JOURNAL OF CLINICAL MICROBIOLOGY, May 2005, p. 2435–2440 Vol. 43, No.

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0095-1137/05/$08.00⫹0 doi:10.1128/JCM.43.5.2435–2440.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Real-Time PCR for Detection and Identification of Plasmodium spp.

Kathy A. Mangold,1,2 Rebecca U. Manson,2 Evelyn S. C. Koay,3,4 Lindsey Stephens,1
MaryAnn Regner,1 Richard B. Thomson, Jr.,1,2 Lance R. Peterson,1,2
and Karen L. Kaul1,2*
Department of Pathology and Laboratory Medicine, Evanston Northwestern Healthcare, Evanston,1 and Northwestern University
Feinberg School of Medicine, Chicago,2 Illinois; Molecular Diagnosis Centre, National University Hospital,
Singapore3; and Department of Pathology, National University of Singapore, Singapore4
Received 16 September 2004/Returned for modification 9 November 2004/Accepted 6 January 2005

Rapid and accurate detection of malaria parasites in blood is needed to institute proper therapy. We
developed and used a real-time PCR assay to detect and distinguish four Plasmodium spp. that cause human
disease by using a single amplification reaction and melting curve analysis. Consensus primers were used to
amplify a species-specific region of the multicopy 18S rRNA gene, and SYBR Green was used for detection in
a LightCycler instrument. Patient specimens infected at 0.01 to 0.02% parasitemia densities were detected, and
analytical sensitivity was estimated to be 0.2 genome equivalent per reaction. Melting curve analysis based on
nucleotide variations within the amplicons provided a basis for accurate differentiation of Plasmodium falci-
parum, P. vivax, P. ovale, and P. malariae. For assay validation, 358 patient blood samples from the National
University Hospital in Singapore and Evanston Northwestern Healthcare in Illinois were analyzed. Of 76
blinded patient samples with a microscopic diagnosis of P. falciparum, P. vivax, or P. ovale infection, 74 (97.4%)
were detected by real-time PCR, including three specimens containing mixed P. falciparum-P. vivax infections.
No Plasmodium DNA was amplified in any of the 82 specimens sent for malaria testing but that were
microscopically negative for Plasmodium infection. In addition, 200 blood samples from patients whose blood
was collected for reasons other than malaria testing were also determined to be negative by real-time PCR.
Real-time PCR with melting curve analysis could be a rapid and objective supplement to the examination of
Giemsa-stained blood smears and may replace microscopy following further validation.

Malaria remains a global concern. The World Health Orga- chromatographic assays based on antigen detection have been
nization estimates 300 to 500 million cases of malaria infec- developed but are also relatively insensitive in cases of low
tions resulting in over one million deaths occurring globally parasitemia (22, 24, 30). In addition, antigenemia may persist
each year. Although the vast majority of these cases are found weeks beyond the actual infection, leading to the false diagno-
in the 100 countries in the tropical regions of Africa, Asia, sis of malaria parasitemia (6, 22). Molecular detection for
Central and South Americas where the disease is endemic, the Plasmodium diagnosis using PCR has resulted in increased
mobile nature of today’s society results in ca. 1,400 cases of sensitivity and species discrimination compared to either mi-
imported malaria reported each year in the United States as a croscopic or immunochromatographic diagnosis of malaria (2,
result of international travel, immigration, and military service 4, 12, 14, 22, 23, 25, 27). However, most published PCR assays
(10). The Centers for Disease Control and Prevention recom- are gel based with (4, 5, 15) or without (2, 3, 9, 23, 25–27, 29,
mends that malaria should be considered in the differential 31, 35, 36) subsequent probe hybridizations, resulting in a
diagnosis of febrile patients who have traveled to a region lengthy procedure not optimal for clinical use. The need for a
where malaria is endemic and in any patients who experience more sensitive and time-efficient assay has led to the develop-
fevers of unknown origin regardless of their travel history (10). ment of molecular assays involving real-time PCR (7, 8, 13,
Provision of these diagnostic services and maintenance of com- 18). Real-time PCR assays have the potential to detect low
petency may pose a challenge to many laboratories. levels of parasitemia, identify mixed infections, and allow for
Species differentiation of Plasmodium is essential for select- precise differentiation of species via melting curve analysis. In
ing the proper treatment. Especially important is differentiat- the present study, we developed and validated a real-time PCR
ing P. falciparum from the others, since this species is respon- assay to detect and identify Plasmodium spp. in a single reac-
sible for ca. 95% of the deaths due to malaria (33). The current tion by using a simple collection method consisting of blood
standard for diagnosis is the microscopic examination of Gi- spotted on treated filter paper.
emsa-stained thick and thin blood smears (12, 16, 21, 22, 28). (This research was presented in part at the 43rd Interscience
This procedure is time-consuming to prepare, read, and inter- Conference on Antimicrobial Agents and Chemotherapy, Chi-
pret the slides. Previous studies have shown that even with cago, Ill., 14 to 17 September 2003, and at the annual meeting
experienced microscopists, misdiagnosis occurs, particularly in of the Association for Molecular Pathology, Orlando, Fla., 22
cases of mixed infection or low parasitemia (12, 28). Immuno- November 2003.)

* Corresponding author. Mailing address: Evanston Northwestern MATERIALS AND METHODS

Healthcare, Department of Pathology and Laboratory Medicine, 2650 Specimens. The study was approved by the Institutional Review Boards of
Ridge Ave., Evanston, IL 60201. Phone: (847) 570-2052. Fax: (847) Evanston Northwestern Healthcare (ENH) in Evanston, Ill., and the National
733-5012. E-mail: [email protected]. University Hospital (NUH) in Singapore. Controls obtained from the American

2435
2436 MANGOLD ET AL. J. CLIN. MICROBIOL.

TABLE 1. Comparison of results between the original microscopic cation was immediately followed by a melt program consisting of 2 min at 95°C,
diagnoses and the microscopic review of a blinded 2 min at 68°C, and a stepwise temperature increase of 0.2°C/s until 90°C, with
subgroup containing discrepant cases fluorescence acquisition at each temperature transition. The fluorescence data
were analyzed by using F1/F2 settings, which improved the detection of P. fal-
Original Final microscopic diagnosis (no. of specimens)a ciparum, and a cutoff of 35 cycles was used to define Plasmodium-positive sam-
microscopic ples in the present study. This assay for Plasmodium species differentiation
diagnosis P. falciparum P. malariae P. ovale P. vivax Negative
required ca. 1 h to complete, in addition to the sample preparation time. Melt
P. falciparum 34ⴱ 0 0 2 0 curve analysis was used to determine the species-specific mean melting temper-
P. malariae 0 0 0 2 0 ature (Tm) based on values determined from the respective plasmid controls.
P. ovale 0 0 1 0 0
P. vivax 0 0 0 40ⴱ 0
Negative 0 0 0 0 82 RESULTS

Total 34ⴱ 0 1 44ⴱ 82

Typical amplification and melt curves depicting Tm detection
for the various species are shown in Fig. 1 to 5. Based on the
a
ⴱ, Includes one specimen of a mixed P. falciparum-P. vivax infection. published genome size for P. falciparum (22.8 Mbp) (11), Avo-
gadro’s number (6.022 ⫻ 1023 molecules per mole), and a
standard nucleic acid molar conversion (1 ␮g of 1-kb DNA ⫽
Type Culture Collection (ATCC; Manassas, Va.) included plasmids containing a
partial 18S rRNA gene sequence from each species (P. falciparum [MRA-177], 1.54 ␮mol), we calculated that 1 ␮g of P. falciparum DNA is
P. vivax [MRA-178], P. malariae [MRA-179], and P. ovale [MRA-180]). In ad- equal to 4.067 ⫻ 107 genome copies or one genome copy in
dition, 10 DNA samples from P. falciparum-infected human red blood cell 24.6 fg of DNA. Control Plasmodium DNA samples obtained
cultures and monkey blood samples infected with P. vivax (five strains) or P. from in vitro culture in human reticulocytes and diluted to 0.2
malariae (one strain) were also obtained from the ATCC. During the develop-
ment of the assay, DNA was isolated from eight blood specimens known to
genome copies (5 fg) per reaction were detected and correctly
contain either P. falciparum or P. vivax from patients at ENH or NUH. For identified in the melting curve analysis; this is possible due to
validation, 200 blood samples from patients whose blood was collected for the presence of more than one copy of the 18S rRNA gene per
reasons other than malaria diagnosis and 158 blinded samples from patients with genome. An excess amount (150 ng) of human DNA previously
fever and a history of recent travel within a region where malaria infection is
tested negative for Plasmodium was included as a negative
endemic that were obtained from ENH and NUH, including specimens from
sources in Malaysia, Myanmar, and Thailand, were tested. Microscopic analysis control to estimate the possible background fluorescence in
was performed by experienced microscopists at each hospital. Thin and thick this SYBR Green assay.
smears from specimens with an initial determination of Plasmodium infection Melting curve analysis permitted the clear identification of
were again blinded and analyzed by a panel of experienced microscopists from each Plasmodium species control, as shown in Fig. 2. The Tm
ENH and Northwestern Memorial Hospital in Chicago, Ill. On the basis of
enlarged infected red blood cells, the occasional presence of Schüffner’s dots,
values for the control plasmids were highly reproducible on
and the rare identification of characteristic schizonts, four cases initially reported eight repeated melt curve runs. Table 2 contains the average
as either P. falciparum or P. malariae were identified as P. vivax in the second melting curve peak Tm for each Plasmodium spp. identified in
analysis; the results of the other blinded specimens were in agreement for both the control plasmids and the other genomic DNA specimens
the initial and second microscopic analyses. The final microscopic determina-
used to develop the real-time PCR assay. Mixed reactions were
tions of species are listed in Table 1 and were compared to the real-time PCR
results. created to mimic a dual infection with P. falciparum and P.
Blood specimen preparation. Since the present study involved an international vivax. Detection of each species was achieved at a ratio equiv-
collaboration, the use of ISOCODE cards (Schleicher & Schuell, Keene, N.H.) alent to 10 P. falciparum: one P. vivax (data not shown), a ratio
containing spotted samples of fresh or EDTA-anticoagulated blood was the that should approximate the number of P. falciparum-infected
requested method for specimen collection. Approximately 40 ␮l of blood was
applied to each circle of an ISOCODE card and allowed to dry completely. DNA
red blood cells that is generally higher in peripheral blood
from a single 6-mm hole punch (equivalent to one-fourth to one-half of an smears. DNA obtained from P. vivax- or P. malariae-infected
ISOCODE card circle) was extracted utilizing one wash with 500 ␮l of nuclease- monkey blood samples and 10 separate isolates of P. falcipa-
free water, followed by a 30-min incubation at 95 to 100°C in 50 ␮l of nuclease- rum genomic DNA from the ATCC confirmed the reproduc-
free water. Twenty blood samples that were clotted or thickened by prolonged
ibility of the melting curve (Fig. 3). Four P. falciparum patient
storage before application to the ISOCODE cards were alternatively processed
by using a Puregene (Gentra Systems, Minneapolis, Minn.) procedure for tissue
samples according to the manufacturer’s instructions. The specimens chosen for
the Puregene procedure were distinguishable as the dried blood and/or clot
remained on the surface of the card and did not penetrate the paper efficiently.
Real-time PCR. Plasmodium detection was performed by using real-time PCR
in the LightCycler (Roche Molecular Systems, Indianapolis, Ind.). The 18S
rRNA gene was chosen as the target since it contains both highly conserved and
variable regions, and at least five copies of the gene are dispersed on separate
chromosomes of the Plasmodium genome (11, 20). Consensus primers were
designed after comparing several partial 18S rRNA gene sequences for each of
four Plasmodium species (PL1473F18 [5⬘-TAA CgA ACg AgA TCT TAA-3⬘]
and PL1679R18 [5⬘-gTT CCT CTA AgA AgC TTT-3⬘]; sequence numbering
from GenBank accession number M19173). A BLAST analysis (1) of GenBank
indicated that amplification with these two oligonucleotides would only occur
when a Plasmodium spp. was available to act as a template (expected value of
0.31 for both oligonucleotides). Each 20-␮l reaction mix contained 2 to 5 ␮l of
sample DNA, 2 ␮l of 10⫻ FastSTART DNA SYBR Green reagent (Roche), FIG. 1. Real-time amplification with SYBR Green fluorescence
6.5 mM MgCl2 (final concentration), and 0.5 mM concentrations of each primer. detection. The plasmid controls for four species, water blank, and
The PCR conditions consisted of an initial denaturation at 95°C for 10 min, negative human control DNA are indicated. The remaining curves are
followed by amplification for 40 cycles of 10 s at 95°C, 5 s at 50°C, and 20 s at patient specimens with various parasitemia levels. The graph was gen-
72°C, with fluorescence acquisition at the end of each extension step. Amplifi- erated by using LightCycler Software v. 3.
VOL. 43, 2005 REAL-TIME PCR FOR PLASMODIUM SPP. 2437

FIG. 2. Melting curve analysis, with the control plasmids, water

blank, and negative human control DNA labeled. Vertical lines indi-
cating the Tm values for each of the plasmid controls are continued in FIG. 3. Melting curve analysis: DNA isolated from blood by using
Fig. 3 through 5. The graph was generated by using LightCycler Soft- the Puregene procedure from monkeys infected with either P. malariae
ware v. 3. or P. vivax (ATCC) and purified P. falciparum genomic DNA (ATCC).
The graph was generated by using LightCycler Software v. 3.

samples and four P. vivax patient samples were also tested

during assay development. We did observe a consistent slight time PCR (Fig. 5). A single patient infected with P. ovale was
shift to a higher Tm (up to 1°C) in the amplicons generated by identified by both microscopic analysis and real-time PCR (mi-
the control plasmids compared to the other specimens tested croscopic identification of P. ovale confirmed by the Illinois
(Table 2). The cause for this shift is currently unknown, al- Department of Public Health). Two specimens microscopically
though we assume sequence differences in the cloned frag- diagnosed as containing P. falciparum with parasitemia levels
ments or variable salt concentrations in the DNA aliquots are of ca. 0.01% (data not shown) did not produce an amplification
responsible. Taking into consideration the Tm values for the curve within the 35 cycle cutoff limit used for the present study,
control plasmids, the infected monkey blood, the infected hu- even when the amount of template DNA used in each reaction
man red blood cell cultures, and the eight patient samples, was increased from 2 to 5 ␮l. Thus, the detection sensitivity for
broad, nonoverlapping Tm ranges were selected to identify the patient samples was estimated to be in the range of 0.01 to
Plasmodium species in the blinded patient specimens. The Tm 0.02% parasitemia. Using the normal range of 4.7 to 6.1 mil-
values chosen were as follows: P. malariae, 73.5 to 75.5°C; lion red blood cells per ml of blood, 0.01 to 0.02% parasitemia
P. falciparum, 75.5 to 77.5°C; P. ovale, 77.5 to 79.0°C; and would be equivalent to 470 to 1,220 parasites per ml, or ap-
P. vivax, 79.0 to 81.0°C. DNA prepared by using the Puregene proximately 1 parasite per ␮l. This sensitivity corresponds fa-
procedure had Tm values universally lower by ca. 1°C than vorably to other published methods, specifically real-time PCR
DNA prepared from identical specimens using the standard assays.
ISOCODE extraction protocol but was still at the lowest end Four samples were determined by initial microscopy to be
of the broad ranges described above. These lower Tm values two P. malariae- and two P. falciparum-infected specimens; all
are most likely due to the salt content in the Puregene DNA four were identified as P. vivax by the real-time PCR assay,
hydration buffer compared to the nuclease-free water of the which is in agreement with the final consensus identification
ISOCODE card protocol. for these four samples. Eighty-two specimens from patients
Parasites in 74 of 76 (97.4%) blinded patient samples with
a positive microscopic diagnosis of malaria were detected by
real-time PCR (Fig. 4), including three specimens containing
mixed P. falciparum-P. vivax infections. Two samples that were
diagnosed by microscopy as only P. falciparum infections were
identified as mixed P. falciparum-P. vivax infections by real-

TABLE 2. Average melting curve peak Tm values

for each Plasmodium sp.
Avg Tm peak ⫾ SD (°C)a in:
Species Plasmid
Patient specimens
clones*

P. falciparum 77.0 ⫾ 0.2 75.9 ⫾ 0.4

P. malariae 75.0 ⫾ 0.4 74.8 ⫾ 0.4† FIG. 4. Melting curve analysis: patient samples infected with P. fal-
P. ovale 78.6 ⫾ 0.3 78.5 ⫾ 0.1ⴱ ciparum, P. ovale, or P. vivax prepared by using the ISOCODE card
P. vivax 80.4 ⫾ 0.3 79.6 ⫾ 0.5 procedure for DNA extraction. Patient sample infected with both
a
ⴱ, single DNA specimens averaged over several runs on separate days; †, no P. falciparum and P. vivax is highlighted with a dashed line, and the
patients infected with P. malariae were identified by using the real-time PCR patient specimen with P. ovale is highlighted with solid circle points.
assay, so this value represents a single P. malariae-infected monkey blood spec- The graph was generated by using LightCycler Software v. 3.
imen obtained from the ATCC and averaged over several runs on separate days.
2438 MANGOLD ET AL. J. CLIN. MICROBIOL.

region of the Plasmodium 18S (small subunit) rRNA gene in

our real-time PCR assay. By targeting a gene with multiple
copies dispersed throughout the Plasmodium genome (11), the
sensitivity of this assay is greater than if a single-copy gene was
chosen. We also chose to target the partial 18S rRNA gene
sequence included in the cloned plasmids available from the
ATCC to permit easy access to individual species-specific con-
trols. Although several fluorescence resonance energy transfer
hybridization probes and different primer sets were tested in
the development of this assay (data not shown), the described
SYBR Green assay provided the best discrimination among
the different species. The sequences bracketed by the primers
FIG. 5. Melting curve analysis: discordant patient samples. Dual are sufficiently divergent in the four species to provide at least
P. falciparum and P. vivax infections not microscopically identified are a 1°C difference between the average Tm values to distinguish
highlighted with dashed lines. The blue dashed line represents a spec- between the species during the melting curve analysis. Single
imen prepared by using the Puregene method, and the red dashed line
base differences theoretically may also exist between strains
is one prepared by using the standard ISOCODE method, exemplify-
ing the slightly lower Tm peaks resulting from the former method. The within a species from different geographic regions, but our
black diamonds indicate microscopically diagnosed P. falciparum spec- results indicate that the variances among individual strains or
imens that amplified beyond the 35 cycle cutoff limit used in the pres- patient specimens prepared by the same DNA purification
ent study. The graph was generated by using LightCycler Software v. 3. procedure are minor in comparison to the differences among
species. Additional specimens from other regions around the
world would be required to confirm these assumptions.
with clinical findings suggesting malaria but microscopically
We did not address the analysis of blood samples collected
negative for Plasmodium and subsequently determined not to
by various methods in the present study. To obtain a diverse
be infected were PCR negative. An additional 200 blood sam-
group of patient specimens, blinded samples from ENH and
ples from patients collected for diagnoses unrelated to malaria
NUH were collected on ISOCODE cards and processed in the
(data not shown) were also determined to be negative by real-
Molecular Diagnostics laboratory at ENH. We overcame po-
time PCR. Using results from the second microscopic analysis
tential problems posed by blood samples that were clotted or
as the gold standard, the sensitivity, specificity, positive and
thickened due to long-term storage at 4°C before application
negative predictive values, and accuracy of the real-time PCR
to cards, thus failing to percolate into the filter paper ade-
assay for each detected Plasmodium spp. are summarized in
quately, by processing the “clumps” using the Puregene pro-
Table 3.
cedure for tissue samples. Samples with significant red blood
cell lysis as a result of freezing were found to be unsuitable for
DISCUSSION
analysis (data not shown). In the present study, DNA was
Although there are many published studies showing the im- isolated from ca. 10 ␮l of blood from each ISOCODE card
proved sensitivity and specificity of PCR-based assays over specimen, and 1/50 to 1/20 of this DNA was used in each
microscopic or immunochromatographic diagnosis of malaria real-time PCR.
(2, 4, 12, 14, 22, 23, 25, 27), only a handful take advantage of We analyzed 76 blood specimens from patients with clinical
the even more sensitive and time-efficient real-time PCR tech- findings suggesting malaria for which a microscopic determi-
nologies. In several studies in which real-time PCR was uti- nation of Plasmodium infection had been identified. We, like
lized, either a single-species was identified (13) or no distinc- others, had some difficulty in arriving at a consensus for the
tion between the four human parasites was made (8, 18). This microscopic diagnosis. Our final microscopic diagnosis used for
distinction is critical in the clinical management of patients, comparison was based on review by a panel of experienced
since the course of treatment varies depending on which spe- microscopists and is reported in Table 1. Real-time PCR anal-
cies is the cause of the infection. Only in the study by de
Monbrison et al. were the four human parasites identified by
real-time PCR (7). This assay included five separate primer TABLE 3. Sensitivity, specificity, positive predictive value, negative
pairs amplified simultaneously in a LightCycler instrument, predictive value, and accuracy of the real-time PCR assay relative
utilizing melting curve analysis to distinguish the Plasmodium to microscopic analysis using the results from the morphologic
spp. In the de Monbrison study, twenty-nine single infections assessment of a panel of experienced microscopists
and four dual infections were identified by real-time PCR. %b
Twelve specimens were negative by both microscopy and real- Parametera
P. falciparum P. malariae P. ovale P. vivax
time PCR.
The single reaction real-time PCR assay described in our Sensitivity 94.1 Unknown 100.0 100.0
study used only a single pair of primers to detect and identify Specificity 100.0 Unknown 100.0 99.1
PPV 100.0 Unknown 100.0 95.5
Plasmodium spp. in 3 h, including standard DNA sample prep- NPV 98.4 Unknown 100.0 98.3
aration, amplification, and detection, with sensitivities equiva- Accuracy 98.7 Unknown 100.0 98.1
lent to microscopy. An additional 1 to 2 h was needed when the a
PPV, positive predictive value; NPV, negative predictive value.
more extensive Puregene DNA preparation was required. As b
Only one patient here was infected with P. ovale, as determined by micro-
with several other studies, we chose to target a species-specific scopic analysis; no patients were infected with P. malariae.
VOL. 43, 2005 REAL-TIME PCR FOR PLASMODIUM SPP. 2439

ysis identified P. falciparum, P. vivax, or P. ovale in 74 (97.4%) using the standard ISOCODE card procedure, PCR amplifi-
of these specimens, including 3 that contained a dual infection cation, and melting curve identification of Plasmodium spp.,
with P. falciparum and P. vivax, for which only 1 had been was completed in less than 3 h. This real-time PCR assay for
microscopically identified as such. Since it is often difficult to the detection and identification of Plasmodium spp. can be
diagnose mixed infections by microscopy, the distinct dual used to confirm microscopic findings and in many settings can
peaks observed in Fig. 5 indicate that the real-time PCR assay be used for the primary identification of an infected patient
may be more accurate than the gold standard of microscopy in without the need for multiple blood specimens or even micro-
these two cases. To date, only a single patient with a P. ovale scopic analysis.
infection had been identified by microscopy and real-time
PCR. Of the two specimens microscopically diagnosed as con- ACKNOWLEDGMENTS
taining P. falciparum that did not produce a positive result for We acknowledge the assistance of Kevin S. W. Tan, Department
any Plasmodium infection in the real-time PCR assay, the of Microbiology, National University of Singapore; May Ann Lee,
amount of parasitemia in these samples was ca. 0.01%, at the Defense Ministry Research Institute, Singapore; and Thiha Nyunt,
Yangon, Myanmar, in obtaining the patient blood specimens used in
lower threshold of sensitivity for this assay. In both cases, the this study. We appreciate the time-consuming microscopic analysis
blood had been stored for several days before aliquots were performed by Linda Kuksuk of Northwestern Memorial Hospital, Chi-
spotted onto the ISOCODE cards. It is also possible that the cago, Ill. We also acknowledge the technical assistance of Wooi Loon
sensitivity of the assay was decreased by the delay in specimen Ng and Mui Joo Khoo of National University Hospital, Singapore.
This study was funded by the Department of Pathology and Labo-
preparation, in spite of using the Puregene procedure. ratory Medicine at Evanston Northwestern Healthcare, Evanston, Ill.
Of the 74 infected specimens detected by both microscopy
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