorbent assay (ELISA)
• Hemagglutination test (HA)
• Immunoblotting (dot blot)

ADVANCES IN DIAGNOSTIC PARASITILOGY –

MOLECULAR DIAGNOSIS
• Nucleic acid-based assays offer greater sensitivity
and specificity than the above mentioned tests. They
allow for direct detection of parasites in samples
including those with very low parasite load from
asymptomatic patients. The use of gene
amplification technology by polymerase chain
reaction (PCR) detects nucleic acid sequences
specific to the parasite in question.
• Molecular Diagnosis in Stool
 Cryptosporidium spp.,
 Cyclospora cayetanensis,
 E. histolytica, and E. dispar,
 Giardia duodenalis and microsporidia
MEDICAL HELMINTHOLOGY AND PROTOZOOLOGY
09/18/2022 (3rd week)
• Molecular Diagnosis of Blood Specimens
 Malaria spp and other protozoa

ADVANCES IN DIAGNOSTIC PARASITOLOGY – RAPID

DIAGNOSTIC TESTS

 RDTs use antibodies (monoclonal or polyclonal) to

detect parasite antigens in blood, stool, urine or
other body fluids. These assays employs
immunochromatographic methods in lateral flow
devices where results are available within 15
minutes. They do not require skilled microscopists
but provide accurate diagnosis in a timely manner
important for prompt and appropriate treatment.