Rapid Diagnosis of Malaria

See Kakkilaya BS. Rapid Diagnosis of Malaria. Lab Medicine. 2003 Aug;8(34):602-608

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.

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.

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.

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 antibodies
against Plasmodium aldolase are pan-specific in their reaction and have been used in a combined
‘P.f/P.v’ immunochromatographic test that targets the pan malarial antigen (PMA) along with
PfHRP2.

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.
RDT Test Format

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 50µL) 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/µL and some of the PfHRP2 tests are said to detect asexual parasitemia of
>40 parasites/µL.
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 forP.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.

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.

Problems with RDTs:

Cross-reactions with autoantibodies: Studies have reported cross reactivity of the various RDTs
with autoantibodies such as rheumatoid factor, resulting in false positive tests for malaria.
Studies in patients with positive rheumatoid factor have shown that the false positive reactions
are higher with the PfHRP2 tests using IgG capture antibody (16.5% to 83% ) compared to the
PfHRP2 tests using IgM antibodies (6.6%) and the pLDH test (3.3%). Cross reactivity of the
PMA antibody with rheumatoid factor does not appear to occur.
Sensitivity: RDTs for the diagnosis of P. falciparum malaria generally achieve a sensitivity of
>90% at densities above 100 parasites per µL blood and the sensitivity decreases markedly
below that level of parasite density. Many studies have achieved >95% sensitivity at parasitemia
of ~500 parasites/µL, but this high parasitemia is seen in only a minority of patients. For the
diagnosis of P. vivax malaria, the PfHRP2/PMA test has a lower sensitivity compared to that for
P. falciparum malaria; however, the pLDH test has an equal or better sensitivity for P. vivax
malaria compared to P. falciparum malaria. For the diagnosis of P. malariae and P. ovale
infections, the sensitivity is lower than that of P. falciparum malaria at all levels of parasitemia
on both the PfHRP2/PMA and the pLDH tests. The specificity appears to be better with the
pLDH test than the PfHRP2/PMA test for both P. falciparum and non-falciparum malaria.

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. The sensitivity of
the pLDH test in field studies was also found to be lower at low parasitemias in field studies. The
comparisons between the PfHRP2/PMA test and the pLDH test in field studies have yielded
variable results, but the pLDH tests were found to have a better specificity for P. vivax. In one
study, PfHRP2, PfHRP2/PMA, and pLDH tests had a sensitivity of <75% at parasitemias of
<1,000/m L. Of concern is the fact that in nonimmune individuals, symptomatic malaria can
occur at parasite densities that are below the detection threshold of currently available RDTS. In
a cross-sectional malaria survey, 84.1% patients with P. falciparum infection had a parasitemia
of <500/µL and the sensitivity of the PfHRP2 test was only 23.3% at this level of parasitemia.
The level of parasitemia encountered in P. vivax infection rarely exceeds 1% (50,000/µL) and
usually is much lower. The level of parasitemia forP. malariae and P. ovale are lower than for P.
vivax, and the affinity of the panspecific antibodies for these parasites is also lower. Lower levels
of parasitemia are also common in nonimmune patients treated with antimalarial
chemoprophylaxis. This would mean that P. falciparum infections with low levels of parasitemia
and a significant proportion of symptomatic, non-immune patients with P. vivax (or other non-
falciparum) malaria may be missed by the RDTs.

Further, the RDTs have been reported to give false negative results even at higher levels of
parasitemia. Therefore, in cases of suspected severe malaria or complex health emergencies, a
positive result may be confirmatory but a negative result may not rule out malaria. Further, a
negative RDT result should always be confirmed by microscopy. It should be emphasized that P.
falciparum malaria, a potentially lethal disease, must not be missed because of a false-negative
dipstick test. It has been suggested that in such cases, 1 in 10 dilution of a negative sample with
0.9% sodium chloride may help to exclude the prozone phenomenon.

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 non-falciparum 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/µl. 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.

Cross reactions between Plasmodia species and problems in identifying non-falciparum

species: Cross reaction of PfHRP2 with non-falciparum malaria could give false positive results
for P. falciparum and mixed infections containing asexual stages of P. falciparum could be
interpreted as negative in about one third of the patients.

Another major difficulty still encountered by the use of RDTs is the correct identification of
Plasmodium species, particularly in areas where nonfalciparum malaria is prevalent. The
PfHRP2 tests can detect only P. falciparum infection and would miss the more common non-
falciparum malaria in areas where other Plasmodium species are co-endemic.

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.

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.

Persistence of antigens: All the antigens targeted by the RDTs are expressed by the asexual as
well as the sexual forms of the parasites and persistent antigenemia can cause positive tests on
RDTs up to one month. With the schizonticidal drugs having no effect on the gametocytes of
P.falciparum (except for the artemisinin compounds), RDTs may not be reliable tools to predict
the therapeutic response.

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.
Lower sensitivity in detecting asymptomatic patients, large numbers of positive tests due to
persistent antigenemia following incomplete treatment, inability to differentiate the mixed
infections and the non-falciparum species, and inability to differentiate between the various
stages of the parasite limit the value of the RDTs in active surveillance in the field. The cost of
the RDTs has also been considered as a major obstacle for their large scale use in field studies.

The RDTs have been evaluated for the diagnosis of malaria in travelers, as self-use kits and at
the laboratories. Studies on self-use by travelers have raised doubts over the reliability of
interpretation of the RDTs by travelers. 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 correctly. High number of false negative results have also been reported.

Comparison of Rapid Diagnostic Tests for Malaria Antigens

PfHRP2 tests PfHRP2 and PMA test pLDH test
Pan-specific
Histidine rich protein 2 of Parasite lactate
Plasmodium aldolase.
P. falciparum,water dehydrogenase. parasite
Target antigen parasite glycolytic
soluble protein expressed glycolytic enzyme
enzyme produced by all
on RBC membrane produced by all species
species and PfHRP2
General test
2 lines 3 lines 3 lines
format
Detects P. falciparum
Capability Can detect all 4 species Can detect all 4 species
only
Detected; Detected; differentiation
Non-falciparum
Not detected differentiation between between the 3 not
species
the 3 not possible possible
Mixed infections Appear as P. Appear as P.
Appear as P. falciparum;
of P.falciparum falciparum; falciparum;
differentiation not
with non- differentiation not differentiation not
possible
falciparum species possible possible
> 100-200 parasites/µL
Higher for P. vivax and
for P. falciparum andP.
Detection limit >40-100 parasites/µL other non-falciparum
vivax; may be higher for
species
P. malariae andP. ovale
Reported; longer for
Post-treatment
pan specific Reported up to 1 -3
persistence of Reported up to 31 days
antigenemia than for weeks
antigens
PfHRP2
Cross-reactivity
between malarial Reported Reported Reported
species
Cross-reactivity Reported, high (up to
Reported. low (3.3%
with auto 83% with rheumatoid Not known
with rheumatoid factor)
antibodies factor)
Indication of Positive test indicates
viability of No No presence of viable
parasites parasitemia
Comparison of Peripheral Blood Smear Examination and RDTs for Malaria
Peripheral Smear Rapid Diagnostic Tests
Format Slides with blood smear Test strip
Equipment Microscope Kit only
Training Trained microscopist ‘Anyone with a little training’
Test duration 20-60 minutes or more 5-30 minutes
Direct visualization of the
Test result Color changes on antibody coated lines
parasites
Detects malaria antigens (PfHRP2/
Detects and differentiates all
Capability PMA/pLDH) from asexual and/or sexual
plasmodia at different stages
forms of the parasite
1 00-500/µL for P. falciparum, higher
Detection threshold 5-10 parasites/µL of blood
for non-falciparum
Cannot differentiate among non-
Species falciparum species; mixed infections of
Possible
differentiation P.falciparum and non-falciparum appear
as P. falciparum
Quantification Possible Not possible
Differentiation
between sexual and Possible Not possible
asexual stages
Availability of equipment and Unpredictable efficiency at low and very
skilled microscopists, high parasitemia; cross reactions among
Disadvantages
particularly at remote areas plasmodial species and with auto-
and odd hours antibodies; persistence of antigens
Status Gold standard Not yet approved by the FDA
Cost per test US$ 0.12-0.40 US$ 1 .20-13.50

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.

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. [See] [Also See]

Further Reading:
 1. Anthony Moody. Rapid Diagnostic Tests for Malaria Parasites. Clinical Microbiology
Reviews 2002;15(1):66-78 Athttp://cmr.highwire.org/cgi/content/full/15/1/66
2. Endeshaw T, Gebre T, Ngondi J et al. Evaluation of light microscopy and rapid
diagnostic test for the detection of malaria under operational field conditions: a household
survey in Ethiopia. Malaria Journal. 2008;7:118 Full Text at
http://www.malariajournal.com/content/pdf/1475-2875-7-118.pdf
3. Msellem MI, Mårtensson A, Rotllant G, Bhattarai A, Strömberg J, et al. Influence of
Rapid Malaria Diagnostic Tests on Treatment and Health Outcome in Fever Patients,
Zanzibar—A Crossover Validation Study PLoS Med2009;6(4): e1000070. Free Full Text
4. Bisoffi Z, Gobbi F, Angheben A, Van den Ende J. The Role of Rapid Diagnostic Tests in
Managing Malaria. PLoS Med. 2009;6(4): e1000063 Free Full Text
5. Jacek Skarbinski et al. Effect of Malaria Rapid Diagnostic Tests on the Management of
Uncomplicated Malaria with Artemether-Lumefantrine in Kenya: A Cluster Randomized
Trial Am. J. Trop. Med. Hyg., 80(6), 2009, pp. 919-926 Available at
http://www.ajtmh.org/cgi/content/abstract/80/6/919?etoc
6. Clinton K. Murray, Jason W. Bennett. Rapid Diagnosis of Malaria: Review Article.
Interdisciplinary Perspectives on Infectious Diseases. Volume 2009, Article ID 415953,
7 pages doi:10.1155/2009/415953. Available at
http://www.pubmedcentral.nih.gov/picrender.fcgi?artid=2696022&blobtype=pdf
7. Laboratory demonstration of a prozone-like effect in HRP2-detecting malaria rapid
diagnostic tests: implications for clinical management Jennifer Luchavez et al.Malaria
Journal 2011;10:286 doi:10.1186/1475-2875-10-286 [Full Text] (Added Oct 9, 2011)

Microscopic Tests
Diagnosis of malaria involves identification of malaria parasite or its antigens/products in the
blood of the patient. Although this seems simple, the efficacy of the diagnosis is subject to many
factors. The different forms of the four malaria species; the different stages of erythrocytic
schizogony; the endemicity of different species; the population movements; the inter-relation
between the levels of transmission, immunity, parasitemia, and the symptoms; the problems of
recurrent malaria, drug resistance, persisting viable or non-viable parasitemia, and sequestration
of the parasites in the deeper tissues; and the use of chemoprophylaxis or even presumptive
treatment on the basis of clinical diagnosis can all have a bearing on the identification and
interpretation of malaria parasitemia on a diagnostic test.

The diagnosis of malaria is confirmed by blood tests and can be divided into microscopic and
non-microscopic tests.

Microscopic Tests

The microscopic tests involve staining and direct visualization of the parasite under the
microscope. For more than hundred years, the direct microscopic visualization of the parasite on
the thick and/or thin blood smears has been the accepted method for the diagnosis of malaria in
most settings, from the clinical laboratory to the field surveys. The careful examination of a well-
prepared and well-stained blood film currently remains the “gold standard” for malaria
diagnosis. The most commonly used microscopic tests include the peripheral smear study and the
Quantitative Buffy Coat (QBC) test.

The simplest and surest test is the time-honoured peripheral smear study for malarial parasites.
None of the other newer tests have surpassed the ‘gold standard’ peripheral smear study.

Remember this:

 Ask for MP test in all cases of fever and related symptoms and also whenever there is
high level of suspicion
 MP test can be done at any time. Do not wait for typical symptoms and signs or for chills
 A negative test DOES NOT rule out malaria. Repeated tests may have to be done in all
doubtful cases. Duration of the illness, level of parasitemia, expertise of the technician
and the method of examination may all have a bearing on the result of the M.P. test

Peripheral smear study for malarial parasites – The MP test

Light microscopy of thick and thin stained blood smears remains the standard method for
diagnosing malaria. It involves collection of a blood smear, its staining with Romanowsky stains
and examination of the Red Blood Cells for intracellular malarial parasites. Thick smears are 20–
40 times more sensitive than thin smears for screening of Plasmodium parasites, with a detection
limit of 10–50 trophozoites/μl. Thin smears allow one to identify malaria species (including the
diagnosis of mixed infections), quantify parasitemia, and assess for the presence of schizonts,
gametocytes, and malarial pigment in neutrophils and monocytes.

The peripheral blood smear provides comprehensive information on the species, the stages, and
the density of parasitemia. The efficiency of the test depends on the quality of the equipment and
reagents, the type and quality of the smear, skill of the technician, the parasite density, and the
time spent on reading the smear. The test takes about 20 to 60 minutes depending on the
proximity of the laboratory and other factors mentioned above. It is estimated to cost about 12 to
40 US cents per slide in the endemic countries.

Before reporting a negative result, at least 200 oil immersion visual fields at a magnification of
1000× should be examined on both thick and thin smears, which has a sensitivity of 90%. The
level of parasitemia may be expressed either as a percentage of parasitized erythrocytes or as the
number of parasites per microliter of blood. In nonfalciparum malaria, parasitemia rarely exceeds
2%, whereas it can be considerably higher (>50%) in falciparum malaria. In nonimmune
individuals, hyperparasitemia (>5% parasitemia or >250 000 parasites/μl) is generally associated
with severe disease.

The smear can be prepared from blood collected by venipuncture, finger prick and ear lobe stab.
In obstetric practice, cord blood and placental impression smears can be used. In fatal cases,
post-mortem smears of cerebral grey matter obtained by needle necropsy through the foramen
magnum, superior orbital fissure, ethmoid sinus via the nose or through fontanelle in young
children can be used.

Preparation of the smear: Use universal precautions while preparing the smears for malarial
parasites – use gloves; use only disposable needles/lancets; wash hands; handle and dispose the
sharp instruments and other materials contaminated with blood carefully to avoid injury.

 Hold the third finger of the left

hand and wipe its tip with spirit/Savlon swab; allow to dry
 Prick the finger with disposable needle/lancet; allow the blood to ooze out
 Take a clean glass slide. Take 3 drops of blood 1 cm from the edge of the slide, take
another drop of blood one cm from the first drop of blood
  Take another clean slide with smooth edges and use it as a spreader and make thick and
thin smears. Allow it to dry
 Slide number can be marked on the thin smear with a lead pencil

Thick smear: The thick smear of correct thickness is the one through which newsprint is barely
visible. It is dried for 30 minutes and not fixed with methanol. This allows the red blood cells to
be hemolyzed and leukocytes and any malaria parasites present will be the only detectable
elements. However, due to the hemolysis and slow drying, the plasmodia morphology can get
distorted, making differentiation of species difficult. Thick smears are therefore used to detect
infection, and to estimate parasite concentration.

Thin smear: Air dry the thin smear for 10 minutes. After drying, the thin smear should be fixed
in methanol. This can be done by either dipping the thin smear into methanol for 5 seconds or by
dabbing the thin smear with a methanol-soaked cotton ball. While fixing the thin smear, all care
should be taken to avoid exposure of the thick smear to methanol.

Staining: A number of Romanowsky stains like Field’s, Giemsa’s, Wright’s and Leishman’s are
suitable for staining the smears. Thick films are ideally stained by the rapid Field’s technique or
Giemsa’s stain for screening of parasites. The sensitivity of a thick blood film is 5-10
parasites/µl. Thin blood films stained by Giemsa’s or Leishman’s stain are useful for
specification of parasites and for the stippling of infected red cells and have a sensitivity of 200
parasites/µl. The optimal pH of the stain is 7.2.

Slides should be clean and dry. It is better to use neutral distilled water.

Thick films: The thick film is first de-hemoglobinised in water and then stained with Giemsa.

Rapid Giemsa: Prepare a 10% Giemsa in buffered water at pH 7.1. Immerse the slide in the stain
for 5 minutes. Rinse gently for 1 or 2 seconds in a jar of tap water. Drain, dry and examine.

Standard Giemsa: Prepare a 4% Giemsa in buffered solution at pH 7.1. Immerse the slide (at
least 12 hours old) in stain for 30 minutes. Rinse with fresh water, drain, dry and examine.

Thin films: Thin film examination is the gold standard in diagnosis of malarial infection.

Giemsa stain: Fix with 1-2 drops of methanol. Cover the film with 10% Giemsa stain and leave
for 30 minutes, wash with distilled water, drain, dry and examine.

Leishman’s stain: Add 7-8 drops of the stain and leave for 1-2 minutes. Then add 12-15 drops of
buffered distilled water, mix thoroughly, leave for 4 – 8 minutes. Then wash off with clean
water, drain, dry and examine.
 Jaswant Singh Battacharya
(JSB) Stain for thick and thin films: This is the standard method used by the laboratories under
the National Malaria Eradication Programme in India.

Preparation of the stain:

JSB I stain: Medicinal methylene blue (0.5 g) is dissolved in 500 ml of distilled water and 3 ml
of 1% sulphuric acid (H2SO4) is gradually added, followed by 0.5 g of potassium dichromate
(K2Cr2O7) when a purple precipitate forms. 3.5 g of disodium hydrogen phosphate dihydrate
(Na2HPO4.2H2O) is next added and when the precipitate has dissolved, the solution is boiled in a
flask with a reflex condenser for 1 hour. The stain is ready for immediate use.

JSB II stain: 1 g Eosin is dissolved in 500 ml tap water.

Buffered water: 0.22 g of disodium hydrogen phosphate dihydrate (Na 2HPO4.2H2O) and 0.74 g
of potassium acid phosphate (KH2PO4) are added to 1000 ml of distilled water or filtered tap
water.

Staining:

After dehemoglobinisation, dip the thick smear in JSB II stain two to three times. Wash it by
dipping in buffer water two to three times. Then keep the thick film dipped in JSB I stain for 40-
60 seconds. Wash it with buffer water. Drain, dry and examine.

Differentiation of Malaria Parasites

 Finding P. falciparum P. vivax P. malariae P. ovale
RBC Size Not enlarged Enlarged Not enlarged Enlarged
Round, sometimes Round or oval, Round or oval,
RBC Shape Round
crenated frequently bizarre often fimbriated
Normal, but may
RBC Colour become darker; may Normal to pale Normal Normal
have a purple rim
Maurer’s spots, appear Schuffner’s dots
Schuffner’s dots, Ziemann’s dots,
as large red spots, (James’s dots).
Stipling appear as small red few tiny dots,
loops and clefts; up to Numerous small
dots, numerous. rarely detected
20 or fewer. red dots.
Black or dark brown; Seen as a haze of
in asexual forms as one fine golden brown Black or brown Intermediate
Pigment or two masses; in granules scattered coarse granules; between P. vivax
gametocytes as about through the scattered and P. malariae
12 rods cytoplasm
Smallest, delicate; Relatively large;
Early sometimes two one chromatin dot, Compact; one Compact; one
trophozoite chromatin dots; sometimes two; chromatin dot; chromatin dot;
(ring) multiple rings often two rings in single single
commonly found one cell
Medium size; compact;
Large; amoeboid; Small; compact; Medium size;
numerous chromatin
numerous few chromatin compact; few
Schizont masses; coarse
chromatin masses; masses; coarse chromatin masses;
pigments; rarely seen
fine pigments pigments coarse pigments
in peripheral blood
Similar to P.
Crescent shaped, larger
vivax, but Like P. vivax, but
Gametocyte and slender; central Spherical; compact
smaller and less smaller
chromatin
numerous
Problems: The exacting needs of the blood smear examination are often not met in certain
remote and poor parts of the world. Detection of low levels of parasitemia, sequestered parasites
of P. falciparum and past infections in aspiring blood donors; ascertaining viability of the
detected parasites; difficulties in maintaining the required technical skills and resultant
misdiagnosis due to poor familiarity and problems in accessing and activating the facility in
emergencies are some of the deficiencies with the blood smear examination.

In falciparum malaria, parasitized erythrocytes may be sequestered in tissue capillaries resulting

in a falsely low parasite count in the peripheral blood (‘visible’ parasitemia). In such instances,
the developmental stages of the parasite seen on blood smear may help to assess disease severity
better than parasite count alone. The presence of more mature parasite forms (>20% of parasites
as late trophozoites and schizonts) and of more than 5% of neutrophils containing malarial
pigment indicates more advanced disease and a worse prognosis. One negative blood smear
makes the diagnosis of malaria very unlikely (especially the severe form); however, smears
should be repeated every 6–12 hours for 48 hours if malaria is still suspected.[1-5]

Sometimes no parasites can be found in peripheral blood smears from patients with malaria, even
in severe infections. This may be explained by partial antimalarial treatment or by sequestration
of parasitised cells in deep vascular beds. In these cases, parasites, or malarial pigment may be
found in the bone marrow aspirates. Presence of malarial pigment in circulating neutrophils and
monocytes may also suggest the possibility of malaria.

Alternative microscopic methods have been tried, including faster methods of preparation,
dark-field microscopy, and stains like benzothiocarboxypurine, acridine orange and Rhodamine-
123. Acridine orange has been tried as a direct staining technique, with concentration methods
such as thick blood film or the centrifugal Quantitative Buffy Coat system and with excitation
filter in the Kawamoto technique. Inability to easily differentiate the Plasmodium species,
requirements of expensive equipment, supplies and special training as well as the high cost limit
the use of these methods.

Parasitemia in blood films:

Thick Blood Film:

Infected erythrocytes are counted in relation to a predetermined number of WBCs and an average
of 8000/µl is taken as standard. 200 leucocytes are counted in 100 fields (0.25 µl of blood). All
parasite species and forms including both sexual and asexual forms are counted together.

If >10 parasites are counted, then the following formulae can be applied:

(No. of Parasites/ No. of WBCs counted) x 8000 = No. of parasites/µl

Or if 200 leukocytes are counted,

No. of parasites counted x 40 = No. of parasites/µl

If the parasites are <9, then 500 WBCs should be counted and the formula will be –

No. of parasites counted x 16 = No. of parasites/µl

In the Earle and Perez method, the number of asexual parasites per known volume of blood
(usually 5µl) spread as a thick film are counted; this is used only in research studies.

Thin Blood Film:

Determining the percentage of parasitaemia will be essential for P. falciparum. The number of
infected red cells (and not number of parasites) in 1000 RBCs is converted to percentage.

This method estimates the percentage of red blood cells infected with malarial parasites. The
smear is scanned carefully, one ‘row’ at a time. The total number of red cells and the number of
parasitised red cells are tabulated separately. If 1000 red cells are counted, then divide the
number of parasitised red cells by 10 to get the percentage (i.e. if 30 out of 1000 cells are
parasitised, then the parasitised red cell count is 3%). If lesser red cells are counted, then divide
the number parasitized by the total number counted and multiply the result by 100 to obtain a
percentage estimate of red blood cells parasitized. If occasional parasites are seen when scanning
the smear, but none are identified during the process of counting 300-500 red blood cells, a
percentage value of less than 1% of red blood cells parasitized is assigned. An estimate of less
than 1% of red blood cells parasitized does not need to be refined, since no clinical predictive
value is gained. It is values of 2-3% or above that are of clinical concern.

The “plus system” is less precise as variation in the thickness of the film results in false variation
in parasite count.

+ = 1–10 per 100 thick fields.

++ = 11-100 per 100 thick fields.

+++ = 1–10 per thick field.

++++ = >10 per thick field.

Further Reading:

1. Andrej Trampuz, Matjaz Jereb, Igor Muzlovic, Rajesh M Prabhu. Clinical review: Severe
malaria. Critical Care 2003;7:315-323 Available at http://ccforum.com/content/7/4/315
2. Moody AH, Chiodini PL. Methods for the detection of blood parasites. Clin Lab
Haematol 2000;22:189-201.
3. Torres JR. Malaria and babesiosis. Therapy of Infectious Diseases (Edited by: Baddour
LM, Gorbach SL). Philadelphia, PA: Saunders 2003;597-613.
4. Nguyen PH, Day N, Pram TD, Ferguson DJ, White NJ. Intraleucocytic malaria pigment
and prognosis in severe malaria. Trans R Soc Trop Med Hyg 1995;89:200-204.
5. White NJ. Malaria. Manson’s Tropical Diseases (Edited by: Cook GC, Zumla AI, Weir
J). Philadelphia, PA: WB Saunders 2003;1205-1295
6. Lee SH, Kara UA, Koay E, Lee MA, Lam S, Teo D. New strategies for the diagnosis and
screening of malaria. Int J Hematol 2002;76(suppl 1):291-293.
7. Moody A. Rapid diagnostic tests for malaria parasites. Clin Microbiol Rev 2002;15:66-
78.
8. Hanscheid T, Grobusch MP: How useful is PCR in the diagnosis of malaria? Trends
Parasitol 2002;18:395-398
9. New Perspectives – Malaria Diagnosis: Report Of A Joint Who/Usaid Informal
Consultation 25–27 October 1999 p 11 At
http://www.wpro.who.int/NR/rdonlyres/3DC6B7D7-711F-4F63-8FF9-
A70DBA99DB7E/0/NewPersectives.pdf
10. Castelli F, Carosi G. Diagnosis of malaria infection In Castelli F, Carosi G ed Handbook
of malaria infection in the tropics. Organissazione per la cooperazione sanitaria
internazionale 1997 pp 114
11. http://www.med-chem.com/procedures/DetofPara.pdf
12. http://www.btinternet.com/~ukneqas.parasitologyscheme/Blood_Scheme/
13. Warhurst DC, William J Laboratory diagnosis of malaria ACP Broadsheet No 148 J Clin
Path 1996;49:533-8
14. Noppadon Tangpukdee, Chatnapa Duangdee, Polrat Wilairatana, Srivicha Krudsood.
Malaria Diagnosis: A Brief Review Korean J Parasitol. 2009 Jun;47(2):93-102.
Available at http://www.parasitol.or.kr/kjp/search.php?
where=aview&id=28586&code=0066KJP&vmode=FULL