Serological Tests For Malaria

Clinical Background of malaria

Malaria is one of the most common diseases in the world. More than half the world population
lives in malaria infected areas. Over 200 million cases annually result in up to 3 million deaths
each year; a majority of which are in young children. In non-endemic areas, it is one of the most
important imported diseases, resulting in a number of deaths in late-diagnosed or unsuspected
cases each year.
The disease is caused by protozoa of the genus Plasmodium, transmitted by the bite of the female
Anopheles mosquito. There are five species causing human malaria: P. falciparum, P. vivax, P.
malariae, P. ovale and P. knowlesi. The disease may also be transmitted by transfusion of
infected blood. Once in the blood the sporozoite makes its way to the liver where for the next 2
weeks merozoites are produced. These are released into the blood where they invade the red cells
and produce more merozoites, causing the cells to rupture. It is this rupturing that is responsible
for the clinical symptoms.
Of the four species, P. falciparum is the most common and the most virulent, causing most
malaria-related deaths. P. vivax is the next most common cause of malaria. Although rarely fatal,
this form of malaria can be accompanied by severe clinical symptoms. People infected with
Plasmodium spp. form antibodies in response.
The life cycle of these species includes both asexual and sexual phases. In the asexual phase,
which occurs in the vertebrate host, slender, spindle-shaped sporozoites inoculated through the
bite of an infected Anopheles mosquito develop into pre-erythrocytic stages in the parenchymal
cells of the liver.
Upon completion of this stage, merozoites are released and penetrate red blood cells. There
follows several rounds of erythrocytic stages through schizogony with subsequent increase of the
number of infected cells in circulation. Some of the ring forms develop into gametocytes of
either sex. When the susceptible mosquito takes its blood meal from the infected host,
gametocytes are ingested, initiating the sexual cycle. Fertilization of the female gamete by the
male takes place in the insect's stomach producing an oocyst, which upon development releases a
large number of sporozoites. After maturation, these sporozoites migrate to the salivary glands
and are eventually inoculated into another susceptible host, thus completing the cycle.

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 History of Malaria
Known since antiquity (oldest disease to mankind ).Early medical writings from India and China
discover periodic fever malaria.Hippocrates described symptoms of malaria in 500 BC. Italians
named malaria as (bad air) in 17th century. Laveran (Charles Louis Alphonse Laveran)
identified parasite of malaria in 1880. Ronald Ross demonstrated mosquito transmission in 1897.
P. malariae described by Laveran in 1881 and Grassiand and Felletti in 1890. P. vivax described
by Grassiand and Felletti in 1890 and Labbe in 1989. P. falciparum described by Welch in 1897
and Schaudinn in 1902. P.ovale described by Stephens in 1922. Garnham described liver stage
1940’s. WHO Launch worldwide malaria eradication in 1955.

Serological tests for malaria

Serology tests help in detecting antibodies in the blood that are produced by the body in response
to a malaria infection. This test cannot diagnose an acute or a complicated infection but it can
certainly help in determining if the person has been previously exposed to this infection.
1. Enzyme-linked immunosorbent assay (ELISA)
2. Indirect Immunoflouresce (IFA)
3. Rapid diagnostic test (RDT)

1. Enzyme-linked immunosorbent assay (ELISA)

Intended use
These kits are intended for use by appropriately trained and qualified personnel for the detection
of antibodies to P.falciparum, P.vivax, P.ovale and P.malariae in human serum and plasma.

Principle of the test

The Malaria ELISA use four recombinant antigens in a sandwich test to produce a test that is
both highly specific and sensitive. The antigens will detect P.falciparum, P.vivax, P.ovale and
P.malariae -specific IgG, IgM, and IgA; enabling the test to detect antibodies during all stages of
infection. All reagents except the Conjugate and Wash solution are supplied ready to use and
colour-coded, and the procedure uses undiluted samples and standard volumes for ease of both
manual and automated use. The assay can be used with both serum and plasma.
The plastic wells are coated with a mixture of P. falciparum and P. vivax recombinant antigens.
The antigenic similarity between Plasmodium species means that antibodies to all species can be
detected. Specific antibodies in serum or plasma specimens combine with these antigens and
with the same antigens conjugated to horseradish peroxidase, when conjugate is added to a well
in which the specimen has been incubated. After unreacted material has been removed by
washing, the presence of bound enzyme indicating the presence in the specimen of specific
antibodies is revealed by a colour change in the substrate/chromogen mixture. The intensity of
the colour is compared to that in control wells to determine the presence or absence of specific
antibody.

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 KIT CONTENTS

Warnings and precautions

For in - vitro diagnostic use only.
The control materials supplied are derived from human serum. They have been tested at donor
level and found negative for Hepatitis B and C, and for HIV 1 and 2. However, they should be
treated as if capable of transmitting disease.
Specimens of human serum and plasma should be treated as micro-biologically hazardous, and
handled in accordance with the applicable regulations.
Do not use the kit after its expiry date.
Do not combine or interchange reagents from kits with different lot numbers.

Storage
Store at 2 – 8 °C when not in use. Store bottles upright.
Do not freeze.
Do not expose substrate to direct sunlight.
Diluted conjugate is stable for 4 weeks at 4 °C
Diluted wash buffer is stable for 4 weeks at 4 °C
Unused coated strips are stable for 4weeks at 4 °C if stored in the re-sealable bag provided.

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 Equipment required
Properly calibrated and maintained pipetting devices capable of delivering volumes of 50
microlitres (specimens and reagents) and approx 300 microlitres (wash fluids).
Plate or strip reader to read at 450 nm and (optionally) at a wavelength between 620 and 690 nm.
37 °C incubator
The Malaria ELISA may be automated for both liquid handling and result interpretation. A
variety of systems have been used for this – please consult the manufacturers of both the kit and
the automation system for advice on automation.
Equipment should be able to support the following tolerances:
Volume dispensed +/- 10%
Incubation
+/- 2 C
temperature
+/- 2
Incubation time
minutes.

Specimens
Serum or plasma (collected into EDTA, sodium citrate, or heparin) specimens should be free of
blood cells and of obvious microbial contamination.
They may be stored at 2-8 °C for up to 7 days before testing. Specimens needing longer storage
should be frozen at –20 °C or lower. Frozen specimens should be thawed and well mixed before
testing.
Assay protocol (manual)
Bring all reagents and specimens to room temperature prior to use.
Dilute Wash Buffer 1 in 20 with distilled or deionised water prior to use
Assay controls
The Negative control must be tested three times with each lot of tests, and the Positive control
twice.
Verification of Specimen addition
Verification is by detecting photometrically the difference between an empty well and a well
containing serum or plasma at a wavelength of 450 nm. Wells containing specimen will have an
A450 reading of between 0.050 and 1.000.

Procedure
1. Add 50 µL of the undiluted sample to a coated well.
Mix on a plate shaker for 30 seconds.
Incubate (covered) at 37o C for 30 minutes.
2. Wash 5 x with working strength wash buffer. A short soak time of about 30 seconds is
recommended between each wash cycle. Tap out excess liquid.

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3. Conjugate Incubation
Dilute conjugate 1 + 10 in Conjugate Buffer. (50 µL + 500 µL per 10 wells)
Add 50 µL diluted conjugate to each well.
Incubate (covered) at 37o C for 30 minutes.
Addition of conjugate is verified by reading at 450/ nm. A well with conjugate added must have
an OD greater than 0.2
4. Wash 5 x with working strength wash buffer. A short soak time of about 30 seconds is
recommended between each wash cycle. Tap out excess liquid.
5. Substrate Incubation
Add 50 µL substrate/chromogen mixture to each well.
Incubate at room temperature for 30 minutes.
There is a clear difference of colour between an empty well and a well containing substrate.
Addition of substrate is verified by reading at 550 nm. A well with substrate added must have an
A550 of greater than 0.080
As the substrate is photosensitive, it is recommended that the plate be protected from light during
this incubation.
6. Stop Colour Development
Add 50 µL 0.5M sulphuric acid to each well. (Blue colour changes to yellow).
7. Read Results
Read at 450 nm (A450) Use of a reference filter at 620 – 690 nm will eliminate effects of
scratches, bubbles, etc

Quality control
For confidence in ELISA results, it is necessary to have three experimental controls for
comparison: a positive control, negative control, and a standard control. Both the positive
and standard controls are known to contain the protein or peptide of interest. The positive control
is to confirm simply if the procedure is performing as intended.

Interpretation
Samples with an A450 value less than the Cut-off value are considered negative by Malaria
ELISA. Samples just below the Cut-off (C.O. –10% A450) should however, be interpreted with
caution. It is advisable to retest the corresponding samples in duplicate when the systems and
laboratory procedures permit. Re-tested samples that are above the cut-off in at least one
duplicate are considered positive and should be investigated further.Samples that are below the
cut-off in both duplicates are considered to be negative.

Performance characteristics
Enzyme-linked immunosorbent assay test has high sensitivity and specificity for malaria test. It
is common for ELISAs to detect antigens at the picogram level in a very specific manner due to
the use of antibodies. ELISA test has also high precision.

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 Summary
In an enzyme-linked immunosorbent assay test for malaria antibodies, antibodies to Plasmodium
vivax and P. falciparum in man are detected using a crude antigen prepared from the malaria
parasite. The test may be suitable for epidemiological study. The Malaria ELISA use four
recombinant antigens in a sandwich test to produce a test that is both highly specific and
sensitive. The antigens will detect P.falciparum, P.vivax, P.ovale and P.malariae -specific IgG,
IgM, and IgA; enabling the test to detect antibodies during all stages of infection.

2.Indirect Immunoflouresce (IFA)

Indirect immunofluorescence assay (IFA) has been used for detection of autoantibodies against
cellular antigens. Malaria antibody detection is performed using the indirect fluorescent
antibody (IFA) test.The IFA procedure can be used to determine if a patient has been infected
with Plasmodium.

Principle of the test

Malaria IFA tests can be made comparable in different places and at different times only if
reference antisera and antigens are exchanged and if the different conjugates used are specified
in terms of established reference conjugates.Blood stage Plasmodium species schizonts
(meronts) are used as antigen. The patient’s serum is exposed to the organisms; homologous
antibody, if present, attaches to the antigen, forming an antigen-antibody (Ag-Ab) complex.
Fluorescein-labeled anti-human antibody is then added, which attaches to the patient’s malaria-
specific antibodies.

Reagents required
0.1 N HC1
Distilled water (dip)
PBS pH 7-7.2
Test serum
Specific conjugated antiglobulin
Mount in buffered glycerin pH 7-7.2
Note: PBS-phosphate-buffered saline

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 Procedure
Serial dilutions of the test plasma or serum samples are made in phosphate-buffered saline (pH
7.2). A dilution of 1: 20 is a commonly used starting point. Measured quantities of the serum
dilutions are then transferred to each antigen spot. The subsequent processing of the slides is
carried out as follows in the range 20°C to 37°C:
1.Incubate with serum dilutions in a humid chamber for 30 min.
2.Wash with phosphate-buffered saline with agitation, 3 changes of 5 min each.
3.Pour off excess saline and dry slides, except for the antigen spots.
4.Apply the diluted fluorescein-labelled antiglobulin conjugate. Incubate in a humid chamber for
30 min.
5.Wash as in (2).
6.Mount slides with coverslips in 10% glycerol in phosphate-buffered saline (or with a
commercial mountant, particularly if permanent preparations are required).
7.Examine with a fluorescence microscope.

Specimen collection and preparation

Serum and plasma can both be used in the tests discussed here. In the many instances where
venepuncture is inadvisable, it is possible to collect blood by finger puncture into heparinized
capillary tubes. It is generally agreed that, wherever possible, the capillary-tube method should
be used for the collection and transport of samples, although under certain conditions the only
practical way may be to use the filter paper technique.
Capillary tubes
The finger is cleaned with an alcohol-moistened swab and is allowed to dry in order to prevent
haemolysis of the sample. A disposable lancet is then used to puncture the finger and the first
drop of blood is wiped off. One or more labelled heparinized capillary tubes (with a minimum
internal diameter of 1.1 mm and a length of 75 mm), preferably containing dried thiomersal, are
then filled. The capillary tubes are sealed at one end by plugging with plasticine or by heat. They
should be centrifuged within a few hours with an electrical centrifuge oreffective alternative. The
capillaries are then cut at the plasma-cellular interface. The plasma section is again sealed at both
ends with plasticine and stored.

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 Filter paper
Under particular conditions in the field, the blood samples collected in capillary tubes may be
absorbed on filter paper from which the serum can be extracted prior to serological testing. In
this case, it will' be necessary to absorb precisely measured quantities of blood on the filter
paper-e.g., by means of capillary tubes.
The impregnated filter papers must not come into contact with fixing agents and should be dried
as soon as possible, avoiding temperatures exceeding 56°C. Although these dried blood samples
on filter paper are known to withstand normal environmental temperatures in subtropical
conditions for at least a fortnight without loss of seroreactivity, it is advisable to store them as
soon as possible at -20°C (experience has shown that they can be preserved at that temperature
for at least a year).
Antigen
The antigen consists of thick or thin blood films on glass slides and is made from the blood of
malariainfected animals or people. From a practical point of view, thick smears are preferable.
Antigen preparations should contain mature schizonts whenever possible, since these give
maximum sensitivity to the test. For the detection of antibodies, the homologous antigens should
be used, except in the case of antibodies to Plasmodium malariae where either the homologous
species or P. brasilianum can be employed. A mixed-species antigen will give maximum
sensitivity, but is very difficult to prepare.
Suitable P. falciparum antigen can be obtained by the in vitro cultivation of heavily infected
human blood.a Suitable P. vivax and P. malariae antigens are less readily obtained direct from
human sources because the parasite densities of these infections are often too low.
Conjugate
The sensitivity and specificity of the IFA test is largely dependent on the conjugate and the
dilution at which it is used. Conjugated antisera prepared against the total immunoglobulins or
against specific immunoglobulins can be used. No chemical or biological parameters have yet
been found to be satisfactory indicators of the potency of such antisera. Conjugates should be
assessed in malaria IFA tests against one of the standardized reagents.

Storage
1.Ideally tests should be carried out on fresh serum.
2.Bacterial growth should be avoided.This can be achieved by handling the serum so as to avoid
gross contamination and by adding sodium azide or thiomersal to the blood or serum sample.
3.Serum should be stored in the cold. For long term storage, it should be frozen (-700C is better
than -20°C).

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4.Thawing and refreezing may be harmful. Therefore all tests should be performed
simultaneously on any batch of thawed serum, or aliquots should be frozen separately to be
thawed as required for tests performed at different times.
5. IgM antibodies or " early " antibodies may be liable to loss of activity on storage.
6.Freeze-drying under the carefully controlled conditions used for the preparation of biological
standards appears to be a good way to preserve the properties of the serum. However, this is not
a method for general application.

Merits of the test

1.The preparation of comparable batches of antigen is relatively simple.
2.The whole infected cell, morphologically identifiable, is used as antigen.
3.Many studies have shown that the test can reflect experience of malaria by an individual or
community.
4.The results of the test can be used to show differences in malarial endemicity between
localities, and to detect transmission.
5.The test is adequately sensitive.
6.At higher titres, the test is virtually always specific for malaria and sometimes can be used to
indicate species-prevalence.

Limitations
1. The reading of results is subjective at present.
2. Malaria-parasite carriers can occasionally give negative reactions. This has been observed
especially with children.
3. The necessity for specialized equipment and personnel limits the test to major laboratories.
4.Antigens are available from only a few centres and their storage requires considerable
refrigeration space.
5.The transport of antigen can present problem

Interpretation
When the slide is examined with a fluorescence microscope, if parasites fluoresce an apple green
color, a positive reaction has occurred. The fluorescence indicates that the patient serum being
tested contains antibodies that are reacting with the antigen prepared. If this color does not exist
a negative reaction occurred.

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 Performance characteristics of IFA
Immunofluorescence antibody testing (IFA) has been a reliable serologic test for malaria. It is
highly sensitive and specific, but on the other hand, time consuming and subjective

Summary
The indirect fluorescent antibody test for malaria antibody can provide a useful measurement of
the immunological status during malarial infection. Variations in the method of performance of
the test, however, may alter its sensitivity and reproducibility. In antigen preparation, special
attention should be directed toward obtaining smears with a- uniform distribution of parasites
and toward maintaining uniformity of size in the area to be stained. The size or stage of the
parasites to be stained will also affect the interpretation of results. Uniform quantities of the
serum dilutions should be used for staining. The sensitivity of the test must be adjusted to the
precision of performance. Use of Evans blue as a counterstain simplifies the reading of these
tests for malarial antibodies.

3.Rapid diagnostic test (RDT)

Malaria antigen detection tests are a group of commercially available rapid diagnostic tests of the
rapid antigen test from patients peripheral blood.

Intended use
Allow quick diagnosis of malaria by people who are not otherwise skilled in traditional
laboratory techniques for diagnosing malaria or in situations where such equipment is not
available. There are currently over 20 such tests commercially available (WHO product testing
2008).

Principle of test
Malaria rapid diagnostic tests (RDTs) assist in the diagnosis of malaria by detecting evidence of
malaria parasites (antigens) in human blood.
Targets
1. Histidine-rich protein 2 of P. falciparum,
2. Parasite specific lactate dehydrogenase (pLDH) and
3. Pan-malarial plasmodium aldolase,

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 Histidine-rich protein 2 of P. falciparum (PfHRP2)
Protein produced by the asexual stages and gametocytes of P. falciparum.Remain in the blood
for at least 28 days after the initiation of antimalarial therapy.Several RDTs targeting PfHRP2
have been developed.
Parasite lactate dehydrogenase (pLDH)
is a soluble glycolytic enzyme produced by the asexual and sexual stages of the live parasites
present in and released from the parasite infected erythrocytesfound in all 4 human malaria
species, and different isomers of pLDH for each of the 4 species exist.
Plasmodium aldolase
Is an enzyme of the parasite glycolytic pathway. Expressed by the blood stages of P. falciparum
as well as the non-falciparum malaria parasites.Monoclonal antibodies against plasmodium
aldolase are pan-specific in their reaction. Have been used in a combined 'p.F/P.V' ICT test that
targets the pan malarial antigen (PMA) along with pfhrp2.

Malaria RDTs included in this evaluation detect one or more of three parasite antigens (HRP2,
pLDH, and aldolase) in various combinations. HRP2 is present only in P. falciparum whereas
aldolase and pLDH are present in all four species and may be used as pan or all-species targets.
Some tests use differences in pLDH sequences between species as a means to differentiate
P. falciparum from P. vivax and other species. There is considerable overlap in the PDS of
products targeting the different antigens in this evaluation. While the products with the highest
PDS for P. falciparum targeted HRP2, a number of pLDH-detecting products demonstrated high
PDS against P. vivax. The thermal stability of tests targeting these different antigens also
overlapped for high parasite density samples.The choice of RDT should take target antigen into
account:HRP2-detecting RDTs should not be used in areas where high rates of HRP2 non-
expression occur.
Tests detecting only HRP2 (without pLDH or aldolase lines) will have limited utility where non-
falciparum malaria is common. pLDH (and possibly aldolase) RDTs may have further
advantages where antigen persistence (common with HRP2) may result in a high false-positive
rate in areas where early retesting in the weeks immediately after treatment is common.The
required sensitivity of a test may also vary with species; less sensitive test may be acceptable for
detection of P. vivax compared to detection of P. falciparum, as severe outcomes due to missed
diagnoses are less likely. Use of a sufficiently sensitive pan-specific-only test may be appropriate
in areas where both P. falciparum and P. vivax occur, if all infections were to be managed
initially as a P. falciparum infection with artemisinin-based combination therapy (ACT), but
species specific monitoring data would be lost.

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 Test Procedure
1.Ensure specimen and test kits are brought to room temperature before testing. ...
2.Open the foil wrapped pouch and remove the cassette. ...
3.Add 5μl of whole blood into the sample well (small well).
4. Add two drops (60μl) of assay buffer into the buffer well.
5. Read the test result in 20 min.

Limitation
Despite high sensitivity and specificity for Plasmodium falciparum infections, RDTs have
several limitations that may reduce their utility in low-transmission settings: they do not reliably
detect low-density parasitaemia (≤200 parasites/µL), many are less sensitive for Plasmodium
vivax infections,
RDT registration
The receipt of each shipment of RDTs at the CDC was recorded in a dedicated RDT register.
Temperature monitoring devices were offered to manufacturers free of charge, to accompany
RDTs shipments to CDC. All RDTs were stored in roomtemperature at ≤ 25°C immediately and
applicable.

Specimen panel registration

All panel specimens were assigned unique identification numbers at the collection sites and
stored in aliquots of 50µL at -70°C until the time of testing. All data pertaining to specimen
identification, storage location and characterization results are stored in a secure, dedicated
database.

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 Interpretation of results
Results of control and test lines were recorded as negative or positive by each technician. Each
test was read against a standard colour chart and the band intensity graded as 0 (no visible band),
1, 2, 3 or 4. If the control line is recorded as absent by either technician, the test is recorded as
invalid.Enzyme-linked immunosorbent assay.

Quality assurance
Product testing follows SOPs developed through prior testing experience. Overall, the quality of
critical steps is controlled, as follows:
i) Quality of the malaria RDTs and their use
ii) Quality and objectivity of the RDT reading results: >40-100 parasites/µL
iii)Quality of the WHO Specimen Bank sample
iv) Quality of the product testing site

Sensetivity
PfHRP2 tests: >40-100 parasites/µL
PfHRP2 and PMA test: Higher for P. vivax and other non-falciparum species
pLDH test: >100-200 parasites/µL for P. falciparum and P. vivax; may be higher for P. malariae
and P. ovale.
Invalid tests
These are the total number of tests that were deemed invalid during testing of both lots, using
samples at 200 parasites/µl and 2000 (or 5000) parasites/µl

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 Summary
A Rapid Diagnostic Test (RDT) is an alternate way of quickly establishing the diagnosis of
malaria infection by detecting specific malaria antigens in a person’s blood. RDT assist in the
diagnosis of malaria by detecting evidence of malaria parasites (antigens) in human blood. RDTs
can be used to distinguish fevers caused by malaria parasites from those caused by other
illnesses, such as meningitis and acute respiratory infection (that cause symptoms similar to
malaria.

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 Reference
Kitchen A.D. et al. Evaluation of a malarial antibody assay for use in the screening of blood and
tissue products for clinical use. Vox Sanguinis (2004) 87, 150 - 155
Bull World Health Organ. 1974; 50(6): 527–535
Seed C.R. et al. The efficacy of a malarial antibody enzyme immunoassay for establishing the
reinstatement status of blood donors potentially exposed to malaria Vox Sanguinis (2005) 88, 98
– 106

Collins, Unit on Primate Malaria, Laboratory of Parasitic Diseases, National Institutes of Health,
Chamblee, Ga., USAAshley, EA, Phyo, AP, and Woodrow, CJ. 2018. Malaria. Lancet. 391:
1608–1621. PMID: 29631781

Phillips, MA, Burrows, JN, Manyando, C, et al. 2017. Malaria. Nat Rev Dis
Primers. 3: 17050. PMID: 28770814

Fairhurst, RM and Wellems, TE. 2014. “Malaria (Plasmodium Species)”. In: Mandell, Douglas,

and Bennett’s Principles and Practice of Infectious Diseases, edited by Bennett, JE, Dolin, R,
Blaser, MJ.

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