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Laboratory investigations

of
Malarial Parasite

Dr. Md Moyez Uddin


Director
Institute of Public Health
Sujan Chandra Mondal
Tests for Malarial Parasites
Microscopic test Non microscopic test
1.Peripheral blood Rapid Diagnostic Tests (RDTs)
film study Para Sight F test
2.Quantitive Buffy OptiMal Assay
Coat (QBC) test The immunochromatographic
test (ICT Malaria P. f. test)
Polymerase Chain Reaction
Detection of antibodies by Radio
immuno assay,
immunofluorescence or enzyme
immuno assay

Sujan Chandra Mondal


Procedure of Diagnosis
♦ Collection of blood

♦Preparation

♦Stain
-Giemsa stain
(1:20 dilute)
30min
-Field’s stain
4-5 second
‘A’ Polychrome methyline blue
‘B’ Eosine

-Leishman stain

Sujan Chandra Mondal


Preparation of PBF
Step 1 Step 2

Hold the third finger of the Prick the finger with


left hand and wipe its tip disposable needle/lancet;
with spirit/Savlon swab; allow the blood to ooze out
allow to dry
Sujan Chandra Mondal
Preparation of PBF
Step 3 Step 4

Take a clean glass slide. Take another clean


Take 3 drops of blood 1 slide with smooth edges
cm from the edge of the and use it as a
slide, take another drop spreader...
of blood one cm from the
first drop of blood Sujan Chandra Mondal
Preparation of PBF
Step 5
Step 6

Prepared smear.
and make thick and thin (Slide number can be
smears. Allow it to dry marked on the thin smear
with a lead pencil.)
Sujan Chandra Mondal
Thick film of MP
♦ Microscopic
view
RBC disappeared

Streaks of blue
cytoplasm with
detached nuclear dot
appearance like
‘coma’ or
‘exclamation mark’

WBC remain unchange

Sujan Chandra Mondal


Thick film
 Advantage
♦ Parasite density
- Rapid detection of parasite (*)
-Number of parasites counted in each - Concentrating parasite 20 fold
microscopic field than thin film
-density considered in thick film - Guide to Rx for testing efficacy
20 or more /field : high density of antimalarial drug
2-19 / field : medium density
1 or less ; low density  Disadvantage
- species cannot detected
-It is important to report parasite
density
( *) 1 microscopic field of thick
film equavalent to 50
microscopic field of thin film

Sujan Chandra Mondal


Thin film
♦ Certain rules before
declared negative
-upper & lower margin
of the tail end of film should
examine thoroughly because
parasite mostly numerous here
-minimum 100 field
must examined
-time taken for examintion
at least 8 to 10 min.

Sujan Chandra Mondal


Diagnostic points:-
♦ Red Cells are not
enlarged.
♦ Rings appear fine and
delicate and there may
be several in one cell.
♦ Some rings may have
two chromatin dots.
♦ Presence of marginal
or accole forms.
♦ It is unusual to see
developing forms in
peripheral blood
films.
♦ Gametocytes have a
characteristic crescent
shape appearance.
However, they do not
usually appear in the
blood for the first four
weeks of infection.
♦ Maurer's dots may be
present.

Sujan Chandra Mondal


Diagnostic points:-
♦ Red cells containing
parasites are usually
enlarged.
♦ Schuffner's dots are
frequently present in
the red cells as
shown above.
♦ The mature ring
forms tend to be large
and coarse.
♦ Developing forms are
frequently present.

Sujan Chandra Mondal


Diagnostic points :-
♦ Ring forms may have a squarish appearance.
♦ Band forms are a characteristic of this species.
♦ Mature schizonts may have a typical daisy head appearance with up to
ten merozoites.
♦ Red cells are not enlarged.
♦ Chromatin dot may be onSujan
theChandra
inner Mondal
surface of the ring.
Diagnostic points :-
♦ Red cells enlarged.
♦ Comet forms
common (top right)
♦ Rings large and
coarse.
♦ Schuffner's dots,
when present, may
be prominent.
♦ Mature schizonts
similar to those of P.
malariae but larger
and more coarse

Sujan Chandra Mondal


P. vivex P.falciparum

P.malariae P.ovale

Sujan Chandra Mondal


QBC test
♦new method
♦involves staining of the centrifuged and
compressed red cell layer with acridine orange
and its examination under UV light source.
♦key feature-centrifugation & concentration of
the red blood cells in predictable area of the
QBC tube, making detection easy and fast.
Parasitized RBC less dense than normal ones
& concentrate just below the leukocytes, at the
top of the erythrocyte column. The float forcs
all the surrounding red cells into the 40 micron
space between its outside circumference and
the inside of the tube. Since the parasites
contain DNA which takes up the acridine
orange stain, they appear as bright specks of
light among the non-fluorescing red cells.
Virtually all of the parasites found in the 60
microliter of blood can be visualized by
rotating the tube under the microscope.

Sujan Chandra Mondal


Fluorescense UV ray microscopy

Sujan Chandra Mondal


Other Rapid test (RDT’s) of MP
♦ new method

OptiMal Assey Kit

OptiMal Assay result


Sujan Chandra Mondal
Immunochromatographic (ICT) Tests for Malaria
Antigens ( HRP-2, LDH, Aldolase)

♦ based on the capture of the


parasite antigens from the
peripheral blood using either
monoclonal or polyclonal
antibodies against the
parasite antigen targets.

♦ can target the histidine-rich


protein 2 of P. falciparum, a
pan-malarial Plasmodium
aldolase, and the parasite
specific lactate
dehydrogenase
.
♦ do not require a laboratory,
electricity, or any special
equipment.

Sujan Chandra Mondal


Comparison between peripheral smear and QBC test for
detecting malaria

Peripheral QBC
smear

Method Cumbersome Easy

Time Longer, 60 - 120 Faster, 15 - 30 minutes


minutes
Sensitivity 5 parasites/µl in Claimed to be more sensitive, at least as good as a thick
thick film and 200 film
/ µl in thin film
Specificity Gold standard ? False positives, artifacts may be reported as positive by
not-so-well-trained technicians

Species Accurate, gold Difficult to impossible


standard
identificatio
n

Cost Inexpensive Costly equipment and consumables

Acceptabilit 100% Not so


y
Availability Everywhere Limited
Sujan Chandra Mondal
Peripheral Smear Rapid Diagnostic Tests

Format Slides with blood smear Test strip


Equipment Microscope Kit only
Training Trained microscopist 'Anyone with a little trai
Test duration 20-60 minutes or more 5-30 minutes
Test result Direct visualization of the Color changes on antibody
parasites lines
Capability Detects and Detects malaria antigens (P
differentiates all PMA/pLDH) from asexual
plasmodia at different sexual forms of the par
stages
Detection threshold 5-10 parasites/µL of 1 00-500/µL for P. falciparum
blood for non-falciparum
Species Possible Cannot differentiate amon
differentiation falciparum species; mixed in
of P. falciparum and non-fa
appear as P. falciparu
Quantification Possible Not possible
Differentiation Possible Not possible
between sexual and
asexual stages Sujan Chandra Mondal
Sujan Chandra Mondal
Sujan Chandra Mondal