Abstract

Malaria is infection with Plasmodium sp, transmitted by an infective female

anopheles mosquito vector.The Plasmodium species known to cause malaria in humans are
P.falcifarum, P.vivax, P.ovale,and P.Malariae.Timely identification of the infecting species is
extremely important, as a P.falcifarum infection can be fatal and is often resistant to standart
chloroquinone treatment.Symptoms are fever (which may be periodic), chills, sweating,
hemolytic anemia, and splenomegaly. Diagnosis is by seeing Plasmodium in a peripheral
blood smear as a gold standart and RDT and PCR as a more accurate gold standart.
Introduction
Malaria is endemic in Africa, much of South and Southeast Asia, North and South
Korea, Mexico, Central America, Haiti, the Dominican Republic, northern South America
(including northern parts of Argentina), the Middle East (including Turkey, Syria, Iran, and
Iraq), and Central Asia. There are 300 to 500 million infected people worldwide, with 1 to 2
million deaths yearly, most in children < 5 yr in Africa. Malaria once was endemic in the US
but has been virtually eliminated. About 1500 cases/yr occur in the US. Nearly all are
acquired abroad, but a small number result from blood transfusions or rare autochthonous
transmission by local mosquitoes that feed on infected immigrants.
In parallel to this, accurate and affordable rapid diagnostic tests for malaria (RDTs)
have become available over the last decade and the 2010 WHO malaria treatment guidelines
now propose that wherever possible all patients suspected of malaria should be tested by
blood slide or RDT and only those with a positive test result should receive anti-malarial
treatment.While this should lead to a number of benefits, there is still relatively little direct
evidence that non-malarial treatment in patient in highly or moderately endemic areas is safe,

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with a concern that a significant number of malaria cases may be missed. The risks of 'test
negative malaria' are consistently raised by clinicians cautious about this approach .In
addition clinicians need more evidence on the probabilities of different non-malarial
diagnoses, especially those that require treatment with antimicrobial agents.
Key messages : Malaria , Diagnostic test for Malaria.
Discussion
Etiology and Clinical Presentation of Malaria
Malaria is infection with Plasmodium sp. Symptoms are fever (which may be
periodic), chills, sweating, hemolytic anemia, and splenomegaly.Individuals with malaria
typically acruired the infection in an endemic area following a anopheles bite.After infection,
the parasites (called sporozoites) travel through the bloodstream to the liver, where they
mature and release another from the merozoites.The parasites enter the bloodstream and
infect red blood cells[picture 1]. (1)

Most symptoms are caused by :

The release of merozoites into the bloodstream

Anemia resulting from the estruction of the red blood cells

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Large amounts of free hemoglobin being released into circulation after red blood cells
break open.

Diagnostic test for Malaria

Although the peripheral blood smear examination that provides the most
comprehensive information on a single test format has been the "gold standard" for the
diagnosis of malaria, the immunochromatographic tests for the detection of malaria antigens,
developed in the past decade, have opened a new and exciting avenue in malaria diagnosis.
However, their role in the management and control of malaria appears to be limited at
present.(1)
Immunochromatographic Tests for Malaria Antigens
Immunochromatographic tests are based on the capture of the parasite antigens from
the peripheral blood using either monoclonal or polyclonal antibodies against the parasite
antigen targets. Currently, immunochromatographic tests can target the histidine-rich protein
2 of P. falciparum, a pan-malarial Plasmodium aldolase, and the parasite specific lactate
dehydrogenase. These RDTs do not require a laboratory, electricity, or any special equipment.
(2)

Histidine-rich protein 2 of P. falciparum (PfHRP2) is a water soluble protein that is produced

by the asexual stages and gametocytes of P. falciparum, expressed on the red cell membrane
surface, and shown to remain in the blood for at least 28 days after the initiation of
antimalarial therapy. Several RDTs targeting PfHRP2 have been developed.(2)
Plasmodium aldolase is an enzyme of the parasite glycolytic pathway expressed by the blood
stages of P. falciparum as well as the non-fa1ciparum malaria parasites. Monoclonal
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antibodies against Plasmodium aldolase are pan-specific in their reaction and have been used
in a combined P.falcifarum/P.vivax immunochromatographic test that targets the pan malarial
antigen (PMA) along with PfHRP2.(2)
Parasite lactate dehydrogenase (pLDH) is a soluble glycolytic enzyme produced by the
asexual and sexual stages of the live parasites and it is present in and released from the
parasite infected erythrocytes. It has been found in all 4 human malaria species, and different
isomers of pLDH for each of the 4 species exist. With pLDH as the target, a quantitative
immunocapture assay, a qualitative immunochromatographic dipstick assay using
monoclonal antibodies, an immunodot assay, and a dipstick assay using polyclonal antibodies
have been developed.(2)
The Rapid Malaria Tests: The RDTs have been developed in different test formats like the
dipstick, strip, card, pad, well, or cassette; and the latter has provided a more satisfactory
device for safety and manipulation. The test procedure varies between the test kits. In general,
the blood specimen (2 to 50L) is either a finger-prick blood specimen, anticoagulated blood,
or plasma, and it is mixed with a buffer solution that contains a hemolyzing compound and a
specific antibody that is labeled with a visually detectable marker such as colloidal gold. In
some kits, labeled antibody is pre-deposited during manufacture and only a lysing/washing
buffer is added. If the target antigen is present in the blood, a labeled antigen/antibody
complex is formed and it migrates up the test strip to be captured by the pre-deposited capture
antibodies specific against the antigens and against the labeled antibody (as a procedural
control). A washing buffer is then added to remove the hemoglobin and permit visualization
of any colored lines formed by the immobilized antigen-antibody complexes. The pLDH test
is formatted to detect a parasitemia of >100 to 200 parasites/L and some of the PfHRP2 tests
are said to detect asexual parasitemia of >40 parasites/L.(3)

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The PfHRP2 test strips have 2 lines, I for the control and the other for the PfHRP2 antigen.
The PfHRP2/PMA test strips and the pLDH test strips have 3 lines, 1 for control, and the
other 2 for P. falciparum (PfHRP2 or pLDH specific for P. falciparum) and non-falciparum
antigens (PMA or pan specific pLDH), respectively. Change of color on the control line is
necessary to validate the test and its non-appearance, with or without color changes on the
test lines, invalidates the test. With color change only on the control line and without color
change on the other lines, the test is interpreted as negative. With the PfHRP2 test, color
change on both the lines is interpreted as a positive test for P. falciparum malaria. With the
PfHRP2/PMA [The immuno chromatographic test (ICT Malaria P. f. /P.v.test)] and the pLDH
tests, color change on the control line and the pan specific line indicates non-fa1ciparum
infection and color change on all the 3 lines indicates the presence of P. falciparum infection,
either as monoinfection or as a mixed infection with nonfa1ciparum species. Also, if the
PfHRP2 line is visible when the PMA line is not, the test is interpreted as positive for
P.falciparum infection. Mixed infections of P. falciparum with the non-falciparum species
cannot be differentiated from pure P. falciparum infections. However, with regard to the
pLDH test, it is claimed that in the presence of P. vivax infection, the genus specific line is
much darker and more intense than the species specific line due to the presence of all the
stages of the parasite in the blood.(4)

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It is also claimed that the rapid diagnostic tests can be performed by individuals with minimal
training. With the different tests that are currently available, the procedure may involve 2 to 6
steps and take 5 to 30 minutes. The cost of the RDT also varies from test to test and from
country to country, ranging from US $1.20 to $13.50 per test. None of the RDTs have been
approved by the Food and Drug Administration for the diagnosis of malaria in the United
States.(4)
Problems with RDTs:
The sensitivity of the RDTs at low levels of parasitemia and for non-immune populations
remains a problem. Compared to microscopy, the PfHRP2/PMA tests were found to be less
sensitive in detecting asymptomatic patients, particularly at low parasitemias.
False Positivity: False positive tests can occur with RDTs for many reasons. Potential causes
for PfHRP2 positivity, other than gametocytemia, include persistent viable asexual-stage
parasitemia below the detection limit of microscopy (possibly due to drug resistance),
persistence of antigens due to sequestration and incomplete treatment, delayed clearance of
circulating antigen (free or in antigen-antibody complexes) and cross reaction with nonfalciparum malaria or rheumatoid factor. Proportion of persistent positivity has been linked to
the sensitivity of the test, type of test, degree of parasitemia and possibly the type of capture
antibody.
False negativity: On the other hand, false negative tests have been observed even in severe
malaria with parasitemias >40000 parasites/l. This has been attributed to possible genetic
heterogeneity of PfHRP2 expression, deletion of HRP-2 gene, presence of blocking
antibodies for PfHRP2 antigen or immune-complex formation, prozone phenomenon at high
antigenemia or to unknown causes.(4)

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Multiple Influences: The performance of the RDTs is reported to be influenced by a multitude

of factors like the type of the parasite and the level of parasitemia; the type of test; the target
antigen and the capture antibody; the expression of the target antigens on the parasites and the
presence of several isomers; the presence of gametocytemia; persistent antigenemia or
sequestration of the parasites; cross-reactions with other malaria species and with
autoantibodies; batch quality variations in test strips; prozone phenomenon; and prior
treatment. The interpretation of the color changes to identify the malaria infection is
influenced by the level of training, the type of instructions, and in case of self-use, by the
state of the patient. The inability to quantify and differentiate between the sexual and asexual
parasitemia could pose problems in the areas of high transmission and in cases of incomplete
treatment.(4)
The sensitivity and specificity of the RDTs, and hence the diagnosis and treatment of malaria
based on the RDTs, are influenced by the positive results due to causes other than malaria
antigenemia, and the negative results due to causes other than low parasitemia. Therefore, the
identification of the color changes on the RDT strips may look simple but the interpretation
of the result would require the knowledge of the malarial dynamics and of the possible errors
with the RDTs. Otherwise, the RDTs may raise more questions than answers, and the
insufficient accuracy of the RDTs could increase the number of incorrect malaria diagnoses.(4)
Interpretation: Although the RDTs have been reported to be useful and easy tools for field
surveys in remote forests and villages, some studies have found that the experience and the
level of training of the field staff can influence the sensitivity and specificity of these tests
and have reported questionable results or failure to interpret the results in 1.7% to 3.75% of
the PfHRP2/PMA test strips. In one study, only 68% of the European tourists to Kenya were
able to perform the test correctly, and 10 out of 11 with malaria failed to diagnose themselves

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correctly. High number of false negative results have also been reported. A potential problem
with the dipstick test is that the circulating antigen will be detectable for many days even
after the elimination of viable P. falciparum from the blood stream. A positive test therefore
may not always indicate an active infection.(4)

Comparison of Rapid Diagnostic Tests for Malaria Antigens(4)

PfHRP2 tests
PfHRP2 and PMA
test
Target antigen
Histidine
rich Pan-specific
protein 2 of P. Plasmodium
falciparum,
water aldolase.
parasite
soluble
protein glycolytic enzyme
expressed on RBC produced by all
membrane
species and PfHRP2
General test format
2 lines
3 lines
Capability
Detects
P. Can detect all 4
falciparum only
species
Non-falciparum
Not detected
Detected;
species
differentiation
between the 3 not
possible
Mixed infections of Appear
as
P. Appear
as
P.
P. falciparum with falciparum;
falciparum;
non-falciparum
differentiation
not differentiation
not
species
possible
possible
Detection limit
>40-100
Higher for P. vivax
parasites/L
and
other
nonfalciparum species

pLDH test
Parasite
lactate
dehydrogenase.
parasite glycolytic
enzyme produced by
all species

3 lines
Can detect all 4
species
Detected;
differentiation
between the 3 not
possible
Appear
as
P.
falciparum;
differentiation
not
possible
>
100-200
parasites/L for P.
falciparum and P.
vivax; may be higher
for P. malariae and
P. ovale
Post-treatment
Reported up to 31 Reported; longer for Reported up to 1 -3
persistence
of days
pan
specific weeks
antigens
antigenemia than for
PfHRP2
Cross-reactivity
Reported
Reported
Reported
between
malarial
species
Cross-reactivity with Reported, high (up Not known
Reported. low (3.3%
auto antibodies
to
83%
with
with
rheumatoid
rheumatoid factor)
factor)
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Indication
of No
viability of parasites

No

Positive
test
indicates presence of
viable parasitemia

Comparison of Peripheral Blood Smear Examination and RDTs for Malaria(4)

Format
Equipment
Training
Test duration
Test result
Capability

Detection threshold
Species
differentiation

Peripheral Smear
Rapid Diagnostic Tests
Slides with blood smear
Test strip
Microscope
Kit only
Trained microscopist
'Anyone with a little training'
20-60 minutes or more
5-30 minutes
Direct visualization of the Color changes on antibody
parasites
coated lines
Detects and differentiates all Detects
malaria
antigens
plasmodia at different stages
(PfHRP2/ PMA/pLDH) from
asexual and/or sexual forms of
the parasite
5-10 parasites/L of blood
100-500/L for P. falciparum,
higher for non-falciparum
Possible
Cannot differentiate among nonfalciparum
species;
mixed
infections of P. falciparum and
non-falciparum appear as P.
falciparum
Possible
Not possible
Possible
Not possible

Quantification
Differentiation
between sexual and
asexual stages
Disadvantages
Availability of equipment and
skilled
microscopists,
particularly at remote areas and
odd hours
Status
Cost per test

Gold standard
US$ 0.12-0.40

Unpredictable efficiency at low

and very high parasitemia; cross
reactions among plasmodial
species and with auto-antibodies;
persistence of antigens
Not yet approved by the FDA
US$ 1 .20-13.50

Histologic Features(5)

Findings
Only

P.falciparum
early Yes

P.vivax

P.ovale

P.malariae

No

No

No

forms present in
peripheral blood
Page 9 of 11

Multiply

Often

Occasionally

Rare

Rare

Young RBCs

Old RBCs

Yes

No

infected RBCs
Age of infected RBCs of all ages Young RBCs
RBCs
Schuffner dots

No

yes

US FDA approves RDT: On June 13, 2007, the U.S. Food and Drug Administration (FDA)
approved the first RDT for use in the United States. This RDT is approved for use by
hospital and commercial laboratories, not by individual clinicians or by patients themselves.
It is recommended that all RDTs are followed-up with microscopy to confirm the results and
if positive, to quantify the proportion of red blood cells that are infected.(2)
PCR (Polymerase Chain Reaction Assay)
Is very specific and sensitive means of determining it species of plasmodium are present in
the blood of an infected individual.PCR assay tests are not available in most clinical
situations.However, they are the World Health Organization (WHO) recommends as a gold
standart(in 2010) and very effective at detecting the Plasmodium species in patients with
parasitemias as low as 10 parasites/mL of blood.(5)
WHO recommendation for diagnostic test malaria
In this situation, The World Health Organization (WHO) recommends prompt parasitological
confirmation by microscopy or rapid diagnostic test (RDTs) for all patients with suspected
malaria before treatment is started. Since both microscopy (as gold standard) and RDTs have
limitations in identifying malaria infection,there is need to use a more accurate gold standard
(such as PCR) in assessing the accuracies of these diagnostic tests.(1)

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Conclusion
High sensitivity of malaria diagnosis is important in all settings, and essential for the
most vulnerable population groups in which malaria infection produces an acute illness that
can rapidly progress to death. The HRP2-based test demonstrated a superior sensitivity
compared to microscopy and presumptive methods in the diagnosis of uncomplicated malaria
in remote health facilities. Based on the current findings, the HRP2-based RDT may be more
suitable for screening of malaria infection in routine practice in primary health care centres,
but as a gold standart to diagnostic malaria still microscopic test and PCR.
References
1. Mtove G, Hendriksen ICE, Amos B,Mrema H,Mandia V,Manjurano A, et al.Malaria
Journal . licensee BioMed Central Ltd.2011;10.
2. Batwala V,Magnussen P, Nuwaha P . licensee BioMed Central Ltd. Malaria Journal
2010;9.
3. Kakkilaya BS. Rapid Diagnosis of Malaria. Lab Medicine. 2003.Aug;8.
4. Jos-Luis Portero et al. Research ArticleAccuracy of an Immunochromatographic
Diagnostic Test. 2010;6.
5. Bailey JW,Williams J,Bain BJ,Parker WJ, Chiodini P.General Haematology Task
Force.Guidline for laboratory diagnostic of malaria.London :British Committee for
Standars in Haematology.2007;19.

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