REVIEW ESSAY
Prospects & Overviews
Diagnosis of Malaria Parasites Plasmodium spp. in Endemic
Areas: Current Strategies for an Ancient Disease
Brian Gitta* and Nicole Kilian*
Fast and effective detection of the causative agent of malaria in humans,
protozoan Plasmodium parasites, is of crucial importance for increasing
the effectiveness of treatment and to control a devastating disease that affects
millions of people living in endemic areas. The microscopic examination
of Giemsa-stained blood films still remains the gold-standard in Plasmodium
detection today. However, there is a high demand for alternative diagnostic
methods that are simple, fast, highly sensitive, ideally do not rely on blood-
drawing and can potentially be conducted by the patients themselves. Here,
the history of Plasmodium detection is discussed, and advantages and disad-
vantages of diagnostic methods that are currently being applied are assessed.
1. Introduction
1.1. Malaria
Malaria (mala aria: “bad air”; a portmanteau word from the 18th
century), ague, or swamp fever (frz. paludisme) is a mosquito-
borne parasitosis that is endemic in 87 countries and causes ap-
proximately 219 million clinical cases and 435 000 deaths per year
(Figure 1).[1] Morbidity and mortality especially affect children
and pregnant women living in the World Health Organization
(WHO) African Region.[1,2]
The causative agents of malaria are protozoan parasites of the
genus Plasmodium.[1] This genus includes more than 200 differ-
ent species that parasitize various hosts such as reptiles, birds,
amphibians, and mammals (53 species total, of which 30 species
parasitize primates).[3] To date, six Plasmodium species have been
identified as human pathogenic, and they cause different types
of malaria: P. malariae (malaria quartana), P. ovale (with two
subspecies P. o. curtisi and P. o. wallikeri causing malaria ter-
tiana), P. knowlesi (zoonotic malaria), P. vivax (malaria tertiana),
B. Gitta
Matibabu
120 Semawata Rd, Ntinda, Kampala 00256, Uganda
E-mail: gitta@matibabu.io
Dr. N. Kilian
Centre for Infectious Diseases
Parasitology Heidelberg University Hospital
Im Neuenheimer Feld 324, 69120 Heidelberg, Germany
E-mail: nicole.kilian@med.uni-heidelberg.de
© 2019 The Authors. BioEssays Published by Wiley Periodicals, Inc. This
is an open access article under the terms of the Creative Commons
Attribution License, which permits use, distribution and reproduction in
any medium, provided the original work is properly cited.
DOI: 10.1002/bies.201900138
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and P. falciparum (malaria tropica), with
the latter two species being responsible for
the majority of clinical cases and deaths
worldwide.[1,4–6]
1.2. Asexual Replication and Host
Erythrocyte Reorganization Can Cause
Severe Complications
The life cycle of Plasmodium parasites
(Figure 2) involves asexual as well as sex-
ual replication that is linked to an obligate
host change from a human intermediate
host to a female mosquito of the genus Anopheles as the final
host.[7,8] During this complex life cycle, Plasmodium parasites
must infect and inhabit multiple cell types to ensure develop-
mental stage progression and progeny production.[7,8] The ery-
throcytic schizogony, which occurs within the erythrocytes of
the human host, involves a sophisticated reorganization of the
terminally differentiated and metabolically reduced host cell in
the process of securing nutrient supply from hemoglobin di-
gestion as well as blood serum uptake, removal of toxic waste,
protection against the host immune response, and life cycle
progression.[1,7,9–13]
During schizogony, P. falciparum establishes the Maurer’s
clefts, a secretory organelle that resides outside the parasite’s
cellular boundaries within the erythrocyte cytoplasm, to facili-
tate host–parasite interaction and sequestration to avoid splenic
clearance of the infected erythrocyte.[14] Sequestration is either
achieved via cytoadherence of the infected erythrocyte to various
receptors presented on the surface of vascular endothelial cells
in the postcapillary venules of different organs or via rosetting
of infected erythrocytes with uninfected erythrocytes.[9,15,16] The
cytoadherence of P. falciparum-parasitized erythrocytes is estab-
lished by members of the diverse var multigene family encoding
several different versions of P. falciparum erythrocyte membrane
protein 1 (PfEMP1), which are exclusively expressed in P. falci-
parum and presented on knob-like protrusions on the erythrocyte
plasma membrane.[17,18]
The causative agent of zoonotic malaria in Southeast Asia, P.
knowlesi, also generates a secretory organelle in the erythrocyte
cytoplasm called Sinton Mulligan’s clefts that appears to play a
crucial role in receiving and harboring proteins produced by the
erythrocyte-residing parasite.[19,20] It has been further reported
in an in vitro study by Fatih et al. that P. knowlesi might be
able to establish a cytoadhesive phenotype that would allow at-
tachment of the host erythrocytes to the inducible human en-
dothelial receptors intercellular adhesion molecule-1 (ICAM-1)
and vascular cellular adhesion molecule (VCAM).[21] However,
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Figure 1. Countries with indigenous cases in 2000 and their status by 2017. Countries with zero indigenous cases over at least the past 3 con-
secutive years are considered to be malaria free. All countries in the WHO European Region reported zero indigenous cases in 2016 and again in
2017. In 2017, both China and El Salvador reported zero indigenous cases. Source: WHO database.[1] Image published with permission from the
WHO.

a more recent study did not observe P. falciparum-like endothe-
lial cytoadherence and subsequent sequestration of P. knowlesi-
infected erythrocytes, but suggested that intravascular hemolysis
might instead be an important factor in the development of se-
vere symptoms.[22]
During an infection with P. ovale and P. vivax, the erythro-
cyte cytoplasm does not contain membrane structures. Instead,
the erythrocyte plasma membrane consists of parasite antigen-
containing caveolae called the Schüffner’s dots.[23,24] To date not
much is known about the establishment and function of the
Schüffner dots, but some evidence currently supports the no-
tion that P. vivax-infected erythrocytes are also able to display
cytoadherence.[18,25] Both P. ovale- and P. vivax-infected erythro-
cytes are able to form rosettes, but rosette formation in vivax and
ovale malaria is, in contrast to P. falciparum, not associated with
severe malaria.[26–28] An infection with P. malariae is not associ-
ated with severe malaria either, although the parasite is still able
to establish a successful erythrocyte infection and reorganization
generating the Ziemann stipplings (intraerythrocytic dots similar
to Schüffner dots) in the process.[29]
Parasite replication, host erythrocyte reorganization, host–
parasite interaction, cytoadherence, and rosetting can cause var-
ious severe symptoms such as severe anemia, jaundice, shock,
multiple organ dysfunction syndrome (MODS), acute pulmonary
edema, acute kidney injury, acidosis and hypoglycemia, cerebral
malaria, and coma, which can ultimately lead to the death of the
patient.[30–33]
1.3. The History of the Malaria Parasite and Vector Discovery
Sets the Stage for Today’s Gold Standard of Malaria Diagnosis
Malaria is a disease that might already have been documented by
the cultures of ancient societies, such as those of ancient China,
Egypt, India, Mesopotamia, and Greece (Figure 3).[34,35] The early
Greeks, including Homer, Plato, Empedocles, and Hippocrates,
were well aware of the characteristic symptoms of malaria such
as distinct fevers, splenomegaly, and generally poor health.[34,36]
In the 17th century, these symptoms were already treated with
“Jesuits’ powder,” a component of the cinchona tree bark, which
is now known as quinine, and is still in use as an antimalar-
ial drug today.[37] However, another century would pass until the
first histopathological discovery of the parasites themselves was
made by military physician Charles Louis Alphonse Laveran in
1880 (Figure 3).[38] By using direct microscopy, Alphonse Laveran
was able to identify granules of black pigment in the erythro-
cytes of a feverish soldier in a military hospital in Constantine
(Algeria).[38,39] Laveran further suspected that mosquitoes were
transmitting the disease, but it was Sir Ronald Ross who veri-
fied this hypothesis and solved the life cycle mystery by examin-
ing birds that were infected with P. relictum almost two decades
later.[36,40] In 1898, Italian malariologists, among them Camillo
Golgi, identified Anopheles mosquitoes as transmitters of human
malaria.[40] Camillo Golgi was further able to connect the dis-
tinct fever curves with the rupture of blood schizonts and mero-
zoite release.[40,41] It took another 50 years until Henry Shortt and

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Figure 2. Life cycle of human-pathogenic Plasmodium parasites. Schizogony and sporogony of human-pathogenic Plasmodium parasites occurring
between the intermediate host (Homo sapiens) and the final host (Anopheles), respectively. An infected female Anopheles mosquito inoculates Plasmodium
sporozoites into the skin of the human host during the blood meal. The sporozoites glide away from the inoculation site to reach a blood vessel that
quickly carries them to the liver sinusoids. The sporozoites leave the bloodstream, infect the hepatocytes, and develop into liver schizonts. The liver
schizonts produce and release thousands of haploid merozoites into the bloodstream after rupture (liver schizogony). P. vivax and P. ovale can further
form a dormant developmental stage (hypnozoites) that is able to persist in the liver, and can cause a malaria relapse even years after the original
Plasmodium infection. The freshly released liver merozoites are able to infect the erythrocytes of the human host. Within the erythrocytes, the parasite
matures from the juvenile ring developmental stage to the mature schizont developmental stage, which consists of various merozoites (erythrocytic
schizogony). These merozoites are released into the bloodstream after schizont rupture and invade new erythrocytes. Eventually, some parasites begin
their differentiation into sexual developmental stages (gametocytes: female macrogametocytes and male microgametocytes). The sexual developmental
stages are then ingested by the next female Anopheles mosquito during the blood meal. Within the midgut of the mosquito, macrogamet and microgamet
are mating and form a zygote. This zygote develops into an ookinete that penetrates the midgut wall, where it further develops into an oocyst. The
oocyst then produces sporozoites, which are released after oocyst rupture and invade the salivary glands of the mosquito. The sporozoites that have
been produced during this sporogony can then again be inoculated into the human intermediate host during the next blood meal via the secretion of
the mosquito’s anticoagulant saliva.

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Figure 3. Milestones of Plasmodium parasite discovery and malaria diagnosis. Cultures of ancient societies might already have observed and described
malaria symptoms. The actual discovery of Plasmodium came centuries later with the use of direct microscopy. Giemsa-stained blood film microscopy is
still the gold standard for malaria diagnosis today. Development of various PCR approaches, several different RDTs, and countless other methods have
provided more diagnostic tools with varying sensitivity, and practical advantages as well as disadvantages.

Cyril Garnham discovered that a pre-erythrocytic liver schizogony always be tested at a hospital by applying the diagnostic tools dis-
has to occur before the schizogony in the erythrocytes can take cussed in the following paragraphs.
place.[36] In 1982, Wojciech Krotoski observed dormant hypno-
zoite stages that were being produced in the liver of the human
host by P. vivax (as well as P. ovale) and caused relapses of malaria
tertiana.[42,43] 2.2. Microscopy of Thick and Thin Blood Films

The microscopic examination of Giemsa-stained blood films re-

2. Diagnosis of Plasmodium Parasites Today: Old
but Gold versus New and Fast
The detection of Plasmodium parasites replicating in the ery-
throcytes of the human host is challenging because the parasite
density of a patient can range from below 1 parasite/µL to tens of
thousands of parasites per microliter.[44] Parasite densities are a
result of pronounced age patterns and reflect lifetime exposure as
well as naturally acquired immunity at a population level.[44] Any
given diagnostic tool must be able to provide a fast and accurate
result to allow an appropriate treatment of the patient.[44,45]
2.1. Clinical Diagnosis
Clinical diagnosis is solely based on the symptoms that a pa-
tient displays. It is used as a diagnostic method when labora-
tory facilities are not available or when the patient is performing
self-diagnosis at home.[46,47] Several tropical diseases cause symp-
toms that are similar to malaria.[48] Self-diagnosis as well as self-
treatment are error-prone procedures that can lead to overdiag-
nosis and overtreatment.[48] Clinical suspicion of malaria should
mains the gold standard method to detect Plasmodium parasites
in the erythrocytes of patients, although devices that automati-
cally analyze the blood of the patients are currently being devel-
oped and tested.[49] Automatic slide reading approaches or vision-
based devices have been developed and tested as well, but the
manual microscopic examination conducted by trained person-
nel can still be seen as the preferred approach when it comes to
thin or thick blood film analysis.[50]
Regardless, proper diagnosis of malaria and the examination
of Plasmodium parasites are still challenging almost over 140
years after the discovery of the parasites by Alphonse Laveran.
In 1880, Laveran was relying on direct microscopy while he ex-
amined the unstained samples of his patients until he discovered
parasite-derived changes in the infected erythrocytes.[35,38] The ex-
amination of the blood samples was simplified after the develop-
ment of various methylene blue-based stains by Paul Ehrlich.[51]
With these newly developed stains, it was discovered that human
malaria is not caused by one Plasmodium species, which was orig-
inally hypothesized by Alphonse Laveran, but by various Plasmod-
ium species.
In 1891, Dimitry Romanowsky further extended and modi-
fied the original staining protocol.[52] By adding eosin to methy-
lene blue, he was able to differentially stain the nucleus and

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the cytoplasm of the Plasmodium parasites replicating within the
erythrocytes.[52] However, preparation and reproducibility of the
staining was difficult because of the required aging process of
the methylene blue.[53] After the proposal and testing of various
additional modifications by numerous contemporary scientists
during that time, Bernhard Nocht and Gustav Giemsa strove to
improve the Romanowsky staining.[53] Bernhard Nocht had dis-
covered that aging of methylene blue leads to the formation of a
new dye (“red from methylene blue”) that could stain the chro-
matin of Plasmodium parasites.[53] Gustav Giemsa was further
able to improve this new staining by identifying the newly dis-
covered dye as azure B and determining that both eosin and
an excess of Azure B needed to be dissolved in a mixture of
glycerol and methanol to achieve the desired stability as well as
reproducibility.[54] Gustav Giemsa’s stain remains, 115 years after
its first publication in 1904, the method of choice for the staining
of Plasmodium parasites.
The gold standard for malaria diagnosis is the routinely used
microscopic examination of thick and thin blood films stained
with Giemsa’s stain.[55,56] To create a thick blood film, a drop of
peripheral blood is removed from the patient’s finger, applied on
a glass slide, and laked before or during the subsequent stain-
ing with Giemsa’s stain, which causes the rupture of the erythro-
cytes and allows only the visualization of leukocytes, platelets,
and parasites. For the thin blood film, the patient’s peripheral
blood is spread on a larger area of the glass slide. The smear is
then briefly fixed in methanol and, after a brief drying period,
stained in Giemsa’s stain. The additional fixation step performed
during the preparation of the thin blood film leaves the infected
erythrocytes intact. While thick blood films are generated to de-
tect the presence of Plasmodium parasites and to determine the
parasitemia, thin blood smears are used to determine the Plas-
modium species responsible for the infection and the develop-
mental stages that are currently circulating within the blood of
the patient. Thick and thin blood films allow a comparatively fast
preparation and examination of the patient’s blood.
However, besides their obvious advantages, both techniques
also have distinct disadvantages. A trained expert is required for
both techniques to perform the entire procedure from the finger
prick to the final microscopic analyses. An untrained examiner
might, for example, confuse the ring stage Plasmodium parasites
with the ring stages of another protozoan parasite, Babesia, or
vice versa.[57] The correct identification of the Plasmodium species
and the determination of the parasitemia are therefore crucial to
define the appropriate treatment, to exclude the occurrence of
complications due to a severe infection, and to ensure the sur-
vival of the patient. Thin blood films are further less sensitive
than thick blood films if the parasitemia is low, but in vitro work
showed that even in the hands of a trained expert, thick blood
films prepared from malaria cultures at known parasitemia con-
sistently underestimate parasite densities.[56] This might be due
to a large number of parasites being lost during staining, which
limits the sensitivity of the method and leads to wrong estimates
of parasite density.[56] Another problem is the detection of the low-
density asymptomatic submicroscopic malaria (SMM) infections
during the dry season in the endemic areas.[58–60] A lack of breed-
ing sites for the Anopheles mosquitoes can lead to less frequent
transmission occurrences. However, many patients can still be
infected without displaying any symptoms, and they can act as
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silent reservoirs by maintaining low-level residual malaria trans-
mission in the community.[59] Detecting SMM infections is there-
fore just as important as detecting infections that cause a high
parasitemia.
Although light microscopy of Giemsa-stained thick and thin
blood films remains the gold standard in the detection of Plas-
modium parasites, there is a high demand for more sensitive and
automated microscopy techniques to provide the proper determi-
nation of the parasitemia of the parasite in the patient’s blood and
the Plasmodium species that is causing the infection.
The world health technology (WHT) autoanalyzer is an auto-
mated malaria slide scanning system and was one of the first de-
vices that was tested and performed at a level comparable to many
human slide readers in a study conducted in 2012.[61] Prescott
et al. acknowledged that minimal additional equipment is needed
to examine blood films that have been stained with standard pro-
cedures and that the device is able to perform with an estimated
limit of detection of 140 parasites µL–1 .[61,62]
A Global Good Fund prototype was tested in 2017 and showed
an estimated limit of detection of 100 parasites µL–1 for P. falci-
parum while scanning 0.2 µL of blood.[62] A more recent approach
uses a prototype digital microscope device, the Autoscope, that
employs an automated microscopy algorithm based on machine-
learning to simplify the Plasmodium species detection as well as
the parasitemia determination.[63] The performance of the de-
vice was comparable to routine microscopy when the slides con-
tained an adequate volume of blood to meet the preset design
assumptions.[63] However, Autoscope and trained personnel both
missed the same low-parasitemia slides that were determined
positive via polymerase chain reaction (PCR).[63]
CellaVision DM96 is another system that digitally determines
the cell morphology and is able to automatically classify leuko-
cytes, as well as different forms of erythrocytes such as para-
sitized erythrocytes, on blood films using its advanced red blood
cell application (ARBCA).[64] Florin et al. examined the speci-
ficity of the CellaVision DM96 and compared it to standard light
microscopy.[64] The device generally displayed a low sensitivity,
but showed a good correlation with microscopy paired with short
turnaround times, which might be applicable in follow-up exam-
inations to determine the parasitemia after initial diagnosis and
treatment.[64]
Another commercialized system, the X-rapid system, has been
exclusively developed for smartphone microscopy and provides a
lens attachment as well as a light-emitting diode (LED) attach-
ment to allow the examination of erythrocytes.[62] Future studies
still need to be conducted to see whether this system can be used
for routine malaria diagnostic procedures.
2.3. Microscopic Examination of Fluorescently Stained
Plasmodium Parasites
Fluorescent labeling followed by microscopic assessment of Plas-
modium parasites is another applied technique in diagnostic pro-
cedures. Usually, the blood of the patients is incubated with
acridine orange that immediately stains DNA and RNA of the
different developmental stages of Plasmodium parasites.[65,66] The
fluorescent parasites are then imaged using either a conventional
fluorescence microscope, a halogen lamp plus interference filter
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system or a fluorescence microscope based on LED.[66,67] The lat-
ter approach has the advantage that it can be used in field ap-
plications because of decreased energy consumption, stronger
brightness, and lower costs.[66,67] A recent study by Kimura et al.
showed that this approach produces superior results relative to
the Giemsa method with a correctly performed revised acridine
orange staining protocol.[66,67] Although this is a feasible method
leading to fast diagnostic results, trained personnel are needed to
correctly label the blood sample of the patient and to perform the
analysis properly with the fluorescent microscope.
2.4. Molecular Approaches to Diagnose Malaria via
Plasmodium-Specific Genes
The PCR was developed in 1983 by Kary Mullis, who won the
Nobel Prize for this work 10 years later (Figure 2).[68,69] The PCR
uses the properties of thermostable DNA polymerases of bacte-
rial origin to amplify even small DNA fragments of interest by
using distinct temperature changes and exposure.
To detect different species of Plasmodium parasites replicat-
ing in the erythrocytes of the patients, multiple PCR approaches
such as nested PCR (nPCR) or semi-nested multiplex PCR
(SnM-PCR) can be applied.[70–73] nPCR and SnM-PCR are de-
fined by two consecutive amplification rounds with a second set
of primer and the previously amplified DNA fragment or frag-
ments representing the new template. The primer selection de-
pends on the copy number of the targeted gene, the amount of ex-
pected Plasmodium-infected erythrocytes, and the body fluid that
is being examined.[72,74,75] For example, the small-subunit 18S
rRNA gene and the highly conserved pfk13, encoding the Kelch13
protein, can be targeted when fresh or even dried patient blood
samples are examined.[71,76–78] A recent publication by Lloyd et al.
probed for cox3 and varATS genes (encoding for the cytochrome
c oxidase subunit 3 and the var gene acidic terminal sequence, re-
spectively) when looking for P. falciparum parasites in the saliva
of the patients.[72]
The PCR approach represents the most sensitive method for
the diagnosis of Plasmodium parasites because it is able to detect
submicroscopic parasitemias that are usually missed by other di-
agnostic approaches.[79]
The application of various PCR approaches is further of partic-
ular importance when epidemiological data are needed for mon-
itoring malaria control and transmission during different sea-
sons, general mass screening and elimination interventions.[79]
Despite the clear advantages of the PCR approaches, the exper-
imental setup can be expensive because of the required material.
Another factor is the time necessary for the preparation of the
samples, setup of the reaction (storage of the reagents), time un-
til the thermocycler has completed the reaction, and the subse-
quent analyses of the results. A trained expert is further needed
to setup and to conduct the experiment, to interpret the results,
and to perform troubleshooting in case the chosen PCR approach
does not work. A PCR-based diagnosis can fail when parasites
have genetically diverse sequences at the target region of the
primers or when the copy number of the target gene is low, which
results in a lower amplification efficiency that will reduce the
sensitivity.[80,81]
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However, various quantitative PCR (qPCR) assays have been
developed to reliably detect Plasmodium parasites in clinical pa-
tients and even in asymptomatic samples.[44] Recently evalu-
ated diagnostic real-time qPCR, comprising a portable device
and the Q3-Plus silicone chip, has been developed to avoid the
use of bulky thermocyclers, temperature-controlled transporta-
tion, and storage of the necessary reagents.[82] However, ab-
solute quantification of parasites by qPCR is challenging be-
cause a standard curve must be established, which can vary
between laboratories.[44] Droplet digital PCR (ddPCR) allows
accurate and absolute quantification by counting DNA molecules
encapsulated in approximately 15 000 discrete, volumetrically de-
fined, water-in-oil droplet partitions that are subjected to end-
point PCR.[44,83,84]
Loop-mediated isothermal amplification (LAMP) might be an
alternative to the various PCR approaches, because the reaction
can simply be conducted in one tube at a constant temperature,
and it does not need a thermal cycler, which is particularly im-
portant for a fast malaria diagnosis in remote areas.[85] LAMP is
also a sensitive method that is able to detect placental malaria in
the peripheral blood of patients.[85]
2.5. Rapid Diagnostic Tests: An Alternative for Fast Malaria
Diagnosis
In areas where microscopy of thin and thick blood films or other
approaches to detect Plasmodium parasites in the blood of pa-
tients cannot be provided, antigen-based rapid diagnostic tests
(RDTs) can be an important alternative for an easy and fast di-
agnosis that should not take longer than 15 min.[67,86] In the
last decade, the amount of available RDTs and the frequency
of their use in epidemic investigations, as well as surveys, have
rapidly increased (Figure 2).[86] RDTs can also be a useful tool of
malaria self-diagnosis when applied by tourists traveling in en-
demic areas.[87]
Currently available RDTs are, for example, able to detect
histidine-rich protein 2 (HRP2), Plasmodium lactate dehydro-
genase (pLDH), or aldolase and provide the patient with a
qualitative, but not a quantitative, result.[33,88,89] It has previously
been suggested that cytoadherent biomass sequestered in the
vascular bed of the inner organs can be estimated from HRP2
concentrations in the blood plasma.[33,89] However, a high rate of
false positives was recently reported in high-transmission areas
on account of the persistent antigenicity of HRP2 even weeks
after the infection.[33,67,90] Aldolase tests have been shown to be
less sensitive when the patient is not infected with P. falciparum
but with another Plasmodium species.[33] pLDH test can also
vary in performance or even fail, depending on the Plasmodium
species the patient is infected with.[91] Plasmodium glutamate
dehydrogenase (pGDH) is another potential RDT candidate,
because the homohexameric protein is being produced by the
parasites throughout the entire schizogony, while being absent
from host erythrocytes. However, there is a high similarity
between the glutamate dehydrogenase produced by the different
Plasmodium species, which can cause cross reaction in areas
with mixed Plasmodium species infections.[92,93] Some studies
further reported the patient serum concentration of the protein
lies usually in nanomolar range.[93,94] Therefore, further tests are
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needed to determine whether pGDH-based RDTs can be applied
to detect Plasmodium parasites in endemic areas.[93]
Another problematic point is that RDTs might give positive re-
sults in febrile patients whose fevers are not caused by Plasmod-
ium parasites.[47] Quality-assured RDTs can be important to con-
firm malaria cases, while the cause of fever in the non-malaria
patients should be followed up and managed appropriately.[46]
A newly developed, highly sensitive RDT (HS-RDT) that has re-
cently been tested for its ability to diagnose malaria in pregnancy
in a low-transmission setting might be a good strategy to better
detect Plasmodium parasites in the future.[95]
2.6. Serological Detection of Plasmodium Parasites Using Serum
from Malaria Patients
The immunofluorescence antibody test (IFAT) has been a reliable
serological test for the presence of Plasmodium parasites.[96] This
approach can be used to detect Plasmodium-specific antibodies in
epidemiological surveys and in the screening procedures of po-
tential blood donors.[96,97] Plasmodium antigen is prepared on a
slide and stored at –30 °C until patient serum is applied, and the
amount of immunoglobulin G and M antibodies can be quanti-
fied using fluorescence microscopy.[96]
A similar method for the detection of Plasmodium-specific anti-
bodies in the blood of patients is the enzyme-linked immunosor-
bent assay (ELISA) that uses different antigens from different
Plasmodium species for antibody detection using a 96-well plate
and an appropriate plate reader.[97] Although both approaches are
relatively simple and moderately sensitive, they are very time con-
suming, and trained personnel are also needed to conduct the
experiments and analyze the results.
2.7. Flow Cytometric Approaches to Identify Infected Erythrocytes
Flow cytometry was initially developed in 1950s and is today used
for the laser-based sorting and measurement of various param-
eters of several thousand cells per second in a rapidly flowing
fluid stream.[98] Various studies reported the successful detection
of Plasmodium species in different flow cytometry assays using
different devices over the years.[99–104] The Sysmex hematology
analyzers (Sysmex Corporation, Kobe, Japan) are promising ad-
junctive diagnostic tools to facilitate a fast detection of malaria
cases in a fluorescence flow cytometry setup by counting and dif-
ferentiating between cell types and Plasmodium species.[100] An-
other promising approach in the detection of Plasmodium par-
asites is the noninvasive in vivo photoacoustic flow cytometry
(PAFC) that irradiates circulating erythrocytes directly in periph-
eral vessels through the skin by using focused linear laser beams
as well as laser-induced photoacoustic waves or fluorescence light
that are detected with ultrasound transducers and photodetec-
tors, respectively.[105] PAFC shows greater sensitivity than con-
ventional flow cytometry and allows the examination of an almost
entire blood volume.[105]
Flow cytometry is a very sophisticated approach to accurately
detect Plasmodium parasites, but the equipment can be expen-
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sive and might need to be operated and carefully maintained by
trained personnel to ensure an appropriate diagnosis.
2.8. Microfluidic Devices: Small but Powerful Tools for Malaria
Diagnosis
Microfluidic devices are portable, easy-to-use, self-contained, and
low-cost diagnostic devices that allow the precise manipulation
of small sample volumes while reducing reagent consumption,
which is crucial for malaria endemic areas with limited access to
healthcare.[106–109] One of the first microfluidic approaches in an
effort to diagnose malaria was conducted by Hou et al. in 2010,
who used distinct changes of erythrocyte plasma membrane stiff-
ness to detect Plasmodium parasites in the erythrocytes of the hu-
man host.[110] In 2016, Xu et al. used origami paper folding for
sequential steps of DNA extraction followed by LAMP and a flu-
orescence readout to detect Plasmodium parasites in finger prick
blood samples within 45 min.[111] A recent study conducted in
Uganda by Reboud et al. again applied a paper-based microflu-
idic device that combined the vertical sample-processing steps of
the origami device with a microfluidic lateral flow LAMP ampli-
fication and a simple visualization system for multiplexed DNA-
based malaria diagnosis from finger prick blood samples.[108]
This approach showed a higher sensitivity than microscopy and
RDTs and is currently being evaluated by the Uganda Vector Con-
trol Division, Ministry of Health, for potential usage in areas
without centralized facilities.[108]
2.9. Aptamer-Mediated Plasmodium-Specific Diagnosis of
Malaria
Aptamers or “chemical antibodies” are short, single-stranded
oligonucleotides such as DNA, RNA, or synthetic xeno nucleic
acids molecules, obtained by in vitro evolution techniques and
defined by high affinity as well as specificity to interact with
any desired corresponding target by folding into distinct tertiary
structures.[112–115] Since the invention of the process of aptamer
selection, called “systematic evolution of ligands by exponential
enrichment” (SELEX) in the early 1990s by Tuerk and Gold, great
efforts have been made to make the application clinically rele-
vant for various diseases such as macular degeneration, cancer,
thrombosis, or inflammatory diseases.[115] The diagnostic appli-
cation of aptamers can be advantageous because the oligonu-
cleotides are cheap and easy to synthetize, have a high stability
in harsh environmental conditions, and can be stored without
functional degradation.[114] The selection for the ideal aptamer
candidate usually begins with a randomized oligonucleotide li-
brary that is incubated with the target molecule followed by affin-
ity selection rounds and PCR amplification steps to determine
the sequence with the highest affinity.[115] Several aptamers have
already been commercialized to date.[115] So far, various aptamer
target proteins of Plasmodium parasites such as pLDH, the high
mobility group Box 1 protein (HMGB1), PfEMP1, and pGDH
have been tested.[112,116–122] The future will tell whether the chem-
ical flexibility of aptamers can further be exploited for binding
optimization and stability in blood, plasma, or samples, but
© 2019 The Authors. BioEssays Published by Wiley Periodicals, Inc
www.advancedsciencenews.com
Box 1
Vaccine Development and Treatment Strategies to Protect Malaria Patients
The development of an effective vaccine against Plasmodium
parasites is crucial to reduce the amount of annual malaria
cases and lift some of the burden off the heavily affected
malaria endemic areas, especially with regard to the two most
prevalent of the human-pathogenic Plasmodium species P. vi-
vax and P. falciparum.[123] However, vaccine development is
scientifically and technically challenging. From all of the can-
didates that are being developed and examined at the mo-
ment, RTS,S/AS01 or MosquirixTM, which targets the P. fal-
ciparum circumsporozoite protein, appears to be the most
promising, and it is currently being tested in a field study to
protect the immunized individuals against P. falciparum.[124]
The non-replicating viral vector candidate ChAd63 MVA ME-
TRAP (chimpanzee adenovirus 63 modified vaccinia virus
Ankara multiple epitope-thrombospondin-related adhesion
protein) and the whole parasite/attenuated sporozoite vaccine
candidate PfSPZ (Plasmodium falciparum sprozoite) both tar-
get P. falciparum-infected hepatocytes and are currently being
investigated in phase IIb clinical trials.[124,125]
aptamers represent a very promising approach for malaria
diagnosis.[117]
2.10. Bloodless Malaria Diagnosis: Examining Bodily Fluids and
Feces for Parasite Detection
Drawing blood for diagnostic purposes can be challenging in en-
demic areas. Bodily fluids such as saliva and urine or fecal matter
represent a good alternative because they can be noninvasively
obtained and tested for the presence of Plasmodium parasites us-
ing different markers and technical approaches such as PCR,
nPCR, lateral flow immunoassay, microfluidics with DNA sen-
sor substrate, and immunochromatography.[80,132–137] The suc-
cessful detection of Plasmodium DNA or proteins depends on
their concentration in the bodily fluid chosen for detection. A re-
cent study conducted in 2017 by Oyibo et al. reported that the
urine malaria test (UMT) developed by Fyodor Biotechnologies,
Inc. (Baltimore, MD, USA) is the only nonblood malaria test that
has undergone a full-scale premarket evaluation trial.[138]
2.11. Raman Spectroscopy to Identify Plasmodium-Infected
Erythrocytes via Different Parasite and Host Erythrocyte
Parameters
Raman spectroscopy measures the wavelength (or wavelength
shift) as well as the intensity of inelastically scattered light
(Raman effect) that has been emitted from a molecule in a
liquid, solid, or gas due to interactions of the incident light
with the molecule’s vibrational energies or signature, which al-
BioEssays 2020, 42, 1900138 1900138 (8 of 12)
www.bioessays-journal.com
Protection against P. vivax could be achieved by targeting
TRAP, the P. vivax circumsporozoite protein (PvCSP) or
the blood-stage antigen Duffy binding protein.[126–130] Vivax
malaria vaccine development and testing is currently ongoing
as well.[126–130]
Until a potent vaccine is developed, people living in endemic
areas have to rely on artemisinin-based combination therapies
(ACTs), primaquine and chloroquine (in case of an infection
with a chloroquine-sensitive P. vivax strain) for the treatment
of malaria.[131] The WHO further suggests the addition of a
single low dose of primaquine to the antimalarial treatment in
order to reduce transmission of the infection in low transmis-
sion areas.[131] Severe malaria, in contrast, is usually treated
with injectable artesunate (intramuscular or intravenous) fol-
lowed by several days of ACT treatment.[131]
Diagnostic approaches must be able to detect the Plasmodium
species that is causing the infection and replicating within the
erythrocytes to ensure a fast and appropriate treatment to pro-
tect the life of the patient.
lows substance identification and the observation of molecular
changes.[139–141] In malaria diagnosis, surface-enhanced Raman
spectroscopy (SERS) is, for example, applied to detect the malaria
pigment hemozoin, an iron-containing paramagnetic byproduct
of the hemoglobin digestion (see below) during asexual replica-
tion, by using silver nanoparticles that enhance the hemozoin
Raman signal by 106 to 107 folds.[142] Hemozoin has already been
the selected molecule for Raman spectroscopy-based Plasmodium
detection for quite a few years.[143]
Another previously published SERS approach has shown that
different stages of Plasmodium-infected erythrocytes, in particu-
lar the SERS spectra from ring stage-infected erythrocytes, differ
from those of uninfected erythrocytes and erythrocytes infected
with later developmental stages of the parasite.[144]
Raman spectroscopy is a sophisticated method that has the po-
tential for a very sensitive detection of Plasmodium infection in
malaria patients. However, like standard medical instruments,
the clinical Raman system should be small in size, easy to trans-
port with a robust calibration and a high sensitivity among
other crucial parameters to become a successful tool in malaria
diagnosis.[145]
2.12. Hemozoin-Based Detection of Plasmodium Parasites
Plasmodium parasites are not able to digest the toxic heme that
remains from the digestion of hemoglobin.[157] Heme is stored
in a metabolically crystallized form called malaria pigment or
hemozoin.[157] Hemozoin does not only serve as heme storage,
but also represents a potent inhibitor of immune cells.[146] There-
fore, hemozoin is an important molecule that can serve as a
marker for malaria diagnosis. Besides the above-mentioned Ra-
© 2019 The Authors. BioEssays Published by Wiley Periodicals, Inc
www.advancedsciencenews.com
man spectroscopy, hemozoin is being investigated as a poten-
tial marker for several different malaria diagnosis approaches
such as magneto-optical detection, laser desorption-time of flight
(LD-TOF) mass spectrometry, hemozoin-catalyzed precipitation,
magnetic purification, etc.[147–154]
Interestingly, hemozoin is also being produced by other par-
asites that feed on blood such as Schistosoma mansoni, Haemo-
proteus columbae, and the vector of the Chagas parasite, Rhodnius
prolixus.[155] These hemozoin molecules also show a similar struc-
tural identity, which is advantageous concerning treatment, but
might potentially be challenging for diagnosis in case of a co-
infection.[155] Hemozoin production is further a hallmark of older
developmental stages of Plasmodium parasites that, in case of P.
falciparum, are able to sequester in a specific organ and can cause
a local hemozoin accumulation there.[15,156] These sequestered
parasites do not circulate in the peripheral blood of the patients
and remain undetected while juvenile ring stages, that have not
yet produced a significant amount of hemozoin, are also not con-
sidered during a hemozoin-based diagnostic procedure.[157]
3. Conclusions and Outlook
Malaria is a curable disease: If you know you are infected.
Many efforts are being made to understand the biology of Plas-
modium parasites and Anopheles mosquitoes to fight and erad-
icate a disease that has strongly shaped human evolution and
history. Early, fast, and accurate diagnosis of Plasmodium para-
sites is crucial for disease management and to avoid suboptimal
care that can lead to presumptive antimalarial over- or undertreat-
ment, causing the emergence of parasite drug resistance.[46,79]
All presented diagnostic methods have advantages and disadvan-
tages and it is hard to determine which method will be the most
promising in the future.
The question is whether there will ever be a “perfect” method
that consists of a device that is cheap to produce and easy to han-
dle while providing sufficient sensitivity. This ideal device should
also be affordable for patients or hospitals, noninvasive, portable,
and with a potential to be applied at the patient’s home, given that
people living in rural areas might be too sick or do not have time
or money to travel to a hospital. The journey towards simplify-
ing malaria diagnosis continues, and for now we have to use the
available methods. However, more innovative ideas are coming
to the forefront in a bid to improve malaria diagnosis, as we have
reviewed here.
Another important point is the documentation of malaria
cases worldwide. This could be achieved with a platform that al-
lows tracking of the geographical distribution and evolution of
cases to improve general disease surveillance and facilitate result
exchange among researchers and physicians in endemic areas.
Time will tell whether the ultimate malaria test can be de-
veloped and whether the observation of clinical cases will help
physicians and researchers monitor the current situation better
and provide adequate help and care.
Acknowledgements
The authors would like to thank the members of the Matibabu team and
Patrick Pitoniak for critically proofreading this manuscript. The authors
BioEssays 2020, 42, 1900138 1900138 (9 of 12)
www.bioessays-journal.com
want to express their gratitude to the partners and further thank the fol-
lowing organizations for their support: UN Women (MSFT Imagine cup),
Resilient Africa Network, ISHOW (American Society of Mechanical En-
gineers), Villgro Kenya, Aspirin Social Award (Bayer Foundation), Royal
Academy of Engineering’s Africa Prize, E4 Impact Challenge, Merck Accel-
erator, Rolex, the Parasitology Unit of the Centre for Infectious Diseases,
Heidelberg University Hospital (Heidelberg, Germany), the Centre for In-
fectious Diseases, Parasitology at Heidelberg University Medical School
(Heidelberg, Germany), and the Malaria Molecular Laboratory, Mulago
National Referral Hospital (Kampala, Uganda).
Conflict of Interest
Brian Gitta is the C.E.O. of Matibabu and discloses financial interest.
Nicole Kilian declares no conflict of interest.
Keywords
host-parasite interaction, infectious diseases, malaria, malaria diagnosis,
medical devices, Plasmodium
Received: August 5, 2019
Revised: November 5, 2019
Published online: December 12, 2019
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