Modern Techniques

in Diagnostic
Parasitology Gaza
Strip
Prof. Dr. Adnan Al-Hindi, PhD
Faculty of Health Sciences
Medical Laboratory Sciences
Department

Outline
-Direct

smear microscopy
-Concentration techniques
-Staining
-Culture
-Antigen detection
-Antibody detection
-Molecular methods

DSM
-This is the conventional method in
intestinal
parasites
detection
worldwide. It is the standard gold
method, where in major cases
give the accurate result.
-The microscopic examination of
stool is needed for the
recognition and identification of
intestinal parasites and useful for
the observation of motile
protozoan trophozoites.

DSM
-Many protozoa can be easily diagnosed
(Entamoeba histolytica/dispar, Giardi
alamblia, Entamoeba coli and others.
-Some types cannt be detected by DSM
(depend on experience) but need
staining like: Cryptosporidium sp.
-It is recommended to do three tests in
three consecutive days.

Photo of protozoa in DSM

Concentration
techniques
In light infection we can do the concentration technique as
. confirmatory test
It increases the possibility of seeing .worm eggs, larvae, and protozoan cysts
The purpose of concentrating stool is to increase possibility to finding ova, cyst,
or larvae in samples that not be able to
seen by direct microscopy

Concentration techniques
Sedimentation method
Modified Formal- Ether sedimentation
technique
Acid- Ether sedimentation technique
Flotation method
Saturated Salt Solution technique
Sheathers Sugar Centrifugal Flotation
technique
Zinc Sulphate Centrifugal Flotation
technique

Protozoa Staining
Iron Haematoxylin Solution A and

Solution B
The background should stain grey with the
protozoa light blue, and the nuclei blue. black
Trichrome for MicrosporidiaMicrosporidial spores are ovoid and
refractile and the spore wall stains bright
pinkish-red. The spores are approximately
1.5 by 0.9. The background debris and
bacteria are counterstained faint green.

Trichrome for Protozoa

May be used to stain fresh stool or
cultured organisms.
Malachite Green is a green
counterstain used to differentiate
bacteria
Modified Z/N Stain Pack (Cold
Kinyoun)
Acid fast organisms stain red, the
background and other organisms stain
blue:
Example: Cryptosporidium sp.

Culture of protozoan
parasites

The in vitro culture of protozoan

parasites involves highly complex
procedures, which are subject to
many

variables.

These

parasites

have very complex life cycles and,

depending on the life cycle stage,
may

require

parameters.

different

culture

When using culture

Only a minority of parasitic
infections are diagnosed routinely
.by cultural techniques
Drugs discovery

However,
in
vitro
cultivation
is
important for many reasons, some of
which include:
Diagnosis,
antigen
and
antibody
production, assessment of parasite
immune modulating capabilities, drug
screening,
improvements
in
chemotherapy,
differentiation
of
clinical
isolates,
determination
of
strain differences, vaccine production,
development of attenuated strains,
and the continued supply of viable
organisms for studying host-parasite
interactions.

Zuhair
Evaluating the
Dardoun effect of selected
ah
medicinal plant
materials and
their synergistic
effect with
Metronidazole
against
Entamoeba
histolytica

2014

Detection of Parasite
Antigens
diagnosis of human intestinal

The
protozoa
depends
on
microscopic
detection of the various parasite stages
in feces, duodenal fluid, or small intestine
biopsy
specimens.
Since
fecal
examination is very labor-intensive and
requires a skilled microscopist, antigen
detection tests have been developed as
alternatives using direct fluorescent
antibody (DFA), enzyme immunoassay
(EIA), and rapid, dipstick-like tests (CDC,
2014) accessed in 30-1-2015.

Antigen
detection
methods
can
be
performed quickly and do not require an
experienced and skilled morphologist. Much
work has been accomplished on the
development of antigen detection tests,
resulting in commercially available reagents
for
the
intestinal
parasitesCryptosporidiumspp.,Entamoeba
histolytica,Giardia
duodenalis,
andTrichomonas vaginalis. In addition,
antigen detection tests using blood or
serum
are
available
forPlasmodiumandWuchereria
bancrofti.

Specimens for antigen

detection
Fresh or preserved stool samples
are the appropriate specimen for
antigen detection testing with
most kits, but refer to the
recommended
collection
procedures included with each
specific kit.

Situations where
immunodiagnosis is
important
In early prepatent and in chronic phases. 1
of infection when the diagnostic stage of the
parasite is scanty and can be missed on
.direct examination
When the parasite cannot be precisely. 2
located for sampling e.g. visceral larva
.migrans

When sampling is impossible or. 3

hazardous e.g. cerebral
. toxoplasmosis and hydatid cyst
.
For differentiation between true and. 4
.spurious infections (e.g. fascioliasis)
.For follow up after treatment. 5
In epidemiological studies where. 6
large numbers of specimens can be
.simultaneously tested

Amebiasis
EIA kits are commercially available for detection
of fecal antigens for the diagnosis of intestinal
amebiasis. Organisms of both the pathogenicE.
histolyticaand the nonpathogenicEntamoeba
disparstrains are morphologically identical.
These assays use monoclonal antibodies that
detect the galactose-inhibitable adherence
protein in the pathogenicE. histolytica. The
primary drawback of these assays is the
requirement for fresh. Several EIA kits for
antigen detection of theE. histolytica/E.
dispargroup are available in the U.S., but only
the TechLab kit is specific forE. histolytic

Manufact
urer Type of
Organism Kit name
distribut
Testb
ora
Crypto
Cellabs
EIA
CELISA
PARAMedical
TECT
Chemical
Cryptospor
EIA
Corporatio
idium
n
Antigen 96
Cryptosp ProSpecT
Remel
EIA

Immunodiagnosis
Serologic methods are availablein cases such as toxoplasmosis,
trichinosis, echinococcosis,
cycticercosis, chronic
schistosamiasis, or extraintestinal amebiasis, where the
organism is not readily
.demonstrated

Serology (ELISA)
-Copro-antigen ELISAs are commonly used
to diagnose canine infection.

Molecular diagnosis parasites

Microscopic examination is still considered
the "gold standard" for the diagnosis of
parasitic diseases. 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.

Why we use molecular

diagnosis for parasites

1. Offer greater sensitivity and

specificity over the existing
diagnostic tests.
2. Differentiation between similar
morphological types.
3. They permit the detection of
infections from very low parasitized
samples including those from
asymptomatic patients samples.

4. multiplexed PCR allows for the detection

of multiple sequences in the same reaction
tube proving useful in the diagnosis of
several parasitic
infections simultaneously.

Polymerase Chain Reaction

(PCR)
The PCR makes it possible to perform
selective
amplification
from
complex
genomes. This technique is based on the
process of denaturing a double-stranded
genomic DNA template using heat. Next, the
temperature is lowered to ensure that
primers can anneal to their complementary
sequences into the template. Thus, the
elongated DNA template follows in both
directions from the primer site by means of
enzymatic catalysis with a thermostable
DNA
polymerase,
generating
doublestranded products .

Real-Time Polymerase Chain

.Reaction (RT-PCR)
It is a system unlike conventional PCR,
allow for the quantification of the
original
templates
concentration
through the use of various fluorescent
chemistries,
such
as
Sybergreen,
Taqman probes, fluorescence resonance
energy transfer (FRET), and Scorpion
primers [7]. The concentration is
measured through comparison to
standard curves.

Loop-Mediated Isothermal
Amplification (LAMP)
Loop mediated isothermal amplification
(LAMP) is a unique amplification method
with extremely high specificity and
sensitivity able to discriminate between a
single
nucleotide
difference.
It
is
characterized by the use of six different
primers specifically designed to recognize
eight distinct regions on a target gene,
with amplification only occurring if all
primers bind and form a product.

In the past, LAMP has been successfully

applied for the rapid detection of both
DNA and RNA viruses such as the West
Nile and SARS viruses . Recently,
parasitologists have adapted the LAMP
approach for the detection of several
parasitic diseases including the human
parasites Entamoeba,Trypanosoma,
Taenia, Plasmodium, and Cryptosporidium,
the animal parasites Theilera, and Babesia
and even to the identification of vector
mosquitoes carrying Plasmodium and
.Dirofilaria immitis parasites

.Luminex xMAP Technology

Luminex
is
a
bead-based
xMAP
technology (multianalyte profiling), a
system that combines flow cytometry,
fluorescent
microspheres
(beads),
lasers and digital signal processing, and
is capable of simultaneously measuring
up to 100 different analytes in a single
sample. It is possible to cover each set
of microsphere beads by utilizing a
reagent specifically designed for a
particular bioassay.

Adapted to the study of parasites, the

Luminex assay could identify multiple
organism or different genotypes of one
particular organism during the same
reaction utilizing very low volume. The
approach could prove useful in the
study of antigenic diversity and drug
resistance alleles and for the diagnosis
of parasitic diseases.

Luminex was applied to the study of

Cryptosporidium. C. hominis and C.
parvum cannot be distinguished using
antigen detection or serology assays.
Only DNA-based approaches have
been successful in doing so by
exploiting
the
single
nucleotide
difference in the microsatellite-2
region (ML-2) of both species.

Ultimately DNA sequencing is the diagnosis

tool of choice but it is costly, labourintensive and time-consuming. In a recent
study, Bandyopadhyay et al. successfully
detected and distinguished C. hominis and
C. parvum in 143 DNA extracts using
Luminex
technology
by
using
oligonucleotide probes specific to the ML-2
regions of each species.
Similarly in other research, Luminex technology
was
able to detect all-blood stage parasite levels of
the four human Plasmodium species (falciparum,
.vivax, malariae, and ovale) simultaneously

.Proteomics
Since proteins are the main catalysts,
structural
elements,
signaling
messengers, and molecular
machines
of
biological
tissues,
proteomic studies are able to provide
substantial clinical relevance. Proteins
can be utilized as biomarkers for
tissues, cell types, developmental
stages, and disease states as well as
potential targets for drug discovery and
interventional approaches.

Random Amplified Polymorphic

Known as AP-PCR
(arbitrarily
DNA
(RAPD)primed PCR),

RAPD has been extensively used for

description of strains in epidemiological
studies. The surveying of genomes of
parasites is enhanced by the advantage that
RAPD is a very simple, fast, and inexpensive
technique that does not require either prior
knowledge of the DNA sequence or DNA
hybridization. (Ex Lesihmania 20 sp.)
RAPD is particularly useful for studying the genetic
structure of populations because it reveals
polymorphisms in the noncoding regions of the
genome

Restriction Fragment Length

Polymorphism (RFLP)
The RFLP technique is currently one of
the most commonly used molecular
methods for diagnosis of species and
genotypes of parasites such as
Toxoplasma gondii. This technique was
first used to detect variations at the
DNA level. This reaction is based on
the digestion of the PCR products by
restriction enzymes or endonucleases.

These enzymes cleave DNA into fragments of

certain sizes, whose analysis on agarose or
polyacrylamide gel results in different
patterns of fragment sizes, enabling the
identification. The RFLP technique is suitable
for environmental samples because it
permits the detection of multiple genotypes
in the same sample. Ex. Cryptosporidum sp.
RFLP technique can also be used in the
differentiation of animal parasites, such as
species of Theileria in sheep. A study
conducted by Zaeemi et al. in Iran was able
to differentiate Theileria lestoquardi, T. ovis,
and T. annulata.

DNA Extraction
It is necessary to extract DNA from the
stool specimens for PCR detection.
Click to view theDNA extraction
protocolsrecommended
for
molecular diagnosis of intestinal
parasites.

PCR Analysis
Molecular
detection
ofCyclospora
cayetanensis,Entamoeba
histolytica,
andE. disparis performed at CDC by both
conventional
PCR
and
real-time
PCR.
Conventional
PCR
is
available
formicrosporidia.

Amplified products detection

Three microlitres of PCR-amplified DNA was
detected by 2% agarose gel electrophoresis
in TAE buffer (0.04 mM Tris-acetate, 0.001
mM EDTA, pH 8.0), stained in a solution of
ethidium bromide (0.5 mgml-1) and
visualized with a UV transilluminator.

PCR

Agarose gel
Electrophoresis

Example E.
histolytica/dispar

Preliminary results for

Ecchinococus granulosus in dogs
1 Kb-----850 Kb--650 Kb--500 Kb--400 Kb--300 Kb-----Kb 200
---Kb 100
Fig. 1 Gel electrophoresis of PCR product generated from Echinococcus ganulosus from
dog

Molecular findings in cattle

650 Kb
500 Kb
400 Kb
300 Kb
200 Kb
Kb 100

Fig.2 Gel electrophoresis of PCR product generated from Hydatid cyst from cattle

The present study included 30 cattle

and sheep samples. The results
showed that 14/30 (46.6%) were
positive for hydatid cyst using
polymerase chain reaction.

Amal Epidemiology of
AlTrichomonas
Maqad vaginalis infection
ma among infertile
women in Gaza
city, Palestine

2014

dentification of two distinct species of Plasmodium responsible for

ovale malaria
People: Colin Sutherland,Mary Oguike, Spencer Polley (HTD), Peter Chiodini
(HTD),Debbie Nolder(MRL), Martina Burke (MRL)

A collaborative study between the UK Malaria Reference Laboratory, LSHTM,

the Hospital for Tropical Diseases London and Mahidol University, Bangkok has
recently demonstrated the existence of a previously unrecognised speciation
between two forms of the Plasmodium parasite causing ovale malaria. We
have named these two forms P. ovale curtisiorP. ovale wallikeri, after two
recently deceased colleagues, Chris Curtis and David Walliker. Extending this
work into the field, Mary Oguike and our collaborators have found that in
some parts of Africa both these species co-exist in the same villages without
inter-breeding or recombining. Future work with African and Asian
collaborators is planned to further unravel the host preferences,
epidemiology, population genetics and transmission biology of these newly
.recognised species
See more at: http://www.lshtm.ac.uk/itd/iid/groups/sutherland- -
hallett_lab/diagnostics/#sthash.pi8CiGNh.dpuf

http://www.lshtm.ac.uk/itd/iid/g
roups/sutherlandhallett_lab/diagnostics/
3-2-2015