Evaluation of rapid diagnostics for Plasmodium falciparum and P. vivax in Mae Sot Malaria endemic area, Thailand.
ABSTRACT Prompt and accurate diagnosis of malaria is the key to prevent disease morbidity and mortality. This study was carried out to evaluate diagnostic performance of 3 commercial rapid detection tests (RDTs), i.e., Malaria Antigen Pf/Pan™, Malaria Ag-Pf™, and Malaria Ag-Pv™ tests, in comparison with the microscopic and PCR methods. A total of 460 blood samples microscopically positive for Plasmodium falciparum (211 samples), P. vivax (218), mixed with P. falciparum and P. vivax (30), or P. ovale (1), and 124 samples of healthy subjects or patients with other fever-related infections, were collected. The sensitivities of Malaria Ag-Pf™ and Malaria Antigen Pf/Pan™ compared with the microscopic method for P. falciparum or P. vivax detection were 97.6% and 99.0%, or 98.6% and 99.0%, respectively. The specificities of Malaria Ag-Pf™, Malaria Ag-Pv™, and Malaria Antigen Pf/Pan™ were 93.3%, 98.8%, and 94.4%, respectively. The sensitivities of Malaria Ag-Pf™, Malaria Antigen Pf/Pan™, and microscopic method, when PCR was used as a reference method for P. falciparum or P. vivax detection were 91.8%, 100%, and 96.7%, or 91.9%, 92.6%, and 97.3%, respectively. The specificities of Malaria Ag-Pf™, Malaria Ag-Pv™, Malaria Antigen Pf/Pan™, and microscopic method were 66.2%, 92.7%, 73.9%, and 78.2%, respectively. Results indicated that the diagnostic performances of all the commercial RDTs are satisfactory for application to malaria diagnosis.
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ABSTRACT: Microscopy is considered as the gold standard for malaria diagnosis although its wide application is limited by the requirement of highly experienced microscopists. PCR and serological tests provide efficient diagnostic performance and have been applied for malaria diagnosis and research. The aim of this study was to investigate the diagnostic performance of nested PCR and a recently developed an ELISA-based new rapid diagnosis test (RDT), NovaLisa test kit, for diagnosis of malaria infection, using microscopic method as the gold standard. The performance of nested-PCR as a malaria diagnostic tool is excellent with respect to its high accuracy, sensitivity, specificity, and ability to discriminate Plasmodium species. The sensitivity and specificity of nested-PCR compared with the microscopic method for detection of Plasmodium falciparum, Plasmodium vivax, and P. falciparum/P. vivax mixed infection were 71.4 vs 100%, 100 vs 98.7%, and 100 vs 95.0%, respectively. The sensitivity and specificity of the ELISA-based NovaLisa test kit compared with the microscopic method for detection of Plasmodium genus were 89.0 vs 91.6%, respectively. NovaLisa test kit provided comparable diagnostic performance. Its relatively low cost, simplicity, and rapidity enables large scale field application.The Korean Journal of Parasitology 08/2014; 52(4):377-81. · 0.88 Impact Factor
© 2011, Korean Society for Parasitology
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Malaria is the major causes of morbidity and mortality in the
world. The disease affects the populations of tropical and sub-
tropical areas worldwide. Of the 5 species of Plasmodium that
cause malaria in humans, Plasmodium falciparum is the most
dangerous and responsible for most morbidity and mortality
. Malaria control relies principally on prompt and accurate
diagnosis and chemotherapy with effective antimalarial drugs
. Prompt and accurate diagnosis is the key to prevent mor-
bidity and mortality while it is avoiding unnecessary use of
antimalarial agents. The traditional malaria diagnosis is based
on the examination of stained blood smears under light mi-
croscope. The method remains the gold standard for malaria
diagnosis as it is inexpensive and sensitive (5-10 parasites/µl
blood) . However, it is labor-intensive, time-consuming, and
more importantly, requires skill and experienced microscopists.
Recently, alternative methods, such as immunochromato-
graphic assay, molecular amplification method, fluorescence
microscopy, mass spectrometry, and flow cytometry have been
developed for malaria diagnosis [3-10]. These methods have
some advantages and also some limitations. PCR is considered
the most sensitive and specific method, but is expensive, re-
quiring PCR machine, relatively sophisticated and time-con-
suming procedure, which may not be applicable for malaria
diagnosis in remote areas. Malaria rapid detection tests (RDTs)
which are based on capture of the parasite antigen by mono-
clonal antibodies incorporated into a test strip provide a possi-
bility to replace microscopic diagnosis. Although there have
been a number of RDTs commercially available, their sensitivi-
ty and specificity remain uncertain. RDTs can be divided into
2 major types. The first type detects histidine-rich protein 2
(HRP2), a protein uniquely synthesized by P. falciparum and
present in the blood stream of an infected individual . Some
HRP2 tests are designed to also detect aldolase enzyme, a pro-
tein synthesized by all 4 human-infecting Plasmodium species
ISSN (Online) 1738-0006
Korean J Parasitol. Vol. 49, No. 1: 33-38, March 2011
Evaluation of Rapid Diagnostics for Plasmodium
falciparum and P. vivax in Mae Sot Malaria
Endemic Area, Thailand
Wanna Chaijaroenkul1, Thanee Wongchai2, Ronnatrai Ruangweerayut2 and Kesara Na-Bangchang1,*
1Pharmacology and Toxicology Unit, Graduate Program in Biomedical Sciences, Thammasat University, Rangsit, Patumthani 12121, Thailand;
2Mae Sot Hospital, Tak Province, Thailand
Abstract: Prompt and accurate diagnosis of malaria is the key to prevent disease morbidity and mortality. This study was
carried out to evaluate diagnostic performance of 3 commercial rapid detection tests (RDTs), i.e., Malaria Antigen Pf/PanTM,
Malaria Ag-PfTM, and Malaria Ag-PvTM tests, in comparison with the microscopic and PCR methods. A total of 460 blood
samples microscopically positive for Plasmodium falciparum (211 samples), P. vivax (218), mixed with P. falciparum and P.
vivax (30), or P. ovale (1), and 124 samples of healthy subjects or patients with other fever-related infections, were collect-
ed. The sensitivities of Malaria Ag-PfTM and Malaria Antigen Pf/PanTM compared with the microscopic method for P. falci-
parum or P. vivax detection were 97.6% and 99.0%, or 98.6% and 99.0%, respectively. The specificities of Malaria Ag-PfTM,
Malaria Ag-PvTM, and Malaria Antigen Pf/PanTM were 93.3%, 98.8%, and 94.4%, respectively. The sensitivities of Malaria
Ag-PfTM, Malaria Antigen Pf/PanTM, and microscopic method, when PCR was used as a reference method for P. falciparum
or P. vivax detection were 91.8%, 100%, and 96.7%, or 91.9%, 92.6%, and 97.3%, respectively. The specificities of Ma-
laria Ag-PfTM, Malaria Ag-PvTM, Malaria Antigen Pf/PanTM, and microscopic method were 66.2%, 92.7%, 73.9%, and 78.2%,
respectively. Results indicated that the diagnostic performances of all the commercial RDTs are satisfactory for applica-
tion to malaria diagnosis.
Key words: Plasmodium falciparum, Plasmodium vivax, malaria diagnosis, rapid detection test (RDT), Thailand
•Received 9 November 2010, revised 5 January 2011, accepted 9 January 2011.
*Corresponding author (firstname.lastname@example.org)
34 Korean J Parasitol. Vol. 49, No. 1: 33-38, March 2011
. The second type detects parasite lactate dehydrogenase
(pLDH), an enzyme produced by all 4 human malaria species
[13,14]. HRP2 test kits have generally shown higher sensitivity
for P. falciparum detection and can be less costly than the pLDH
[15-18]. Nevertheless, several studies have demonstrated that
HRP2 remains in the blood stream for an extended time fol-
lowing successful eradication of the parasite with effective an-
timalarial treatment, contributing to false positive results and
limited specificity [19,20]. In areas along the Thai-Myanmar
border with a high annual malaria incidence, malaria diagno-
sis is an important tool in controlling disease morbidity and
mortality . RDTs would be an effective alternative diagnos-
tic tool or used as an adjunct to microscopy for successful ma-
In the present study, 3 commercial RDTs (SD BIOLINE: Stan-
dard Diagnostics, Seoul, Republic of Korea), i.e., Malaria Anti-
gen Pf/PanTM, Malaria Antigen Ag-PfTM, and Malaria Antigen
Ag-PvTM, were assessed for their diagnostic performance for P.
falciparum and P. vivax malaria. Ag-PfTM detects HRP2 specific
to P. falciparum, whereas Pg-PvTM detects pLDH specific to P.
vivax, and Pf/PanTM detects both HRP2 and pLDH specific to
Plasmodium species in human blood. The assessment of their
diagnostic performance was performed in comparison with
the gold-standard microscopic and reference PCR methods.
MATERIALS AND METHODS
The study was cross-sectional and was conducted during May
2008- April 2009 at Mae Sot General Hospital, Mae Sot District,
Tak Province, Thailand, an area along the Thai-Myanmar bor-
der with a high annual malaria incidence (http://www.thaivbd.
org/cms/index.php). The inclusion criteria were blood sam-
ples obtained from febrile patients (oral temperature >37.5˚C)
with acute uncomplicated malaria and healthy subjects who
had no previous sign of fever for at least weeks. The exclusion
criteria were blood samples from those having previous anti-
malarial treatment or presence of clinical signs and symptoms
of severe malaria. P. falciparum accounts for 50-60% of the Plas-
modium species in this region. A total of 584 blood samples,
500 µl each for microscopic examination and 100 µl finger-
prick blood for PCR and RDTs, were collected for malaria di-
agnosis by 3 methods, microscopic, PCR, and RDTs. Among
them, 460 were collected from patients with signs and symp-
toms of malaria, 72 were from patients with fever related to
other infections (10 typhus, 17 scrub typhus, and 45 dengue
hemorrhagic fever), and 52 normal blood samples obtained
from blood bank of Mae Sot General Hospital. Blood smears
of 460 malaria samples were confirmed by 2 microscopists;
211, 218, 30, and 1 samples were identified as infections with P.
falciparum, P. vivax, mixed infection with P. falciparum and P.
vivax, and P. ovale, respectively.
Thick blood smears were prepared for all blood samples
and stained with 10% Giemsa. The malaria parasite was de-
tected under light microscopy. The number of parasites was
counted against 200 leucocytes and parasite density was esti-
mated by assuming 8,000 leucocytes/μl blood. Samples were
considered negative when no parasite was detected after exam-
ining 100 microscopic fields. The malaria microscopic exami-
nation was performed by 2 independent experienced micros-
copists from Mae Sot General Hospital and Pharmacology and
Toxicology Laboratory, Thammasat University, Thailand. Each
blood slide was blinded and the result was masked to both of
the 2 microscopists. In order to check for inter-observer vari-
ability, a double blind cross reading of a random sampling of
100 blood slides was carried out by the senior microscopist.
Rapid diagnostic tests (RDTs)
Malaria diagnosis by 3 commercial RDTs, i.e., Malaria Anti-
gen Pf/PanTM (Catalogue No.05FK60), Malaria Ag-PfTM (Cata-
logue No. 05FK50), and Ag-PvTM (Catalogue No. 05FK70) (SD
BIOLINE) were performed in all blood samples (finger-prick
blood) using the method described by the manufacturer (http:
//standardia.com/html_e/mn03/mn03_01.asp). The presence
of both the control and test lines indicated a positive result for
P. falciparum and P. vivax, whereas the presence of only the con-
trol line indicated a negative result. All RDT kits were stored as
directed by the manufacturer and the quality of package desic-
cant was checked before use.
PCR analysis was performed in a total of randomly selected
129 (20%) finger-prick blood samples. The fresh blood sam-
ple (20 µl) was spotted onto a filter paper (Whatmann No. 3),
and the dried blood spot paper was stored in a zipper plastic
bag containing desiccant, and transported to he Pharmacology
Unit, Graduate Program in Biomedical Sciences, Thammasat
Chaijaroenkul et al.: Rapid diagnostics for P. falciparum and P. vivax in Mae Sot, Thailand 35
University for PCR analysis. Genomic DNA was extracted us-
ing Chelex-resin (Biorad, Hercules, California, USA) according
to the method of Wooden et al. . The previously published
nested PCR methods were employed to detect malaria species
specific reactions . The method is highly specific and can
differentiate P. falciparum, P. vivax, and other malaria species.
The performances of all RDTs were evaluated based on the
following criteria: sensitivity, specificity, positive prediction
value (PPV), negative prediction value (NPV), false positive
rate, and false negative rate, in 2 separate analyses; (i) diagnos-
tic performance of the 3 RDTs in comparison with the micro-
scopic method (gold standard), and (ii) comparative diagnos-
tic performances of the 3 RDTs in comparison with the micro-
scopic method and PCR (reference). The sensitivity of the test
was calculated as (number of true positives/[number of true
positives+number of false negatives])×100, and the specificity
of the test was calculated as (number of true negatives/[number
of true negatives+number of false positives])×100, PPV and
NPV were determined from (number of true positive/[number
of true positive+number of false positive])×100, and (number
of true negative/[number of true negative+number of false
negative])×100, respectively. The false positive and the false
negative rates were determined from 1–specificity, and 1- sen-
sitivity, respectively. The detection limit was calculated from
the sample with the lowest parasitemia with the true positive
result, and was confirmed by the laboratory clone P. falciparum
culture with different dilutions of parasitemia.
Statistical analysis was performed by the chi-square test at a
statistical significance level of P=0.05, using the SPSS version
Diagnostic performance of RDTs in comparison with
The parasite density of P. falciparum and P. vivax were 2-70,000
and 2-27,200 parasites/µl, respectively. A double blind cross
reading of a random sample of 100 slides showed 1% inter-
The diagnostic performances of all the 3 commercial RDTs
were evaluated in 584 blood samples, in comparison with the
microscopy method. Table 1 summarizes the number of blood
samples with positive and negative detection of P. falciparum
and P. vivax. A total of 229 and 211 samples, respectively, show-
ed positive for P. falciparum by Ag-PfTM and the microscopic
method. P. vivax was detected in 219 and 218 samples by Ag-
PvTM and the microscopic method, respectively. P. falciparum
and P. vivax , respectively, were detected in 232 and 200 sam-
ples by Pf/panTM. The sensitivity, specificity, PPV, NPV, false
positive rate and false negative rates of the 3 RDTs compared
with the microscopic method are shown in Table 2. The sensi-
tivity of Ag-PfTM and Pf/PanTM compared with the microscopic
method for detection of P. falciparum was 97.6% and 99.0%,
respectively. The sensitivity of Ag-PvTM and Pf/PanTM for detec-
Table 1. Detection of P. falciparum and P. vivax by Malaria Ag-
Pf, Ag-Pv, and Malaria Antigen Pf/Pan testsa in comparison with
the microscopic method
Species (microscopy positive)Ag-Pf kitAg-Pv kitP.f/Pan
P. falciparum (211)
P. vivax (218)
P. falciparum and P. vivax mixed (30)
Negative or P. ovale (125)
Total (584) 584584584
aData are presented as the number of positive samples by the 3 RDTs
and microscopic method (in parenthesis).
Table 2. The test performancea of Malaria Ag-Pf, Ag-Pv, and Mal aria Antigen Pf/Pan for detection of P. falciparum and P. vivax in com-
parison with the microscopic method
Ag-Pf Ag-Pv Pf/Pan
Sensitivity for P. falciparum
Positive predictive value (PPV)
Negative predictive value (NPV)
False positive rate
False negative rate
Detection limit (parasites/µl)
97.6 (94.7-98.9)-99.0 (96.9-99.7)
Pf> 2-5, Pv>2
- 98.6 (96.5-99.6)
aData are presented as percentage (95% confidence interval; CI).
36 Korean J Parasitol. Vol. 49, No. 1: 33-38, March 2011
tion of P. vivax was 98.6% and 99.0%, respectively. The speci-
ficity of Ag-PfTM, Ag-PvTM, and Pf/PanTM was 93.3%, 98.8%, and
94.4%, respectively. All the 3 RDTs showed significant correla-
tion with the microscopic method in detecting malaria para-
site species (P<0.001). Parasitemia of the false negative sam-
ples for Ag-PfTM and Ag-PvTM were 2-1,446 and 4-62 parasite/
µl, respectively. Parasitemia of the false negative samples by Pf/
panTM for detection of P. falciparum and P. vivax were 41-1,466
and <20 parasites/µl, respectively. The detection limit for P.
falciparum and P. vivax was >2 parasites/µl for all RDT tests.
For the laboratory strain P. falciparum dilution, the detection
limit was 5 parasites/µl.
The sensitivity of the 3 RDTs categorized by parasite density
is summarized in Table 3. Results clearly showed lower sensi-
tivity of all RDTs in detecting both P. falciparum and P. vivax
with parasite densities of less than 50 parasites/µl.
Diagnostic performance of RDTs, microscopy, and PCR
PCR was performed in a total of 129 randomly selected bl-
ood samples. The sensitivity, specificity, PPV, NPV, false posi-
tive and false negative rates of the 3 RDTs, and the microscopic
method in comparison with the PCR method are shown in
Table 4. Ag-PfTM showed the highest false positive rate. The sen-
sitivity of Ag-PfTM, Pf/PanTM, and the microscopic method for P.
falciparum detection were 91.8%, 100%, and 96.7%, respective-
ly. The sensitivity of Ag-PvTM, Pf/PanTM, and the microscopic me-
thod for P. vivax detection were 91.9%, 92.6%, and 97.3%, re-
spectively. The specificity of Ag-PfTM, Ag-PvTM, Pf/PanTM, and the
microscopic method were 66.2%, 92.7%, 73.9%, and 78.2%,
respectively. All the RDTs and the microscopic method showed
significant correlation with the PCR method in detecting cer-
tain species of malaria (P<0.001). Based on PCR results, the
microscopic method provided the best diagnostic performance
compared with the RDTs for detection of both P. falciparum
and P. vivax.
The present study demonstrated that the 3 RDTs, Malaria
Table 3. The sensitivitya of Malaria Ag-Pf, Ag-Pv, and Malaria Antigen Pf/Pan tests for detection of P. falciparum and P. vivax in com-
parison with the microscopic method, categorized according to parasite density
Parasite density (parasites/μl) Ag-PfAg-PvPf/Pan
aData are presented as percentage (95% CI).
Table 4. The test performancea of Malaria Ag-Pf, Ag-Pv, Malaria Antigen Pf/Pan, and the microscopic method for detection of P. fal-
ciparum and P. vivax compared with the PCR method
Sensitivity for P. falciparum
Positive predictive value (PPV)
Negative predictive value (NPV)
False positive rate
False negative rate
aData are presented as percentage (95%CI).
Chaijaroenkul et al.: Rapid diagnostics for P. falciparum and P. vivax in Mae Sot, Thailand 37
Antigen Pf/PanTM, Malaria Antigen Ag-PfTM, and Malaria Anti-
gen Ag-PvTM showed good test performances for detection of
both P. falciparum and P. vivax. Pf/PanTM is a one-step test for
detection of HRP2 specific to P. falciparum and pLDH pan-spe-
cific to Plasmodium species in the human blood sample. Ag-
PvTM is a one-step detection of pLDH specific to P. vivax, and
Ag-PfTM is a one-step detection of HRP2 specific to P. falciparum
in the human blood sample. Ag-PfTM and Ag-PvTM are specific
for detection of P. falciparum and P. vivax, respectively. Both sho-
wed excellent sensitivity and specificity.
The performance of the Ag-Pf test observed in the present
study was in agreement with previous studies with other HRP2-
based commercial RDTs, i.e., BinaxNow MalariaTM (Binax, Inc.,
Inverness Medical Professional Diagnostic, Scarborough, ME,
USA) , Paracheck PfTM (Orchid Biomedical system) [22,23],
and ParaHit PFTM (Span Diagnostic Ltd) [22,23]. The tests also
showed good performance when compared with pfLDH-based
commercial RDTs, such as CareStart Malaria testTM (AccessBio
Inc.) , OptiMAL stripTM (DiaMed AG) , and OptiMAL-
ITTM (DiaMed AG) [22-24]. Malaria Ag-PvTM which detected
pvLDH showed very good performance similarly to other com-
mercial RDTs, such as OptiMAL stripTM (DiaMed AG) , and
SD FK70 Malaria Antigen Pv testTM (Standard Diagnostics, Seoul,
Republic of Korea) . The excellent performance of Pf/PanTM
for detection of both P. falciparum and P. vivax observed in this
study was also similar to that reported with OptiMALTM (Di-
aMed AG) . Markedly variable sensitivity and specificity
have been reported for commercially available RDTs [13,22-
35]. It is difficult to directly compare the diagnostic performan-
ces of these tests since results reported may be influenced by
many factors, such as environmental conditions, the use of
different gold-standards, as well as possible geographic varia-
tion in malaria antigens. All these factors should be taken into
consideration when selecting RDTs for application to malaria
When PCR was the reference method, the microscopic meth-
od showed a low specificity (78.2%). The false positive was
21.7%; this was an interesting data. The explanation for this
could be other species of Plasmodium spp. (P. knowlesi) that
have been reported in Thailand [22,23]. The PCR method used
as the reference could not differentiate this P. knowlesi. There-
fore, the PCR result showed negative data. However, the 5 bl-
ood slides from these samples showed malaria parasites in red
blood cells. False negative results of RDTs were observed and
have been attributed to possible genetic heterogeneity of HRP2
or LDH expression, deletion of HRP2 or LDH gene, presence
of blocking antibodies, or immune-complex formation. On
the other hand, false positive tests can occur even in samples
with high parasitemia, which could be due to several reasons,
including viable asexual-stage parasitemia below the detection
limit of microscopy (possibly due to drug resistance), persis-
tence of antigens due to sequestration and incomplete treat-
ment, delayed clearance of circulating antigen (free or in anti-
gen-antibody complexes), and cross reaction with non-falci-
parum malaria or rheumatoid factor.
In conclusion, the test performance of these 3 RDTs as ma-
laria diagnostic tools are promising. The tests could detect Plas-
modium species with high accuracy, sensitivity, and specificity.
This work was supported by the Commission on Higher Ed-
ucation, Ministry of Education, Thailand, Thailand National
Research University (NRU), and Standard Diagnostics (Repub-
lic of Korea). We thank Ms. Kulaya Ruengweerayut for her kind
support for sample collection.
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