Amir et al.

Parasites & Vectors (2018) 11:53

DOI 10.1186/s13071-018-2617-y

REVIEW Open Access

Diagnostic tools in childhood malaria

Amirah Amir, Fei-Wen Cheong, Jeremy R. De Silva and Yee-Ling Lau*

Abstract
Every year, millions of people are burdened with malaria. An estimated 429,000 casualties were reported in 2015, with the
majority made up of children under five years old. Early and accurate diagnosis of malaria is of paramount importance to
ensure appropriate administration of treatment. This minimizes the risk of parasite resistance development, reduces drug
wastage and unnecessary adverse reaction to antimalarial drugs. Malaria diagnostic tools have expanded beyond the
conventional microscopic examination of Giemsa-stained blood films. Contemporary and innovative techniques
have emerged, mainly the rapid diagnostic tests (RDT) and other molecular diagnostic methods such as PCR,
qPCR and loop-mediated isothermal amplification (LAMP). Even microscopic diagnosis has gone through a paradigm shift
with the development of new techniques such as the quantitative buffy coat (QBC) method and the Partec rapid malaria
test. This review explores the different diagnostic tools available for childhood malaria, each with their characteristic
strengths and limitations. These tools play an important role in making an accurate malaria diagnosis to ensure that the
use of anti-malaria are rationalized and that presumptive diagnosis would only be a thing of the past.
Keywords: Childhood, Malaria, Diagnosis, Microscopy, PCR, RDT

Background Generally, children with malaria often present with

First described in 2700 BC and recognized as one of the fever, chills, headache, myalgia, vomiting and anorexia
oldest known diseases on our planet, malaria is still a [3, 10]. Although severe malaria is often associated with
cause of devastation in many parts of the world [1]. Mal- P. falciparum, P. vivax monoinfection and mixed (P.
aria is caused by a protozoan parasite of the genus Plas- falciparum and P. vivax) infection can also develop into
modium [2]. The five Plasmodium species which cause severe malaria in children, as they too demonstrate re-
malaria in humans are P. falciparum, P. vivax, P. malar- spiratory distress, anemia and neurological manifestation
iae, P. ovale and P. knowlesi [3]. The former three spe- [11]. While severe malaria has been reported in adults in-
cies are distributed across Africa, Asia and South and fected with P. knowlesi [12], little is known in children.
Central America, whereas P. ovale is found in Africa and Existing data showed that most children present with
P. knowlesi in Asia [4]. Despite sharing the same geo- thrombocytopenia and anemia but did not show any pro-
graphical distribution, P. vivax infection is less fre- gression into severe malaria [5, 13]. On the other hand,
quently found in Africa compared to P. falciparum, but the least common P. malariae and P. ovale generally cause
is the dominant species causing malaria in many regions fever but the child does not appear ill [14].
outside Africa [4]. All five species are known to infect Due to the non-specific manifestation of malaria infec-
children [3, 5–7], with falciparum malaria being respon- tion, it is sometimes mistaken for gastroenteritis, pneu-
sible for the majority of malaria-related deaths. Although monia or sepsis [3, 15–17]. In contrast, since the
the World Health Organization (WHO) has reported a symptoms of malaria are also exhibited by other micro-
fall in incidence and malaria deaths among populations bial infection presenting with acute febrile illness, this
at risk between the years 2010 and 2015, the estimated may result in overestimation of malaria burden [18].
number of malaria deaths in 2015 remained high with a Diagnosis made based on clinical findings alone (pre-
number close to 429,000, of which more than two thirds sumptive diagnosis) may be unreliable and confirmatory
were in children under the age of five years old [8, 9]. laboratory diagnosis using diagnostic tools is crucial to
ensure the accuracy of malaria diagnosis, thus, allowing
* Correspondence: [email protected]
administration of appropriate treatment [19]. Presump-
Department of Parasitology, Faculty of Medicine, University Malaya, 50603 tive diagnosis has been shown to lead to over diagnosis
Kuala Lumpur, Malaysia

© The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0
International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and
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(http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
Amir et al. Parasites & Vectors (2018) 11:53 Page 2 of 12

and over treatment of malaria in children aged 1–5 years facilities as it helped to improve the appropriate man-
[20, 21]. Inappropriate use of antimalarial drugs in the agement of non-malarial fevers and reduced the pre-
past has led to the emergence of resistant malaria para- scription of antimalarial drugs to children in Tanzania.
sites [22, 23]. The newer ‘magic bullet’, artemisinin is not In ideal conditions, at least three negative serial blood
spared as P. falciparum in Myanmar, Cambodia, smears (repeated every 12 h for 48 h) are needed before
Thailand, Vietnam, Lao People’s Democratic Republic malaria diagnosis can be excluded [38, 39]. The detec-
are seen to develop resistance [22, 24]. Furthermore, the tion limit of a well-trained microscopist can be as low as
latest WHO guidelines for the treatment of malaria has 5 parasites/μl blood, while average laboratory personnel
recommended artemisinin-based combination therapy may only report a positive blood smear at 50–100 para-
(ACT) for adults and children [25]. Therefore, the WHO sites/μl of blood [14, 40]. The sensitivity and specificity
has come up with several recommendation under the of microscopic diagnosis varies greatly and are
Global Plan for Artemisinin Resistance Containment dependent on many factors. Ngasala et al. [37] showed
(GPARC) which include improving access to affordable that when 934 slides were examined by different micros-
and quality-assured malaria diagnostic tools to ensure copists, the overall sensitivity and specificity for detec-
that only confirmed cases receive treatment [26]. Sug- tion of childhood malaria is 74.5% (95% CI: 69.8–78.7%)
gestions have been made to change the policy from pre- and 59.0% (95% CI: 54.9–62.9%), respectively, with a
sumptive malaria treatment to laboratory-confirmed positive predictive value of 53.4% (95% CI: 49.0–58.0%)
diagnosis and treatment [27–30]. In addition to being and a negative predictive value of 78.6% (95% CI: 74.0–
able to avoid unnecessary adverse reaction to antimalar- 82.0%). A study performed in Tanzanian children dem-
ial drugs, a laboratory-confirmed diagnosis and treat- onstrated that by comparing microscopy with RDTs and
ment also reduces drug wastage and minimizes the risk PCR, the sensitivity of conventional microscopy to de-
of parasite resistance development [31]. Over the years, tect pediatric malaria ranged from 26.3 to 100%, with
malaria diagnostic tools have expanded, from the con- the specificity ranged from 91.7 to 100% [41]. Discrep-
ventional microscopic examination of Giemsa-stained ancy has also been found when microscopy and RDT
blood films to a myriad of serological and molecular yielded a positive result of 17 and 30%, respectively, in
methods which include the more commonly used rapid 515 Kenyan primary school children [42], whereas mi-
diagnostic tests (RDTs) and polymerase chain reaction croscopy detected a 30% malaria prevalence in 230 Ni-
(PCR) [19]. This review explores the different diagnostic gerian children compared to 24.3% using RDT [43].
tools currently available for childhood malaria with the Besides, the lack of malaria microscopist experts is
aim of updating clinicians and researchers alike. With another crucial factor in contributing to false reporting
the array of diagnostic tools available, it is important and errors in species identification. Plasmodium
that we work towards reducing presumptive diagnosis. knowlesi, the fifth human Plasmodium could easily be
misdiagnosed as P. malariae or P. falciparum under mi-
Microscopic diagnosis croscopy due to their morphological similarities [44]. Some
Accurate diagnosis with rapid and effective treatment is children who were initially diagnosed with P. malariae in-
particularly important in children with malaria as most fection via microscopy were confirmed to have P. knowlesi
deaths occur within the first 24 h after admission to the infection instead after PCR [5, 13]. These misdiagnoses
hospital [32]. Direct microscopic observation of patients’ could result in treatment delay and even fatal complica-
blood to detect malaria parasite remains the gold stand- tions, as P. knowlesi can cause hyperparasitaemia within a
ard for malaria diagnosis [33, 34]. Microscopic examin- short period of time and it causes a more severe disease
ation of a stained thick blood film is done to determine compared to P. malariae. Sitali et al. [45] demonstrated
the presence or absence of malaria parasite, whereas that children under five years old have a higher frequency
microscopic examination of a stained thin blood film al- of acquiring mixed infection (infection with more than one
lows Plasmodium species identification and parasitaemia Plasmodium species). However, mixed infections are often
quantification. Microscopic examination could also pro- unrecognized or under-estimated by microscopists due to
vide pathophysiological and prognostic information that the tendency of one species dominating the other. This
can serve as indicators for the severity of disease, such could lead to inadequate antimalarial drug treatment, par-
as the morphological characteristics of the parasites, the ticularly hypnozoites of P. vivax and P. ovale, the dormant
maturity of asexual stages of the parasite and circulating form of parasites that can remain in the liver for many
pigment-containing phagocytes [35, 36]. The micro- years, which can cause relapse. Difficulties in detecting par-
scopic technique is widely used as it requires only a asites in low parasite density samples, overloaded laboratory
small volume of blood and it is cost effective compared personnel, ineffective quality control and assurance, poor
to molecular techniques. Ngasala et al. [37] showed that condition of microscopes, and improper slide preparation
microscopic diagnosis is useful in primary healthcare can also lead to unreliable microscopy results [34, 46–48].
Amir et al. Parasites & Vectors (2018) 11:53 Page 3 of 12

Besides the conventional bright field microscopic (95% CI: 56.3–67.8%) and 61.8% (95% CI: 55.9–67.4%),
examination, additional techniques have been designed respectively, for detection of malarial infections, with
to improve malaria diagnosis via microscopy. The quan- their specificities of 96.0% (95% CI: 92.3–98.3%) and
titative buffy coat (QBC) method is used to identify mal- 98.0% (95% CI: 95.0–99.5%), respectively. In a study of
aria parasites in peripheral blood by staining the DNA of 541 Cameroonian schoolchildren using light microscopy
parasites with acridine orange. QBC was found to be as reference, PT was found to be 91.3% sensitive and
able to detect malaria in samples with low parasite num- 86.7% specific for detection of malaria [59]. Another
bers, as low as 5 parasites/μl blood [49]. Several studies study with 107 Plasmodium sp.-positive samples by mi-
proposed that QBC has a higher sensitivity compared to croscopy demonstrated that both the sensitivity and spe-
conventional microscopic examination in detecting mal- cificity of PT for detection of childhood malaria [100%
aria infection. Bosch et al. [50] demonstrated that QBC (95% CI: 96.6–100%) and 97.4% (95% CI: 93.6–99.3%),
has a 100% sensitivity compared to microscopy examin- respectively] are higher than the sensitivity and specifi-
ation in 37 indigenous children in Venezuela. Another city of RDT Binax Now® [97.2% (95% CI: 92.0–99.4%)
study evaluated QBC on 720 schoolchildren and re- and 93.6% (95% CI: 88.5–96.9%), respectively] [60].
vealed a 99.6% sensitivity and 81.7% specificity on this Comparable sensitivity and specificity of PT to micro-
technique, with 5.5% more sensitive than thick-film scopic examination and RDT thus led to the proposal of
microscopic examination [51]. Similar results were ob- using PT as an alternative malaria diagnostic tool in en-
tained by Oloo et al. [52] where the overall sensitivity demic areas. However, species differentiation could not
and specificity for QBC was 98 and 84% respectively, in be done using this test. Besides, the presence of non-
a malaria survey performed on 360 Western Kenyan specific artefacts, nuclei-containing cells such as reticu-
schoolchildren, with an accuracy of 92% and negative locytes, leukocytes and bacterial cells are very likely to
predictive value of 98%. Possible drawbacks of QBC are lead to false positive results.
the difficulties in parasite species differentiation and Conventional microscopy examination encounters
subjective parasite quantification [53, 54]. Nonetheless, many challenges, particularly the lack of trained malaria
with its relatively reliable high sensitivity and specificity, microscopists which could then lead to false diagnosis.
QBC could still be useful as a supportive malaria diag- To overcome this deficit, large efforts have been made in
nostic tool together with blood film microscopic screen- invention of computer-vision-based techniques and sys-
ing in endemic field, as the processing of QBC is easier, tems. These systems could function as “automated mi-
faster, and requires less-trained personnel as compared croscopists” and could greatly improve the speed,
to conventional microscopic examination [55–57]. For consistency and accuracy in malaria diagnosis. For in-
example in large-scale malaria screening, QBC could be stance, digital imaging scanning can be performed by
performed prior to conventional bright-field microscopic SightDx first generation P1 and second generation P2
examination to detect the presence of malaria parasites. systems in which blood samples on test slides would be
Once malaria diagnosis has been established by the scanned automatically by the device with pre-set algo-
QBC, thin blood smears can then be used for accurate rithms to detect the malaria parasites. These systems
species identification and quantification of parasitaemia. were able to scan the entire slide within few minutes,
Another microscope-related technique, the Partec determine the parasitemia level and identify the Plasmo-
Rapid Malaria Test (PT) utilizes the Partec CyScope® dium species, with the performance result on par with
(Partec GmbH, Münster, Germany), a microscope that human microscopist experts and many commercial
has an extra incident UV light for detection of fluores- RDTs [61, 62]. Recently, the latest commercial device
cence light. The test slides used is readily labelled with Parasight platform was evaluated and demonstrates im-
an unspecific DNA binding fluorescent dye, 4′-6-diami- proved accuracy over the previous prototype P1 and P2
dino-2- phenylindole (DAPI), that binds to intraerythro- devices [63]. Despite above mentioned advantages, these
cytic Plasmodium DNA resulting in fluorescence. This computerized malaria diagnostic systems have yet to be
method is easy and rapid, less labour intensive and re- evaluated on large scale.
quires less training time for laboratory personnel. Be-
sides, the Partec CyScope® operates with integrated Rapid diagnostic tests
rechargeable batteries, which is convenient to be used in The RDT is an effective and important tool in malaria
fields without electricity supply. Furthermore, it only re- diagnosis and is increasingly seen as a complement to
quires few microliters of blood sample, which is ideal in traditional diagnosis by microscopy. It forms the main-
paediatric patients. By using real-time PCR as gold stay of diagnosis in resource-poor areas which do not
standard, comparison study performed by Nkrumah et have access to a laboratory or electricity and in these set-
al. [58] in 489 children in Ghana reported the sensitiv- tings may supersede microscopy for diagnosis of malaria.
ities of PT and microscopy examination were 62.2% According to the WHO, there are more than 200
Amir et al. Parasites & Vectors (2018) 11:53 Page 4 of 12

malaria RDT products in the market [64] many of which infection. The prevalence of parasites with the HRP-2
have been assessed by independent studies according to gene deletions may, however, vary in differing localities.
WHO guidelines. According to the World Malaria Re- HRP-2 deletions leading to false negative reports have
port 2015 [65], there were a total of 314 million RDT been published in Mali [75], Rwanda [76], Colombia
sales in 2014 and numbers are expected to increase over [77], Ghana [78], Kenya [79], the Democratic Republic
the years as the efficiency of these marketed RDTs of Congo [80] and India [81]. Guidelines issued by the
increase. WHO, however, indicate that deletion of the HRP-2
Most RDTs work on the principle of capillary action. gene is not likely to be the main cause of false-negative
A capture and a separate detection antibody are used to results in RDTs and point towards other more probable
provide a visual result where the capture antibodies are causes including poor transport and storage conditions,
laid as a stripe on the membrane and the detection anti- operator errors, or the use of poor quality RDTs or the
body is conjugated to an indicator, typically gold parti- use of a wrong comparator such as poor-quality micros-
cles, that bind to the parasite antigen. This antigen- copy for cross-referencing of negative diagnostic results
detection antibody complex binds to the capture anti- [82]. The guidelines do state that HRP-2 deletion should
body producing a visible line if the targeted antigen is be suspected in a specific number of instances and PCR
present in the clinical sample [66]. Most RDTs are able may be used to confirm the diagnosis and the deletion
to detect malaria antigens in 5–15 μl of blood and re- of the HRP-2 gene.
sults can be obtained in 5–20 min depending on the The use of RDTs for malaria diagnosis in children suf-
manufacturer’s instructions. The detection limits for fer the same limitations that affect the use of the diag-
RDTs vary depending on individual manufacturers and nostic method in adults. The primary limitations of this
the quality and the sensitivity of the RDTs depend on test include reduced sensitivity for non-falciparum mal-
such factors as storage conditions, temperature and time aria, and a lack of accuracy in extreme environmental
of the assay [67]. conditions such as those found in field situations where
There are currently three established antigens used for it ironically is used the most [83, 84]. However, the
detection of Plasmodium in RDTs: P. falciparum histi- RDT’s ease of use and interpretation and its ability to be
dine rich protein II (HRP-2), Plasmodium lactate de- deployed in the field makes it an invaluable tool for
hydrogenase (pLDH) and aldolase. The HRP-2 protein is diagnosis of malaria in children in rural areas. A study
specific for P. falciparum detection while the pLDH and by Smart et al. [85] on pediatric inpatients in a two Tan-
aldolase antigens are pan-malarial. Thus, most RDTs in- zanian referral hospitals found moderate agreement be-
corporate two of the three antigens to allow users to dis- tween the use of microscopy and RDT for diagnosis of
tinguish falciparum from non-falciparum infections. patients but suggested that RDT are a better initial test
HRP-2 is a water soluble protein produced by asexual in diagnosing malaria among pediatric patients. The au-
stages and young gametocytes of P. falciparum [68]. The thor also argues that the use of RDT would reduce the
pLDH enzyme on the other hand is produced by the rate of overtreatment in hospitals in Tanzania which in-
sexual and asexual stages of Plasmodium and different directly would provide a significant cost saving. Further-
isomers for this protein have been detected in various more, withholding antimalarial drugs from children who
Plasmodium species [69]. Aldolase is an antigen utilized test negative in an RDT test is a safe practice in an out-
in the parasite glycolytic pathway and like pLDH is pan- patient setting [86]. The use of RDT in hospitals would
malarial as well [70]. also decrease turn-around time providing physicians
The HRP-2 antigen has been shown to have better with feedback and results in a timely manner. Overtreat-
sensitivity compared to pLDH although specificity was ment of children was also reported in Samara hospital,
found to be better with pLDH [71]. Furthermore, HRP-2 Nigeria where 57% of febrile children admitted to the
is less expensive, has a lower detection threshold and is hospital received antimalarial medication for treatment
stable at a wider range of temperatures thus making it of presumed malaria before the presentation [87]. When
the more widely used antigen [72]. The drawbacks of evaluating the sensitivity and specificity of RDT for diag-
this antigen however, are that it only is able to detect P. nosis of pediatric malaria, the authors found that RDT
falciparum infection and any antigenic variation may had a sensitivity of 40.3% and a specificity of 89.6%.
give a false-negative result [73]. Furthermore, HRP-2 an- However, it was of note that the sensitivity of the test
tigens are known to still be in the circulation of the pa- dropped drastically with lower parasite densities. These
tient weeks after clearance of the parasite [74] thus results were contradictory to a previous study on the ef-
making it unsuitable to be used in assays for monitoring fectiveness of RDT in febrile children in Sokoto, Nigeria,
response to drug treatment. where sensitivity of RDT was found to be 93% [88].
More recently, there have been reports of deletions of Recent advancements in RDTs for malaria diagnosis
the HRP-2 in several areas endemic for P. falciparum include the development of the ultra sensitive rapid
Amir et al. Parasites & Vectors (2018) 11:53 Page 5 of 12

diagnostic test (uRDT) which has higher sensitivity com- 108 parasites/ml blood. Similarly in Tanzania, 13–14 sam-
pared to the conventional RDTs. A recent study by Das ples were found positive with molecular methods with
et al. [89] on the Alere™ Malaria Ag P.f uRDT showed a parasite counts ranging from 9 to 170,000 parasite/ml
greater than 10-fold lower detection limit for the HRP-2 blood. Additionally, molecular methods have the potential
antigen compared to a conventional RDT in both a high to detect malarial parasites in asymptomatic infections.
and low transmission setting. The study also indicates These undetected sub-microscopic infections, though less
high specificitiy for this uRDT relative to quantitative common in children than in adults, may still be able to in-
PCR (qPCR) and histidine-rich protein II (HRP2) en- fect mosquito vectors and could reintroduce malaria into
zyme linked immunosorbent assay (ELISA). Further- certain regions. Semi-nested PCR based on the 18S small
more, the uRDT was able to detect new infections subunit ribosomal RNA (ssrRNA) gene permitted the
1.5 days sooner indicating overall improved diagnostic identification of a high number of children (80%) in-
performance characteristics when compared to conven- fected with P. falciparum. These children had all ini-
tional RDTs [89]. tially tested negative with the microscopy method
Overall, studies on the sensitivity and specificity of [104]. Interestingly, others have found the high copy
RDT in diagnosing malaria have been positive in both a number of cytochrome b PCR to be more sensitive than
laboratory and outpatient clinical setting [90–92] and 18S rDNA PCR [41, 105, 106]. Hsiang et al. [107] de-
thus should be considered a useful tool for pediatric tected three times as many infections with cytochrome
malaria diagnosis. b nested PCR than by microscopy (15/472 vs 4/472)
among asymptomatic children with lowest detection
Molecular diagnostic methods limit of 10 parasites/μl. This technique also performed
Molecular techniques, such as PCR, have gained much better than single-round PCR and real-time methods
attention and significance in malarial diagnosis, espe- [107]. A more recent study by Mahende et al. [108] de-
cially after the discovery of knowlesi malaria in humans termined 21 malaria RDT-positive samples from Tanza-
[93, 94]. This method enables the specific identification nian children while microscopy was negative; six
of malarial parasites up to the species level. Further- samples were detected positive by 18S rDNA PCR
more, it is highly sensitive when compared to micros- [108]. Of note is that eight samples that were RDT-
copy. The theoretical detection limit of PCR can be as negative but microscopy-positive were confirmed to
low as 0.02 parasites/μl [95, 96] with nested PCR being possess P. falciparum species through PCR. Three-
the most sensitive nucleic acid amplification technology quarters of the 867 malaria patients from this study had
thus far [41, 97] versus an experienced microscopist, low levels of parasitaemia. Therefore, PCR is a worthy
which is said to have a detection limit of approximately alternative as the interpretation of the results either by
5 parasites/μl [40]. With other diagnostic methods fall- agarose gel observation or Ct value does not require
ing short in terms of practicality, cross-reactivity prob- specialized skill and is not altered by the subjectivity of
lems, or having incomplete coverage of all medically the observer [95]; this is the precise opposite to micros-
important Plasmodia [98, 99], PCR-based methods seem copy where specific training is required for species
promising as the new gold standard in malarial diagno- differentiation.
sis, especially in cases with low parasitemia or in the The specificity of PCR has also been demonstrated by
case of mixed species [100]. Nsobya et al. [109]. Upon enrollment, 55 (17%) of 316
The molecular amplification of the small subunit, 18S, asymptomatic children in Uganda were found to be in-
of ribosomal RNA (18SrRNA) was first carried out by fected with P. falciparum via microscopy (parasite densities
Snounou et al. [101] using a nested PCR technique, the was 16–71,840 parasites/ml). By using species-specific
most widely-used PCR method in malarial diagnostic re- nested PCR, the prevalence of malaria was observed to be
search. The sensitivity of this molecular method was 148 (47%), of which 36% were P. falciparum, 18% P. falcip-
found to be greater than RDTs and microscopy in a arum mixed infection, 10% P. ovale, 7% P. vivax, 4% P.
study conducted by Mens et al. [102]. In their work, 338 malariae, and 3% non-P. falciparum mixed infection. Two
children with the clinical symptoms of Plasmodium infec- children were PCR negative but microscopy positive [109].
tion in Tanzania and Kenya were analyzed with micros- Real-time quantitative PCR (qPCR) and nucleic acid
copy, RDT, or molecular method. Molecular testing found sequence-based amplification (QT-NASBA) assays can
a substantially higher amount of positive samples com- also be utilized to determine parasite density. The ad-
pared with RDTs and microscopy, confirming the elevated vantage of qPCR over other molecular techniques is the
sensitivity of PCR [103] which enabled the identification of quantification of parasitic densities. By correlating quan-
more children with low parasitemia. Around 40–42% the tification by two commercial qPCR (PrimerDesign Ltd.,
samples collected in Kenya were found positive with mo- Alicante, Spain) kits, Santana-Morales et al. [109] con-
lecular methods, with parasite counts ranging from 16 to firmed that qPCR is an accurate means to quantify
Amir et al. Parasites & Vectors (2018) 11:53 Page 6 of 12

parasitic densities. One case of imported falciparum mal- histidine protein or fragments present in the urine of fe-
aria was reported in France in a boy that visited his family brile patients. Similar to most RDTs, UMT works on the
in Africa during a summer break. qPCR quantified P. principle of capillary action. This method involves dip-
falciparum DNA levels in an effort to monitor the parasit- ping the UMT strip into 200 μl urine for 2 min and re-
emia under treatment and to determine chloroquine re- sults read after 20 min based on the lines appearing on
sistance. qPCR was able to detect and quantify infections the strip. Initial assessment on patients in South-East
that have very low infection (0.001%) [110]. qPCR assays Nigeria showed that it was comparable with the micros-
targeting the high-copy telomere-associated repetitive copy technique [119]. Another multicenter UMT trial in
element 2 (TARE-2) and the var. gene acidic terminal se- Lagos state, Nigeria, managed to include patients in-
quence (varATS) were also developed for ultra-sensitive fected with P. falciparum, P. vivax, P. malaria and P.
detection of P. falciparum. Compared to TARE-2 or var- ovale [120]. Here, they found that UMT sensitivity and
ATS qPCR, 18S rRNA gene qPCR was unable to identify specificity was 93 and 83%, respectively, when used
48 infections in 498 samples from a malaria survey under- among febrile children under the age of five. The per-
taken in Tanzania [111]. formance of the UMT in this study was found to be
In a recent report, a reverse transcription-polymerase comparable with that of BinaxNow, a blood-based mal-
chain reaction (qRT-PCR) was developed for the asexual aria RDT.
18S rRNAs of P. falciparum and P. vivax. qRT-PCR Several studies involving a mixed population consist-
demonstrated high sensitivity as compared to qPCR by ing of adults and children have tried using urine and sal-
detecting 34/52 symptomatic patients and 13/36 asymp- iva as alternative DNA sources for malaria diagnosis by
tomatic patients [112]. Other molecular techniques de- PCR with promising results [121, 122]. Furthermore, sal-
veloped for malaria diagnosis include loop-mediated iva has also been used for the quantitative detection of
isothermal amplification (LAMP), flow cytometry, and P. falciparum HRP-2 antigen using ELISA with encour-
microarray [113–115]. However, the efficiency of these aging outcome [123, 124].
methods has not been established for childhood malaria. Another fascinating method recently developed was
A lack of clear consensus on standardized methods for the transdermal detection of vapor nanobubbles around
PCR makes it difficult to interpret and compare results. intraparasite hemozoin using a prototype device [125].
There is a need to develop guidance on indications for This method involves delivering laser pulses to blood
use, assay selection, and quality assurance/control for through the skin using a probe after which, the respond-
PCR and other molecular diagnostic techniques for the ing acoustic traces are collected simultaneously with
specific conditions in which employing malaria diagnos- laser irradiation and analyzed.
tic tools may be appropriate. To ensure the data ob- These non-invasive methods serve as a potential alter-
tained from PCR-based method are reliable, users are native tool in malaria diagnosis especially where there are
strongly encouraged to follow the WHO external quality difficulties in obtaining blood samples (particularly in chil-
assurance scheme for malaria nucleic acid amplification dren), problems with carrying out conventional diagnostic
testing [116]. Another drawback is that PCR is cumber- method or when safety concerns are expressed. However,
some, expensive, and requires well-trained staff with most of these non-invasive tests are still at their infancy
stringent laboratory cleanliness. Such criteria may not be and there is a need for optimization of the technology and
fulfilled in certain laboratory settings, especially those in further testing to also include field-acquired infection and
remote areas in developing countries [117]. paediatric populations.

Non-invasive tests Conclusions

The future of malaria diagnostic tools is exciting as we The characteristic features of microscopic diagnosis,
start to see the development of new non-invasive rapid diagnostic test and molecular diagnostic methods
methods. One such method is by detecting specific thio- are summarized in Table 1. Despite the progress and ad-
eter levels in human breath which acts as biomarkers. vancement of various malaria diagnostic tools, the mi-
Exhaled breath is analyzed using gas chromatography- croscopy technique remains the gold standard for
mass spectrometry. An early clinical trial of this method malaria diagnosis. RDTs prove to be valuable as it can be
on adult volunteers infected with P. falciparum showed undertaken without any basic laboratory infrastructure
promising results with thioether levels appearing to have or trained personnel. The ability of RDTs to produce re-
the same periodicity as the parasite’s 48 h erythrocytic sults within a few minutes help reduce over-treatment
life-cycle [118]. or misdiagnosis from presumptive diagnosis. Neverthe-
The urine malaria test (UMT) kit, a recombinant less, because of the variable sensitivity and specificity of
monoclonal antibody based test was also recently devel- different RDTs, WHO procurement guidelines should be
oped to detect P. falciparum specific HRP-2, a poly- followed when procuring malaria RDTs and a good
Table 1 Summary of the three main diagnostic methods in childhood malaria with their characteristic features
Method Key characteristics Parasite species Advantages Disadvantages Reference
detectable
Microscopic diagnosis Conventional bright field Giemsa-stained thick blood Plasmodium genus- - Small amount of sample - Difficulties in detecting [5, 13, 14, 33–48]
microscopic examination film to determine the specific (blood) is required; parasites in low parasite density
presence or absence of - Able to quantify parasitaemia; samples (50–100 parasites/μl);
malaria parasite - Provides prognostic information - Malaria microscopist expert/
that serves as indicator for well-trained personnel is needed
Giemsa-stained thin All human Plasmodium
disease severity (morphological to interpret the result (high
blood film to identify the species characteristics of the parasites, morphological similarities
Plasmodium species the maturity of asexual stages of between P. falciparum,
the parasite); P. malariae and P. knowlesi
- Cost effective compared to could lead to misdiagnosis and
molecular techniques treatment delay)
Amir et al. Parasites & Vectors (2018) 11:53

Quantitative buffy coat Detection of malaria parasites Plasmodium genus- - Higher sensitivity (5 parasites/ - Difficulties in parasite species [49–57]
method (QBC) in centrifuged peripheral specific μl) compared to bright field differentiation and subjective
blood by staining the microscopic examination; parasite quantification;
parasite DNA with acridine - Fast and easy to be performed; - Specific equipment
orange and examination - Interpretation of the result is (fluorescence microscope)
under fluorescence microscope simple and requires less-trained is required
personnel
Partec Rapid Malaria Detection of malaria parasites Plasmodium genus- - Easy and rapid, less labour- - Difficulties in species [58–60]
Test (PT) using test slide that is readily specific intensive and requires less differentiation;
labelled with 4′-6-diamidino- training time for laboratory - False positive results due to the
2- phenylindole (DAPI) which personnel; presence of non-specific artefacts
binds to intraerythrocytic - Could be used in the field or nuclei-containing cells
Plasmodium DNA, resulting in without electricity supply; (reticulocytes, leukocytes and
fluorescence under Partec - Small amount of sample bacterial cells);
CyScope® (fluorescence (few μl) is required -Specific equipment (Partec
microscope) CyScope®) is required
Rapid diagnostic OptiMAL® Detection of malaria via the P. falciparum and P. vivax Ease of use, rapid diagnosis and Less sensitive compared to [83]
test (RDT) pLDH antigen result interpretation, sensitive, molecular diagnostic methods,
field-deployable heat sensitive, reduced sensitivity
ParaSight-F test Detection of malaria via the P.falciparum [84]
for non-falciparum malaria, false-
HRP-2 antigen
negative results due to low-level
Immunochromatographic Detection of malaria via the P.falciparum expression or deletion of target [84]
test (ICT) Malaria PF test HRP-2 antigen antigen genes (pfhrp2)
SD Bioline Malaria AG Detection of malaria via the P. falciparum (HRP-2), [85]
Pf/Pan HRP-2 and pLDH antigen pan-malarial (pLDH)
CareStart™ Malaria Detection of malaria via the P. falciparum (HRP-2), High specificity and PPV Low sensitivity at low parasite [87]
HRP-2 and pLDH antigen pan-malarial (pLDH) densities
Malaria pf Rapid device Detection of malaria via the P. falciparum Sensitivity and specificity [88]
HRP-2 antigen comparable to those for light
microscopy
Ultra sensitive RDT (uRDT) Detects HRP-2 antigen of P. falciparum Higher sensitivity, specificity and Similar to conventional RDTs, is [89]
P. falciparum malaria ability to detect new infections less sensitive compared to
faster than conventional RDT molecular diagnostic methods
Page 7 of 12
Table 1 Summary of the three main diagnostic methods in childhood malaria with their characteristic features (Continued)
Method Key characteristics Parasite species Advantages Disadvantages Reference
detectable
Molecular diagnostic Nested PCR Targeting 18S rRNA gene Plasmodium genus- Elevated sensitivity compared to Cumbersome, expensive, and [101]
methods specific RDTs and microscopy requires well-trained staff with
stringent laboratory cleanliness
Targeting 18S rRNA gene Plasmodium genus- More sensitive than microscopic [108]
to minimize risk of contamination
specific followed by examination for identification of
nested species-specific asymptomatic malaria
PCR
Targeting cytochrome b gene Plasmodium genus- Detection limit of 10 parasites/μl, [41, 104, 105, 107]
specific better than single-round PCR and
real-time methods
Semi-nested PCR Targeting 18S rRNA gene P. falciparum and P. vivax More sensitive than microscopic [103]
Amir et al. Parasites & Vectors (2018) 11:53

examination for identification of

sub-microscopic infections
Quantitative nucleic Targeting 18S rRNA gene. P. falciparum - Fast, sensitive, reliable, and [101, 102]
acid sequence-based Quantification was achieved quantitative;
amplification (QT-NASBA) by co-amplification of the - Allowed for the sub-microscopic
RNA in the sample with one quantification;
modified in vitro RNA as a - Detection limit of 10 parasites/μl
competitor
Multiplex PCR Targeting 18S rRNA gene P. falciparum and P. vivax - Detection limit of 0.1 parasites/μl; [106]
- No cross-reaction between
Plasmodium spp.;
- Able to detect the mixed
infection
Real-time quantitative Targeting plasmepsin 4 in P. falciparum and P. vivax - Quantification of parasite [103]
PCR (qPCR) P. falciparum and the aspartic densities;
protease PM4 in P. vivax - More sensitive than microscopic
examination;
- Detection limit of 5.6 copies/μl;
- Able to detect and quantify
infections that have very low
infection (0.001%)
qPCR Targeting telomere-associated P. falciparum - Detection limit of 0.03 to 0.15 [110]
repetitive element 2 and the parasites/μl;
var. acidic terminal sequence - 10× more sensitive than
standard 18S rRNA qPCR
Reverse transcription- Targeting 18S rRNA P. falciparum and P. vivax Able to detect and differentiate [111]
polymerase chain submicroscopic malaria
reaction (qRT-PCR) infections as low as 10 parasites/
ml and 18 copies/μl
Page 8 of 12
Amir et al. Parasites & Vectors (2018) 11:53 Page 9 of 12

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