Commentary provided by:

Carlo Ledesma, MS SH(ASCP)QLS, MT(ASCPi), MT(AMT)

Program Director, CLT/Phlebotomy
Midwest City, OK

EDUCATIONAL COMMENTARY – MALARIA

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Pathology (ASCP). To obtain FREE CME/CMLE credits, click on Earn CE Credits under Continuing
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**Florida licensees, please note: This exercise will appear in CE Broker under the specialty of
Microbiology.

Learning Objectives

On completion of this exercise, the participant should be able to

• discuss the epidemiology of malaria;

• identify the five malarial species that affect humans;

• discuss the plasmodia life cycle;

• discuss the effect and pathogenesis of the malarial infection; and

• recognize microscopic features of plasmodia.

Introduction and History

Malarial infection remains a global threat. Malaria is a mosquito-borne disease that is uncommon in
developed countries, where the disease occurs mainly in travelers who have returned from endemic
regions. According to the Centers for Disease Control and Prevention (CDC), about 1,700 cases of
malaria are diagnosed in the United States each year. The vast majority of cases in the United States
occur in travelers and immigrants returning from countries where malaria transmission occurs, many from
sub-Saharan Africa and South Asia.

Malaria is transmitted primarily by the bite of infected anopheline mosquitoes. It can also be transmitted
by inoculation of infected blood and congenitally. Anophelines (Figure 1) feed at night and their breeding
sites are primarily in rural areas. Therefore, the greatest risk of malaria is from dusk to dawn, in rural
areas. In many malaria-endemic areas, there is little or no risk in urban areas because of improved
housing, drainage of Anopheles breeding sites, household mosquito proofing, and expanded personal
protection.1 However, urban transmission is still common in some parts of the world, especially Africa.2

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Plasmodia Species

Malarial infections are caused by plasmodia species.

Only 5 of the more than 100 species of plasmodia are
infectious to humans. Most cases of malaria and almost
all malarial deaths are caused by Plasmodium
falciparum. Plasmodium vivax, Plasmodium ovale,
Plasmodium malariae and Plasmodium knowlesi cause
Figure 1. An Anopheles mosquito. Image
less severe disease. More than 90% of all malaria cases courtesy of Emily Lund, Harvard University T.H.
Chan School of Public Health.
occur in Africa and most are caused by P falciparum. This
species is also most prevalent in Haiti and the Dominican Republic. In Mexico, Central and South
America, the Mediterranean, Asia, and Oceania, both P falciparum and P vivax are endemic. Disease
caused by P ovale and P malariae is relatively rare.3 Plasmodium knowlesi can cause acute, severe
illness but mortality rates are low.

Pathophysiology and Clinical Manifestations of Malaria

The life cycle of the malaria parasite, spanning its mosquito and human hosts, is shown in Figure 2.
Malarial infections are transmitted by the nocturnal Anopheles mosquitoes. Tropical climates create the
most favorable conditions for mosquitoes to breed and it prolongs their survival.

After the infective bite by the Anopheles mosquito, an incubation period that lasts between 7 to 30 days
occurs before symptoms start to appear. Shorter incubation periods are observed with P falciparum
infections and longer incubation periods are seen with P malariae infections.

The most characteristic symptom of malaria is fever and the classical malaria attack lasts between 6 and
10 hours. The triad of malarial symptoms often are defined as: cold stage, hot stage and the sweating
stage. Symptoms of malarial infection include chills, headache, myalgias, nausea, and vomiting. Diarrhea,
abdominal pain, and cough are occasionally seen. As the disease progresses, some patients may
develop the classic malaria paroxysm with bouts of illness alternating with symptom-free periods.

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Figure 2. The plasmodia life cycle. The human (asexual) stage of the life cycle begins with the
exoerythrocytic phase. When an infected mosquito bites a human, sporozoites in the mosquito's
saliva enter the bloodstream (1). The sporozoites travel to the liver, where they invade hepatocytes
(2); over a period of up to 4 weeks, the infected hepatocytes mature into schizonts. In Plasmodium
vivax and Plasmodium ovale infections only, some schizonts may remain dormant as hypnozoites
(3) for weeks to years before causing clinical relapses. With schizont rupture, merozoites are
released into the bloodstream (4). In the erythrocytic phase, merozoites invade erythrocytes and
either undergo an asexual cycle of reproduction (5) or develop into non-multiplying sexual forms
(gametocytes) (6). These gametocytes are crucial for perpetuating the life cycle, as they are
ingested by a feeding mosquito (7) and undergo sexual reproduction within the mosquito midgut;
thousands of infective sporozoites (8) are produced, which then migrate to the salivary glands,
ready to initiate another life cycle.3 Photo: Lianne Friesen and Nicholas Woolridge.

Malarial Paroxysm Stages

Symptoms follow 3 successive stages:
1. Cold stage: A stage lasting an hour, usually characterized by shivering.
2. Hot stage: A stage lasting 2 to 6 hours in which there is fever, sometimes reaching 105.8°F,
flushed, dry skin, and often headache, nausea, and vomiting.
3. Sweating stage: Finally, there is a sweating stage lasting 2 to 4 hours, during which the fever
drops rapidly and the patient sweats.

The periodic febrile response in all types of malarial infections are due to the rupture of mature schizonts
and release of merozoites into the bloodstream. In P vivax and P ovale malaria, a brood of schizonts
matures every 48 hours, so the periodicity of fever is tertian (“tertian malaria”), whereas in P
malariae disease, fever occurs every 72 hours (“quartan malaria”). The fever in P falciparum malaria may

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occur every 48 hours but is usually irregular and shows no distinct periodicity. These classic fever
patterns are usually not seen early in the course of malaria; therefore, the absence of periodic,
synchronized fevers does not rule out a diagnosis of malaria.4

In an uncomplicated malaria, a variety of laboratory abnormalities can be seen: normochromic,

normocytic anemia, thrombocytopenia, leukocytosis or leukopenia, hypoglycemia, hyponatremia,
elevated liver and renal function tests, proteinuria, and laboratory evidence of disseminated intravascular
coagulation (although clinically important bleeding is rare). Eosinophilia is not seen. Patients with
complicated malaria occasionally show evidence of massive intravascular hemolysis with
hemoglobinemia and hemoglobinuria. If there is a delay in diagnosis especially with P falciparum
infections, these cases can potentially lead to death. One of the complications caused by malaria is
severe anemia with cerebral involvement but the most serious complication caused by malaria is death.

Malarial Development and Pathogenesis

The stage in the malarial life cycle that is infective to humans is the uninucleate, lancet-shaped sporozoite
(approximately 1 × 7 μm). Sporozoites are produced by sexual reproduction in the midgut of the
anopheline mosquitoes and migrate to the salivary gland.

When an Anopheles mosquito bites a human, she may inject sporozoites into small blood vessels then
the sporozoites enter liver parenchymal cells. In the liver, the parasite evolves into multinucleate liver-
stage schizont which contains 2,000 to 40,000 uninucleate merozoites. This process of enormous
amplification is called exoerythrocytic schizogony. Upon rupture of the schizonts, each merozoite can
infect a red blood cell. Within the red blood cell, the merozoite develops to form either an erythrocytic-
stage (blood-stage) schizont (by the process of erythrocytic schizogony) or a gametocyte. The mature
erythrocytic-stage schizont contains merozoites, each 5 to 10 μm long, which are released into the blood
when the schizont ruptures. These merozoites proceed to infect another generation of erythrocytes. The
time required for erythrocytic schizogony-which determines the interval between the releases of
successive generations of merozoites-varies with the species of plasmodium and is responsible for the
classic periodicity of fever in malaria.4 The gametocyte, which is the sexual stage of the plasmodium, is
infectious for mosquitoes that ingest it while feeding. Within the mosquito, the gametocytes undergo
fertilization and then develop across 2 to 3 weeks into sporozoites that can infect humans. Clinical illness
is caused by the erythrocytic stage of the parasite.

Several factors contribute to the severity of clinical disease. High parasite burdens combined with the
unique ability of infected erythrocytes to adhere to host endothelium contribute to microvascular
occlusion, metabolic derangement and acidosis, which lead to the manifestations of severe malaria
(acute respiratory distress syndrome, renal insufficiency and cerebral malaria). Cytokine response to

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parasite proteins released during schizont rupture can contribute to adverse clinical outcomes.
Manifestations of disease may also be related to intravascular hemolysis and parasite consumption of
glucose. Host factors such as sickle cell disease and glucose-6-phosphate dehydrogenase deficiency can
modify the severity of disease. Heterozygotes for the sickle cell gene are relatively protected against
malaria, while patients who are homozygous for the sickle cell gene, suffer from sickle cell disease and
are highly prone to the lethal effects of malaria.7 Infections caused by P vivax, P ovale, and P
malariae are generally milder than P falciparum malaria; symptoms are related to parasite burden and
cytokine release, because vaso-occlusive phenomena do not occur.3

Laboratory Diagnosis of Malaria

Rapid diagnosis is essential to optimize the outcome of patients infected with malaria. Thick and thin
peripheral blood smears, stained with Giemsa stain (or, alternatively, Wright or Field stains), remain the
criterion standard for routine clinical diagnosis. Malaria smears permit both species identification and
quantification (expressed as a percentage of erythrocytes infected or as parasites per microliter) of
parasites. Figure 3 shows P falciparum images and Figure 4 is a chart with characteristics of four of the
species. Malaria should not be excluded until at least three negative blood smears have been obtained
within 48 hours. Rapid malaria tests, which require minimal skill to perform and interpret, have been
developed to overcome the problems of malaria smears. The most practical of these are the rapid antigen
detection tests, which detect parasite proteins in finger-prick blood samples. Malarial antigens are
suitable for rapid antigen detection tests, these antigens are HRP-2, pLDH, and Plasmodium aldolase.
HRP-2 is a water-soluble protein produced by asexual stages and young gametocytes of P falciparum. It
is expressed on the red blood cell membrane surface, and because of its abundance in P falciparum, it
was the first antigen to be used to develop a test for its detection (rapid antigen detection test).
Plasmodium lactate dehydrogenase (pLDH), an enzyme found in the glycolytic pathway of the malaria
parasite, is produced by sexual and asexual stages of the parasite. Different isomers of pLDH for each of
the four Plasmodium species infecting humans exist and their detection constitutes a second approach to
rapid antigen detection test development. Several other enzymes of the malaria parasite glycolytic
pathway, notably aldolase, have been suggested as target antigens for RDT for species other than P
falciparum.5

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Figure 3. Stages in the life cycle of Plasmodium falciparum. A, Ring forms (early
trophozoites). B, Mature schizont, rarely seen in peripheral blood smears because of
microvascular sequestration. C, Gametocyte, demonstrating the classic banana
shape. Source: Division of Parasitic Diseases, US Centers for Disease Control and
Prevention, Atlanta, Georgia. Photo: CDC.

Preparation of Thick and Thin Smear

To prepare a thick blood film, a drop of blood is stirred in a dime-sized (1-2cm) circular motion in the
corner of the slide, taking care not make the preparation too thick, and allowed to dry without fixative.
After drying, the spot is stained with 5% Giemsa stain for 20 min, and washed by placing the film in
buffered water for 3 minutes. The slide is allowed to air-dry in a vertical position and examined using a
light microscope. Because they are unfixed, the red blood cells lyse when a water-based stain is applied.

A thin blood film is prepared by placing the smooth edge of a spreader slide in a drop of blood, adjusting
the angle between slide and spreader to 45° and then smearing the blood with a swift and steady sweep
along the surface. The film is then allowed to air-dry and is fixed with absolute methanol. After drying, the
sample is stained with 5% Giemsa for 20 minutes and washed by briefly dipping the slide in and out of a
jar of buffered water (excessive washing will decolorize the film). The slide is then allowed to air-dry in a
vertical position and examined under a light microscope.6

Thick smears consist of a thick layer of lysed red blood cells, which provides better opportunity to detect
parasitic forms against a more transparent background. However, they do not permit an optimal review of
parasite morphologic features. Thin smears consist of blood spread in a layer such that the thickness
decreases progressively toward monolayer. It allows optimal assessment of the morphologic features of
any parasitic forms that may be present.

Although this method requires a trained microscopist, and sensitivity and specificity vary compared with
recent technical advances, it is inexpensive and reliable. Quick and convenient rapid tests are currently
implemented in many remote settings but they are costly and improved quality control is needed.
Serologic tests are useful for epidemiologic surveys but are not suitable for the diagnosis of acute
malaria.

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Figure 4. The microscopic findings of four of the Plasmodium species that are known to cause malaria in humans.8

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Summary

Malaria is a mosquito-borne disease that remains to be a global threat. Delay in diagnosis and treatment
can lead to death, therefore timely and accurate laboratory testing is of utmost importance. Rapid testing
is quick and convenient, especially for remote locations, however it is costly. Microscopic review of thick
and thin blood smears by trained individuals remains to be the criterion standard for routine clinical
diagnosis. Trained microscopists must be able to recognize the characteristics of the various forms of
Plasmodia species in order to make an accurate identification.

References

1. Wilson ML, Krogstad DJ, Arinaitwe E, et al. Urban malaria: understanding its epidemiology,
ecology, and transmission across seven diverse ICEMR network sites. Am J Trop Med Hyg.
2015;93(3 suppl):110-123.

2. Baron S, ed. Medical Microbiology. Galveston, TX: University of Texas Medical Branch at
Galveston; 1996.

3. Suh KN, Kain KC, Keystone JS. Malaria. CMAJ. 2004;170(11):1693-1702.

4. Crutcher JM, Hoffman SL. Malaria. In: Baron S, ed. Medical Microbiology. Galveston, TX:
University of Texas Medical Branch at Galveston; 1996.

5. Moody A. Rapid diagnostic tests for malaria parasites. Clin Microbiol Rev. 2002;15(1):66-78.

6. Tangpukdee N, Duangdee C, Wilairatana P, Krudsood S. Malaria diagnosis: a brief

review. Korean J Parasitol. 2009;47(2):93-102.

7. Luzzatto L. Sickle cell anaemia and malaria. Mediterr J Hematol Infect Dis. 2012;4(1):e2012065.

8. Microscopic tests. Malariasite.com. https://www.malariasite.com/microscopic-tests/. Updated

March 27, 2015.

© ASCP 2019

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