M

A
L
A
R
I
A

C
O
N
T
R
O
L

P
R
O
G
R
A
M

2
0
0
8
 Learning Objectives

M At the end of the session, the participants should be

A
L able to:
A
R
I
A

C
O Enumerate the various laboratory diagnosis for malaria.
N
T
R
Discuss the importance of laboratory based diagnosis
O
L over clinical diagnosis.
P Discuss the use and principle of Rapid Diagnostic Test
R
O
G
Demonstrate skills in performing the RDT
R
A Explain the advantages and disadvantages of RDT
M

2
Discuss the proper way of handling, storing and
0
0
transporting RDTs
8
Interpret correctly and act accordingly to the result of
RDT.
M
A
L
A
R
I
A

Diagnosis
C
O
N
T

for
R
O
L

Malaria
P
R
O
G
R
A
M

2
0
0
8
M
A
L
A Rapid and accurate diagnosis
is key to effective treatment
R
I
A

C
and management of malaria.
O
N
T
R
O
L
Conventional approaches
most often do not allow
satisfactory diagnosis of
P
R
O
G malaria.
R
A
M

2 1. Clinical assessment
0
0
8
2. Microscopy
 Conventional methods of malaria diagnosis
M
A
L Clinical assessment Microscopy
A
R
I
A

C
gold standard
O
N
Based on signs and based on demonstration
T
of parasites in blood films
R
O symptoms and history of
L
travel to a malaria- requires high degree of
P expertise for reliable
R
O
endemic area results
G
R
A Done by trained health needs special equipment
M
(microscope) and good
2
workers in areas where reagents
0
0 microscopy is not
labor-intensive and time
available
8

consuming

Not very specific

 MALARIA RAPID DIAGNOSTIC TESTS
(RDTs)
M
A
L
A
R
I
A

C
O
N
T
R
O
L

P
R
O
G
R
A
M

2
0
0
8
M Malaria RDTs
A
L
A
R
I
A

C
O Also called dipsticks or malaria rapid diagnostic
devices.
N
T
R
O
L
Lateral flow immuno-chromatographic antigen-
P
detection tests, in dipstick or cassette format
R
O
Detect antigens (or proteins) produced by the
G
R
malaria parasites in human blood.
A
M Some RDTs detect P. falciparum only
2 (Ex. Paracheck Pf ).
Some detect P. falciparum + P.vivax or P.malariae
0
0
8
or P. ovale (OptiMAL, ICT).
Single test takes about 10-20 minutes.
They come in cassette, dipstick or card formats.
 Guidelines on the use of Rapid
M
Diagnostic Test
A
L
A
1. In remote areas where there is no microscopy
R
I center.
A

C
2. In areas where clients take more than 2 hours travel
O
N to the nearest microscopy centers as in inaccessible
T
R coastal or island areas.
O
L
3. In epidemic situations where microscopy is not
P
R
O
available, RDT maybe used.
G
R 4. In selected hospitals without a trained microscopist
A
M in emergency situation, RDT may be used at the
2
0
same time, take blood film for confirmation.
0
8 5. Do not use RDT as diagnostic method in afebrile and
follow-up of patients.
 Mechanism of action
M
A
L
A
R
I
A 1. Dye-labeled specific antibody (Ab) is pre-deposited
C
O
on nitrocellulose strip or in a plastic well. Ab specific
N
T
for target antigen (Ag) is bound to the strip in test
R
O line, and Ab specific for labeled Ab, or Ag is bound at
L
the control line.
P
R
Test band
O
G
(bound Ab)
R
A
M
Control band
2
Labeled Ab (bound Ab)
0
0 unbound bound Ab
8
labeled Ab
 Mechanism of action
M
A
L
A
R

2. Blood is lysed with buffer to release more proteins

I
A

C
O
(Ag). Parasite antigen, if present, binds to the labeled
N
T
antibody. Buffer flushes the labeled antigen-antibody

R
O complex along the test strip.
L

P
buffer
R Parasite Ag captured Parasitised
O
G by labeled Ab blood
R
A
M

2
0
0
8

Blood and labeled Ab flushed

along strip.
 Mechanism of action
M
A
L
A
R
3. Some labeled Ag-Ab complex is trapped on the test
I
A line. Excess labeled Ab is trapped on the
C
O
control line.
N
T
R Labeled Ag-Ab Labeled Ab
O
L complex is captured captured by
P by bound Ab of test bound Ab of
R
O band control band
G
R
A
M

2 Captured labeled Captured

0
0 Ag-Ab complex labeled Ab
8
 Target Antigens
M
A
L
1. HRP II (Histidine-Rich Protein II)
A
R - produced by P. falciparum only
I
A
- persists after parasite death
C
O
N
2. pLDH (parasite Lactate Dehydrogenase enzyme)
T
R - produced by all 4 Plasmodium species
O
L
- asexual and sexual stages
P
R
O
- closely reflects parasite viability
G
R - ? potential for monitoring treatment efficacy
A
M

2 - Pv, Po, Pm-specific Mabs developed

0
0
8 3. Aldolase
- pan-specific
- closely reflects parasite viability
 ParaCheck Pf Procedure: Step
1
M
A
L
A
R
I
A 1) Before doing the test, do the
C
O
following:
N
T
a. Explain to the patient what
R
O you are going to do.
L

P
b. Prepare the things you will
R
O need: new lancet, cotton,
alcohol, pencil or pen for
G
R
A
M
labeling, record book
2
0 b. Check the expiration date
0
8 of the RDT at the back of
pouch.
DO NOT use expired RDTs!
 ParaCheck Pf Procedure: Step
2
M
A
L
A
R
I
A

C
O Juan dela Cruz
N
T

Malaria
R

P.f.
C T A B
O
L

P
R
O
G
R
A
M

2
0 2) Open the RDT pouch just before use and
0
8
take out cassette or device. Label with
patients name or ID number.
 ParaCheck Pf Procedure: Step
3
M
A
L
A
R
I
A

C
O
N
T
R
O
L

P
R
O
G
R
A
M 3) Do a finger prick on the patient. Touch the
2
0
0
surface of the blood with one end of the
8
collecting tube or sample loop to get 5L
volume. DO NOT collect too much blood as this
may affect the test result.
 ParaCheck Pf Procedure: Step
4
M
A
L
A
R
I
A

C
O Juan dela Cruz
N
T

Malaria
R

P.f.
O C T A B
L

P
R
O
G
R
A
M

2
0
0
4) Slowly deliver the blood from the collecting
8
tube or sample loop on to the square
sample well marked A.
 ParaCheck Pf Procedure: Step 5
M
A
L
A
R
I
A

C
O
N
T Juan dela Cruz
R

Malaria
O

P.f.
L
C T A B

P
R
O
G
R
A
M

2
0
0
5) Dispense 6 drops of clearing buffer into
8
the round well marked B by inverting the
buffer bottle vertically.
 ParaCheck Pf Procedure: Step
6
M
A
L
A
R 12
I
A

C
O
N
T
9 3
R
O
L

P
R
6
O
G
R
A
M
6) Wait for 15 minutes before reading and
interpreting the results.
2
0
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8
 ParaCheck Pf Procedure: Step
7
M
A 7) Interpret results as follows:
L
A
Juan dela Cruz
R
I Negative for P. falciparum
A
- Only one pink band in the
C
O control window C
N
T
R
O Positive for P. falciparum Juan dela Cruz

P
L
- Pink band in the control +
R window C plus another
O
G pink band in the test window
R
A T
M
Juan dela Cruz
2
0 Invalid results - No band in x
0
8 the control window C or no
bands at all (test may be
repeated with a new RDT)
M
A
Best practice guidelines when using RDT
L
A
R
I
A

C
Some limitations of RDTs are the following:
O
N
T
R
O Persistence of antigenaemia with the HRP II antigen
L

P
tests despite parasite clearance following treatment,
R
O
limiting their use in monitoring treatment.
G
R
A
Parasite density cannot be measured by most
M
malaria RDTs; intensity of test band does not reflect
2
0 parasite load.
0
8
Most antigen tests cannot detect mixed malaria
species infections.
 Best practice guidelines when using RD
M
A
L
A Limitations .
R
I
A

C
False-negative results, even in the face of high parasitaemias, have
O
N
been described.
T
R
O
L False-positive results, especially in patients who are Rh factor-
P
positive, are possible.
R
O
G
R Most antigen tests that claim to detect P. falciparum and P. vivax,
A
M in fact do not specifically detect P. vivax, but a pan-malarial antigen
2
common to all four human malaria species.
0
0
8
The sensitivity of RDTs decreases at low parasite densities (<100
parasites/uL blood). False negative results have also been reported in
higher parasite densities.
 Potential sources of
M variation
A
L
A
in RDT performance
R
I
A

C
O
N
Antigenic variation (extensive in PfHRP2, less
T
R
O
in pLDH and aldolase)
L

P
Environmental conditions (heat and humidity)
R
O
G
Poor preparation and interpretation by user and
R
A
M
failure to act on results appropriately
2
0
Quality of manufacture
0
8
(physical condition and age)
M
A
These can be addressed by:
L
A
R
I
A

C
Good storage and distribution
O
N
T
R
Good training of HWs
O
L Good management algorithms
P
R
O
Monitoring of outcomes in remote areas
G
R
A
Good QC testing to check sensitivity using
M

2
local parasites
0
0
8
 Advantages of RDTs
M
A
L
A
R
I Simple to perform and interpret
A

C Needs minimal training only; no need for electricity

O
N and special equipment
T
R
O
Detect circulating antigen; can detect sequestered P.
L
falciparum
P
R
O
Detection limits comparable w/ high quality
G
R microscopy
A
M

2
0
0
8

CCMOVBD CCMOVBD
M
Disadvantages of RDTs
A
L
A
R
I
A

C Reduced sensitivity at low levels of parasitemia

O
N (<100p/uL)
T
R
O
Some RDTs
L

P
- can detect P. falciparum only
R
O - may give (+) results up to 2 weeks following
treatment and parasite clearance
G
R
A
M
Cant estimate parasite density
2
0
0
Cant distinguish between Pv, Pm and Po
More expensive than microscopy (US$ 0.60 US$
8

2.50)
 Handling, storage and transport of
RDTs
M
A
L
A
R
I Maintain cool chain during shipment of RDTs;
A
transport RDTs in air-con vehicles, whenever possible
C
O
N
Factors reported to cause poor sensitivity:
T
R - exposure to high temperatures during transport
O
L (i.e., very hot airport tarmacs, vehicles parked in the
P
R
sun)
O
G - freezing (i.e., during air transport)
R
A
M
- prolonged exposure to humidity during RDT
2
preparation
0
0 Storage at central and final destinations should be
8
within recommended temperatures (2oC to 30oC);

In remote areas, storage conditions should be as cool

(Source: Malaria Rapid Diagnosis: Making It Work, 2003)

as possible and exposure to direct sunlight and

M
A
L
A
R
Acknowledgements:
I
A

C
O National Center for Infectious Diseases, DOH
N
T
R Centers for Health Development
O
L
Global Fund Malaria Project
P
R
O WHO Roll Back Malaria Project
G
R
A
ACTMalaria Foundation Inc.
M

2
0
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8