Experimental Parasitology 100 (2002) 131134

www.academicpress.com

Research Brief

Plasmodium vivax: parasitemia determination by

real-time quantitative PCR in Aotus monkeys
Juan Carlos Polanco, Josefa Antonia Rodr
guez, Vladimir Corredor,
and Manuel Alfonso Patarroyo*
Fundaci

on Instituto de Inmunolog
a de Colombia (FIDIC), Departamento de Biolog
a Molecular, Carrera 50 No. 26-00 Bogot

a, Colombia
Received 8 August 2001; accepted 4 March 2002

Abstract

Plasmodium vivax and Plasmodium falciparum are the two prevalent human malaria species. A Colombian P. vivax wild strain has been adapted in
Aotus nancymaae monkeys for use in further biological and immunological studies. We present data validating a real-time PCR assay quantifying P.
vivax parasitemia, using the small subunit ribosomal RNA genes as an amplication target. P. vivax species-specic primers were designed on the 18S
ribosomal gene V8 region, for amplifying both asexual and sporozoite ssrRNA genes. The assay detects amplication products bound to uorescent
SYBR-Green I dye using PerkinElmer GeneAmp-5700-SDS. Linear range standard curves from 6 DNA concentration logs (+0.99 correlation
coecients) were obtained. Standard curves were constructed using a plasmid containing target gene for real-time PCR amplication. This P. vivax
specic assay is very sensitive, having a three parasite detection limit, and is reproducible and accurate. It involves a closed-tube PCR, avoids time-
consuming post-PCR manipulation, and decreases potential PCR contamination.

Index Descriptors and Abbreviations: Plasmodium vivax; Aotus nancymaae; real-time quantitative PCR; SYBR Green I; parasitemia; small subunit
ribosomal RNA (ssrRNA); polymerase chain reaction (PCR); deoxyribonucleic acid (DNA); ribosomal DNA (rDNA); threshold cycle (Ct); coef-
cient of variation (CV). 2002 Elsevier Science (USA). All rights reserved.

Plasmodium vivax is the most widely distributed malarial parasite in
the world and the predominant species in the Americas and Asia
(Mendis et al., 2001). A wild Colombian P. vivax strain has been
adapted in our Institutes facilities to the Aotus nancymaae monkey so
that this parasites biology can be better understood. A peculiar
characteristic of P. vivax is its preference for invading primarily young
red blood cells, known as reticulocytes, normally accounting for only
about 1% of the red blood cell population, a factor believed to control
parasitemia (Galinski and Barnwell, 1996). The standard test for
Plasmodium spp. diagnosis and parasite quantication is microscope
examination of Giemsa-stained thick and thin blood smears. Although
this method is a rapid and inexpensive diagnostic test, it depends on
the microscopists experience; achieving high sensitivity requires
counting up to 100 microscope elds (Barker et al., 1992; Vu et al.,
1995), which is time-consuming and labor-intensive.
The development of assays having higher sensitivity and repro-
ducibility than microscope examination for determining P. vivax par-
asitemia is a priority for gaining a deeper knowledge of P. vivax
biology in the Aotus experimental model. Molecular techniques are
presently being applied to the diagnosis of malaria. Species-specic
regions have been exploited in developing sensitive and specic diag-
nostic procedures (Lal et al., 1989; Waters and McCutchan, 1989a),
*
Corresponding author. Fax: +57-1-3244674.
E-mail address: mapatarr@mail.com (M. Alfonso Patarroyo).
following characterization of Plasmodium small subunit ribosomal
RNA (ssrRNA) genes (McCutchan et al., 1988; Waters and McCut-
chan, 1989b). The ssrRNA gene sequence is composed of a mosaic of
conserved and variable regions. This type of arrangement allows the
design of genus-specic primers (based on conserved sequences) and
the internal variable regions allow designing species-specic primers
for the dierent Plasmodium species (Li et al., 1995). The ssrRNA
genes are few in number (ranging between four and eight rDNA units)
(McCutchan et al., 1995), being genetically unlinked and dispersed
throughout the Plasmodium genome (Wellems et al., 1987).
This report presents the validation of a real-time-PCR-based assay
for determining P. vivax parasitemia in A. nancymaae monkey pe-
ripheral blood. We chose ssrRNA genes as amplication target for
achieving acceptable sensitivity, due to their moderate repetitiveness
and the possibility of designing species-specic primers for P. vivax.
Alignment of all human malaria parasite ssrRNA genes complete and
partial sequences (reported in the GenBank) was performed to design
the forward (50 -TTATTAAAATTACGATTCAGCTTGCTG-30 ) and
reverse (50 -AATTGCAATAATCTATCCCCATCAC-30 ) primers.
These primers are specic for both asexual and sporozoite stage-spe-
cic ribosomal genes and specically amplied a 100 bp product only
when a P. vivax ribosomal RNA gene was used as DNA template (data
not shown).
The recent advent of real-time quantitative PCR has improved
PCR-based DNA and RNA quantication. Reactions are character-
ized by the point in time during cycling when PCR product ampli-

0014-4894/02/$ - see front matter 2002 Elsevier Science (USA). All rights reserved.
PII: S 0 0 1 4 - 4 8 9 4 ( 0 2 ) 0 0 0 1 0 - 3
132 J. Carlos Polanco et al. / Experimental Parasitology 100 (2002) 131134
cation is rst detected rather than the amount of PCR product accu-
mulated after a xed number of cycles. The Ct parameter (threshold
cycle) is the basis for real-time-PCR-based quantication accuracy and
reproducibility (Bustin, 2000; Heid et al., 1996). Higuchis pioneer
work (Higuchi et al., 1992, 1993) demonstrated that an initial target
copy numbers log plot for a set of standards versus Ct is a straight
line. The amount of target in unknown samples can be quantied by
measuring Ct and using the standard curve to determine the starting
copy number.
It was decided to quantify the amount of rDNA units in P. vivax
genomic DNA samples from the Aotus monkeys by using standard
curves constructed from plasmid clone p596-41 serial dilutions. Pla-
smid p596-41 was obtained by cloning in pUC-18 vector an asexual-
type P. vivax ssrRNA gene amplied by PCR using ribosomal uni-
versal primers (Corredor and Enea, 1994). The maximum parasitemia
exhibited in our Aotus model has been 3707 parasites=lL, which is
very low for achieving standard curves from several DNA concentra-
tion logs. The assay uses uorescent SYBR Green I dye binding spe-
cically to double-stranded-DNA. Real-time quantitative PCRs were
prepared with reagents from a PerkinElmer SYBR Green PCR Core
Reagents kit and performed with the GeneAmp 5700 Sequence De-
tection System (PE Applied Biosystems).
Genomic DNA was isolated from 300 lL peripheral blood using
the Wizard Genomic DNA Purication System (Promega). Blood
samples from the femoral vein of A. nancymaae monkeys (infected with
an adapted P. vivax strain) were collected in tubes containing EDTA.
Triplicate DNA extractions were done with every blood sample to
determine the eciency of DNA isolation. Real-time PCR was per-
formed with each triplicate DNA sample to determine the parameter
Ct using the ribosomal primers designed for this work. Ct was found to
be strongly reproducible within DNA triplicate samples, indicating
that no internal control should be used to compensate variations be-
tween DNA extractions (data not shown).
A potential risk of quantifying genomic samples on a plasmid-
constructed standard curve is that their rDNA copy numbers could be
under-estimated, basically because the same target in plasmid form can
have a higher PCR eciency than as a complex sample, such as ge-
nomic DNA. Fig. 1 shows that a standard curve constructed with
plasmid p596-41 serial dilutions exhibits the same PCR-amplication
eciency as a standard curve constructed with P. vivax genomic DNA
serial dilutions. Both curves showed a similar slope and colinearity,
demonstrating identical amplication eciency throughout the tested
range. The quantication range is wider with p596-41 serial dilutions
than with P. vivax genomic DNA, because our experimental model did
not develop high parasitemia (a maximum of 3707 parasites=lL to
date). Only one cycle separated these two curves and suggested that
under-estimation could be minimum, which is in accordance with re-
sults shown below.
No primer dimers were observed within a range of 107 102 rDNA
copies. Primer dimers were conspicuous and aected linear regression
by diminishing correlation coecients in 101 and 100 orders of mag-
nitude, increasing the assays variation coecients and standard errors.
Our results demonstrate that the assay was linear over a range of 107
and 102 rDNA copies, where standard curve correlation coecients
were over 0.99.
Three identical experiments were performed to assess our real-time
PCR assays reproducibility and reliability. Standard curves were
constructed from tenfold plasmid p596-41 serial dilutions within the
quantication range (with no primer dimer presence) to determine the
amount of rDNA input copy number in P. vivax unknown genomic
DNA samples. Each PCR was performed in triplicate to assess the
assays intra- and inter-variation. Unknown P. vivax samples had been
previously quantied for parasites by microscope examination of Gi-
emsa-stained thin blood smears for comparing our results from real-
time PCR with the results obtained by the microscope test. The pro-
portion of every asexual stage in the blood samples was established. A
distinctive feature between P. vivax and P. falciparum is that the for-
Fig. 1. Eciency of PCR amplication of target DNA (ssrDNA units)
in genomic DNA form and in plasmid p596-41 form. Tenfold Plas-
modium vivax genomic DNA and plasmid clone p596-41 serial dilu-
tions were prepared in triplicate. A master mix, containing all reagents
necessary for PCR, was prepared and distributed for 50 lL individual
reactions. Final primer concentration was 0:2 lM and template DNA
was added individually. 40 PCR cycles (95 C for 15 s and 60 C for 1
min) were performed, following initial activation of AmpliTaq Gold
DNA polymerase at 95 C for 10 min. The threshold cycle value (Ct),
indicative of the quantity of target gene at which the uorescence ex-
ceeds a pre-set threshold, was determined. The Ct values obtained are
plotted against log of initial input copy number. r P. vivax genomic
DNA; j plasmid clone p596-41.
mer does not sequester maturing blood-stage forms from circulation;
consequently all the asexual stages are visible in blood by microscopy.
Polynucleated stages (such as schizonts) vary their proportion in blood
samples, depending on parasite development stage. Schizonts can
contain between 14 and 24 nuclei or merozoites (Jawetz et al., 1991),
meaning that two blood samples with the same parasitemia, but with
dierent and substantial proportions of schizonts, can show a very
dierent gene copy number. This biological variability does not allow
direct correlation between parasitemia and gene copy number without
taking into account determination of each asexual blood-stage pro-
portion in the samples (Table 1).
The three standard curves exhibited a similar slope and colinearity
in the tested DNA concentration range (Fig. 2). The Cts displayed
intra-assay coecients of variation (CVs) between 0.3% and 2.4%.
Inter-assay Ct CVs were between 1.3% and 6.2%. Although Ct values
in the three validation experiments showed some variation, input copy
numbers were very reproducible, due to a similar shift in Ct values in
the included standard curve (Fig. 2). Statistical analysis of variance
(ANOVA) was performed to measure the homogeneity of data gen-
erated with our assay. With a 0:05, analysis demonstrated that our
assay is homogeneous for every source of variability analyzed, sup-
porting the reproducibility deduced from the calculated coecients of
variation.
Table 1 shows a brief comparison between the results obtained by
microscope test and our real-time PCR assay quantifying rDNA copy
numbers in P. vivax genomic DNA samples from the experimental
model. As can be seen, the quantities obtained by real-time PCR are
slightly lower than those obtained by microscopy (columns 6 and 8 in
Table 1). The orders of magnitude are conserved; however, the amount
of quantitative data obtained by microscope test was twice that ob-
tained by real-time PCR assay. Likewise, we were able to quantify
samples by real-time PCR that gave a negative result by microscopy.
Identication of amplication products by melting curves (Ririe et al.,
1997) allowed it to be ascertained that negative results by microscopy
but positive by PCR were neither PCR artifacts nor contamination,
since primer dimer uorescence is the sole product obtained with
negative controls, such as uninfected blood samples and no-template
reactions.
A P. vivax DNA serial dilution was subjected to real-time PCR
analysis by using a standard curve constructed with tenfold plasmid
 J. Carlos Polanco et al. / Experimental Parasitology 100 (2002) 131134 133

Comparison of Plasmodium vivax genomic DNA sample rDNA copy number quantication by microscope test and real-time quantitative PCR (NTC: no template control, blood (): uninfected

rDNA obtained by
Copy number of

real-time PCR
4.28E + 5
3.37E + 5
4.75E + 4
3.15E + 4
0
0
real-time PCR
obtained by
Mean Ct

Fig. 2. Standard curves obtained from our assays three reproducibility

21.75
22.09
25.46
25.77
36.60
35.51
validation experiments. Tenfold plasmid clone p596-41 serial dilutions
(range: 1.65E6 to 1.65E2 copies of rDNA units) were performed in
triplicate. The obtained Ct values are plotted against initial input copy
number log. The three standard curves are colinear and show a similar
rDNA calculated by

slope. r Experiment 1; j experiment 2; N experiment 3.

Copy number of

microscope test

p596-41 serial dilutions to determine our methods detection limit. It

9.61E + 5
9.44E + 5
8.37E + 4

was possible to detect and quantify (with no primer dimer presence) a

DNA concentration equivalent to 3 parasites (i.e., 1 parasite  1 in-
fected-cell). Although we detected less than three parasites, the con-
0
0
0

spicuous presence of primer dimers did not make the quantities

obtained by our assay reliable.
added to each PCR
Individual parasites

In spite of primer dimer presence below 102 rDNA copies, our

assays detection and quantitation limit was 3 parasites, an extremely
sensitive, accurate and reproducible methodology, which does not have
inherent microscope reading limitations. The method described here is
160,146
157,383
13,950
0
0
0
reaction

very reproducible within the established range of linearity and, al-

though Ct values showed some variation between dierent experi-
ments, the input copy numbers were reproducible because a similar
shift occurred in standard curve Ct values included in each experiment
(Fig. 2). It was thus possible to quantify P. vivax samples considered to
be negative by microscope test (Table 1). A similarity was found be-
Parasite number corrected

proportion parasites=lL

tween data generated by real-time PCR and microscopy. We consider

the data generated by real-time PCR to be more reliable because the
according to schizont

copy numbers calculated with microscope test are based on the average
ribosomal gene copies in Plasmodium (McCutchan et al., 1995) and
the average number of nuclei in schizonts, which are not absolute
quantities and are inuenced by biological variability. It is worth
1550
17,794
17,487

0
0
0

mentioning that rDNA copy numbers can be converted into parasite

numbers by using the average number of ribosomal genes in each
nucleus or individual parasite as a conversion factor, as can be inferred
from Table 1.
Ribosomal genes have been considered as being an ideal target for
Microscope parasitemia

diagnosis and quantication (Li et al., 1995). This works results and
those of others (Bru~ na-Romero et al., 2001; Hermsen et al., 2001)
conrm the utility of ssrRNA genes for parasite quantication of
per infected cell
parasites=lL)

dierent malaria species by real-time PCR. We have thus validated a

real-time quantitative PCR assay for determining P. vivax parasitemia.
The technique is fast, reproducible, accurate and sensitive enough and
3707
2800

0
0
0
600

has a decreased potential for PCR contamination as it involves

closed-tube reaction not requiring post-PCR manipulation.
microscope
Result of

Acknowledgments
test

The Colombian Presidents oce and the Ministry of Public Health

+
+
+
)
)
)

supported this research. Blood samples of A. nancymaae monkeys in-

fected with P. vivax were provided by Raul Rodriguez from our In-
Blood ()

stitutes Amazonian branch. We are thankful to Juan Camilo Santana

Sample

NTC
Table 1

for his help in the statistical analysis of data, and Professor Manuel
blood)

G1
L1

F1
E1

Elkin Patarroyo, Yago Pico de Coa~ na, and Jason Garry for critically
reviewing the manuscript.
134 J. Carlos Polanco et al. / Experimental Parasitology 100 (2002) 131134
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