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Diagnostic Microbiology and Infectious Disease 59 (2007) 415 – 419
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Parasitology
A new polymerase chain reaction/restriction fragment length
polymorphism protocol for Plasmodium vivax circumsporozoite protein
genotype (VK210, VK247, and P. vivax-like) determination
Renata Tomé Alves a,d , Marinete Marins Póvoa b , Ira F. Goldman c , Carlos Eugênio Cavasini d ,
Andréa Regina Baptista Rossit d,e , Ricardo Luiz Dantas Machado d,e,⁎
a
Universidade Estadual Paulista Júlio de Mesquita Filho, 15054-000 São José do Rio Preto, São Paulo State, Brazil
b
Instituto Evandro Chagas, MS/SVS, 67030-000 Ananindeua, Pará State, Brazil
c
Centers for Disease Control and Prevention, Atlanta, GA 30333, USA
d
Centro de Investigação de Microrganismos, Departamento de Doenças Dermatológicas, Infecciosas e Parasitárias, Faculdade de Medicina de São José do
Rio Preto, 15090-000 São José do Rio Preto, São Paulo State, Brazil
e
Fundação Faculdade de Medicina de São José do Rio Preto- FUNFARME, 15090-000 São José do Rio Preto, São Paulo State, Brazil
Received 20 March 2007; accepted 27 June 2007
Abstract
For the molecular diagnosis of Plasmodium vivax variants (VK210, VK247, and P. vivax-like) using DNA amplification procedures
in the laboratory, the choice of rapid and inexpensive identification products of the 3 different genotypes is an important prerequisite. We
report here the standardization of a new polymerase chain reaction/restriction fragment length polymorphism technique to identify the 3
described P. vivax circumsporozoite protein (CSP) variants using amplification of the central immunodominant region of the CSP gene
of this protozoan. The simplicity, specificity, and sensitivity of the system described here is important to determine the prevalence and
the distribution of infection with these P. vivax genotypes in endemic and nonendemic malaria areas, enabling a better understanding of
their phylogeny.
© 2007 Published by Elsevier Inc.
Keywords: Plasmodium vivax genotypes; PCR/RFLP; Diagnosis; Circumsporozoite protein; Plasmodium vivax-like; VK210; VK247
1. Introduction
Plasmodium vivax is the second most prevalent malaria
parasite affecting more than 75 million people each year
(Imwong et al., 2005). The circumsporozoite protein (CSP) of
the infective sporozoite is the main target for the development
of recombinant malaria vaccines (Qari et al., 1993; Gonzales
et al., 2001; Herrera et al., 2007). Nevertheless, the data
generated require special considerations because of the
discovery of sequence variations in the central portion of
⁎ Corresponding author. Centro de Investigação de Microrganismos,
Departamento de Doenças Dermatológicas, Infecciosas e Parasitárias,
Faculdade de Medicina de São José do Rio Preto, 15090-000 São José do
Rio Preto, São Paulo State, Brazil. Tel.: +55-17-3201-5913; fax: +55-173201-5736.
E-mail address: [email protected] (R.L.D. Machado).
0732–8893/$ – see front matter © 2007 Published by Elsevier Inc.
doi:10.1016/j.diagmicrobio.2007.06.019
the CSP gene (Gopinath et al., 1994). Rosenberg et al. (1989)
described a variant of P. vivax in Thailand (VK247), and Qari
et al. (1993) reported on the P. vivax-like variant in Papua
New Guinea, which morphologically resembles the classic
form (VK210) and VK247 but has a distinctive repeated
portion of the central region of the CSP gene. Another
important issue generated by the existence of these genotypes
is the possibility of differential variant-linked responses to
treatment. The first evidence that P. vivax develops resistance
to chloroquine (CQ) was reported in Papua New Guinea
(Rieckmann et al., 1989) and, consequently, studies carried
out by Kain et al. (1993) suggested that the response to CQ
can vary depending on the P. vivax genotypes. In addition,
Machado et al. (2003) confirmed a significant correlation
between parasite clearance and its 3 genotypes.
By serological and/or molecular approaches, different
authors evaluated the global occurrence of these variants.
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The proportion of positive sera, specific for the VK210 and
VK247 variants, ranged from 28% to 66% in Thailand
(Wirtz et al., 1990). Nevertheless, VK247 genotype was
identified in 58% of all patients infected with both genotypes
(Kain et al., 1992, 1993). In Brazil, all variants were
genotyped, but only VK210 was found as a single agent of
infection, whereas the other 2 occurred as mixed infections
(Machado and Póvoa, 2000; Silva et al., 2006). On the other
hand, serological approaches had shown higher levels of
positivity for antibodies against the 3 variants in Brazilian
endemic and nonendemic areas (Arruda et al., 1996;
Oliveira-Ferreira et al., 2004). VK247 variant was mainly
found as a single infection in West Africa and the Indian
subcontinent. In addition, the majority of the studied
individuals had mixed infections with both variants, the
predominant and VK210 (Kain et al., 1991; Gonzales et al.,
2001). In Southern Mexico, it was observed that all patients
were infected with VK210 and most of them also had VK247
(Rodriguez et al., 2000). All variants were detected in field
isolates from malarious regions of Papua New Guinea,
Indonesia, and Madagascar, although no pure P. vivax-like
isolate was verified (Qari et al., 1991, 1993).
Kain et al. (1992) developed a genotype-specific polymerase chain reaction (PCR) technique by 32P-end–labeled
oligoprobes to detect the VK210 and VK247 variants,
whereas Kho et al. in 1999 investigated the polymorphisms
of the CSP gene in isolates from Korea by the PCR/
restriction fragment length polymorphism (RFLP) technique.
The first methodology developed to identify P. vivax
genotypes was PCR/hybridization, which also uses radiolabeled oligoprobes, but the technique is expensive and
time-consuming, and also requires an adequate laboratorial
structure for elimination of the oligoprobes proper disposal
(Qari et al., 1993). Six years ago, Machado and Póvoa (2000)
optimized the Glass Fiber Membrane (GFM)/PCR/enzymelinked immunosorbent assay (ELISA) method; however, it
needs much time, as well as uses initiating biotinylated
primers and digoxigenin-labeled probes, raising the cost of
the procedure. In 2006, a protocol of nested-PCR/RFLP was
standardized for the diagnosis of 2 of the 3 genotypes:
VK210 and VK247 (Zakeri et al., 2006).
The analysis of RFLPs of PCR products is a fast and
simple technique (Trost et al., 2004) normally used in
molecular biology laboratories in malaria endemic countries,
requiring only basic equipment (Tahar et al., 1998). Here, we
report on the standardization of a new PCR/RFLP for the
identification of the 3 described P. vivax CSP gene variants.
2. Materials and methods
2.1. Samples
For PCR standardization, we used 3 different plasmids
(BlueScript, Stratagene, La Jolla, CA), one for the
characteristic CSP repetitive region of each variant
(VK210, VK247, and P. vivax-like), and 45 frozen plus 10
fresh blood samples collected in different endemic areas of
the Brazilian Amazon region, all with positive results for
P. vivax thick blood films (TBFs). TBFs were examined by
independent experienced microscopists who were unaware of
each result as recommended by the World Health Organization. Furthermore, molecular confirmation of P. vivax was
performed for all samples according to the method described
by Kimura et al. (1997). The protocol for this study was
reviewed and approved by the Ethics Research Board of the
Medicine School in São José do Rio Preto, Brazil.
2.2. Target DNA sequences and design of synthetic
oligonucleotides
DNA was extracted from blood samples by the phenol–
chloroform method (Pena et al., 1991). To amplify the CSP
gene, 2 sets of forward and reverse primers were designed
based on the conserved central portion of the CSP gene.
The CSP sequences are available in the GenBank database
(VK210, accession number 11926; VK247, accession
number M69061; P. vivax-like, accession number
L13724). The sequences were amplified using the following set of primers: PR1 (5′-ACT TTT ATT CGA CTT TGT
TGG TC-3′) and PR2 (5′-ATG GAC TCC ATG CAG TGT
AAC C-3′). The optimal specificity was achieved using the
BLAST program (http://www.ncbi.nlm.nih.gov/BLAST/).
A conformational analysis was made to investigate the
possibility of secondary structure formations (primer
dimer). All oligonucleotide primers were synthesized by
the Integrated DNA Technologies (Coralville, IA).
2.3. PCR standardization
Different PCR conditions were tested, varying PCR mixer
concentrations, primer annealing, and number of cycles.
After optimization, DNA (1.5 μL) was amplified in a total
reaction volume of 25 μL consisting of 1× PCR buffer
(10 mmol/L Tris–HCl, pH 8.3, 50 mmol/L KCl), 1.5 mmol/L
of MgCl2, 1.0 μmol/L of each primer, 200 μmol/L
deoxyribonucleotide triphospate (dNTPs), 2.5 U ampli-Taq
DNA polymerase, 1% betaine, and water (25 μL). Twentyfive cycles of amplification were performed in a thermocycler (DNA MasterCycler, Eppendorf, Hamburg, Germany)
after initial denaturation of DNA at 94 °C for 5 min. Each
cycle consisted of a denaturation step at 93 °C for 60 s, an
annealing step at 41 °C for 90 s, and an extension step at
72 °C for 2 min, with a final extension at 72 °C for 10 min
after the last cycle. The PCR products were analyzed by
electrophoresis using 1.5% agarose gels and stained with
ethidium bromide.
2.4. Restriction digests of PCR products
The selected enzymes were required to have at least 1
cleavage site in the amplification of each variant, resulting in
DNA fragments that are easily visible in polyacrylamide gel.
Restriction digests were set up with 10 μL of PCR product
and 1 U of the respective enzyme (AluI and DpnI, Promega,
R.T. Alves et al. / Diagnostic Microbiology and Infectious Disease 59 (2007) 415–419
417
San Diego, CA), incubated for 1 h at 37 °C. Restriction
fragments were separated by electrophoresis in 12.5%
polyacrylamide gels. The gels were stained with ethidium
bromide and analyzed with a Gel Doc 2000 illuminator (BioRad, Hercules, CA).
2.5. PCR sensitivity threshold
Three P. vivax blood samples from patients with
parasitemia ranging from 300 to 12,500 parasites per
microliter were used. These samples were serially diluted
in blood from an uninfected donor to a final level of
parasitemia corresponding to 10−6 and further processed
for PCR amplification. After that, a parasitologic evaluation was performed to compare the sensitivity among the
PCR products.
2.6. PCR specificity
As a negative control, blood samples obtained from 20
molecularly diagnosed Plasmodium falciparum-infected
patients and from 10 blood donors living in the same areas
with negative molecular results for Plasmodium were used.
2.7. P. vivax CSP gene amplification control
A single amplification of a CSP gene fragment using a set
of previously described oligonucleotide primers (AL60 5′GTC GGA ATT CAT GAA GAA CTT CAT TCT C-3′and
AL61 5′-CAG CGG ATC CTT AAT TGA ATA ATG CTA
GG-3′) was performed for all DNA samples (Machado and
Póvoa, 2000).
3. Results
3.1. Amplification of the P. vivax CSP gene fragment
As shown in Fig. 1, DNA from all samples of P. vivax
included in this study were amplified with the PR1 and PR2
primers. PCR products had lengths of 694 bp (VK210),
721 bp (VK247), to 736 bp (P. vivax-like), as expected from
the BLAST program analysis. No amplifications were observed
Fig. 1. P. vivax CSP gene PCR products. Lane 1, 100-bp DNA ladder
(Invitrogen, Carlsbad, CA); lane 2, VK210 plasmid; lane 3, VK247 plasmid;
lane 4, P. vivax-like plasmid: lanes 5–6, P. vivax DNA from blood samples;
lanes 2–6, P. vivax CSP gene amplified with PR1 and PR2 primers; lanes
7–8, P. vivax CSP gene amplified with AL60 and AL61.
Fig. 2. P. vivax CSP gene RFLP patterns after enzymatic digestion with AluI
(from lanes 2 to 4) and DpnI (from lanes 5 to 7). Lane 1, 50-bp DNA ladder
(Invitrogen); lanes 2 and 5, VK210 plasmid; lanes 3 and 6, VK247 plasmid;
lanes 4 and 7, P. vivax-like plasmid; lane 8, 100-bp DNA ladder.
with human DNA alone or with samples containing only
P. falciparum parasites. The sensitivity of PCR was determined
by serial dilutions of P. vivax blood samples with known
parasitemia. PCR of the P. vivax CSP gene detected levels of
parasitemia corresponding to 0.0069 parasites per microliter.
3.2. RFLP analysis
To distinguish among the 3 P. vivax genotypes, RFLP
using AluI identified fragments of 10, 27, 38, 54, 106, and 135
bp for VK210 and 10, 38, and 673 bp for VK247, whereas
P. vivax-like showed an unique fragment of 10 and 726 bp. In
respect to RFLP, using DpnI, we observed fragments with
sizes of 27, 42, 54, 81, 108, and 301 bp (VK247), whereas
P. vivax-like presented as 2 fragments (39 and 697 bp). The
second enzyme has no restriction site for VK210 (Fig. 2).
Other fragments below 38 bp, not considered for variant
determination, were also formed after the RFLP procedure.
4. Discussion
Malaria is one of the most prevalent severe infectious
diseases in tropical and subtropical regions worldwide.
Because P. vivax malaria has been endemic in many
countries and its CSP genotypes are found worldwide, its
effective diagnosis is very important. Indeed, P. vivax
malaria variants may have different characteristics with
respect to the intensity of symptoms, the response to drugs,
and vector preference, which could cause drug resistance and
failure of control measures (Gopinath et al., 1994).
A new PCR/RFLP system was developed to identify
P. vivax genotypes. PCR primers were designed to amplify
the central immunodominant region of the CSP gene of this
protozoan. In our method, PCR primers were optimized to
achieve easily distinguishable restriction fragments. The
choice of restriction enzymes was also influenced by our
objective of creating an efficient test with optimal resolution
of restriction profiles. Based on the sequence analysis of
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P. vivax variants available in GenBank, the AluI and DpnI
endonucleases were found to be the most suitable enzymes
for this purpose. AluI showed optimal discriminatory power
to distinguish VK210 and P. vivax-like, but it was not adequate
to identify VK247 in mixed infections with the P. vivax-like
variant. We solved this problem by adding a second
restriction step of the PCR product using DpnI, thereby
unequivocally separating the VK247 and P. vivax-like
variants, which allowed us the detection of mixed infections.
The high cost and the need for adequate laboratory
conditions are the most frequently used arguments against
using PCR in developing countries (Torres et al., 2006).
However, PCR-based assays have advantages over microscopic tests because of their great capacity to distinguish P.
vivax genotypes, as all 3 variants are morphologically similar
(Qari et al., 1993). Our method is sensitive and specific to
detect P. vivax variants in both fresh and frozen samples. A
previous PCR/RFLP assay described by Zakeri et al. (2006)
can only detect VK210 and VK247 and is inadequate for
complete large-scale studies. In addition, our methodology
saves time and reduces the cost by more than one-half of the
price of the GFM/PCR/ELISA method developed by
Machado and Póvoa (2000). Moreover, it does not use
oligonucleotide primers labeled with radioisotopes as
described by Qari et al. (1993).
RFLP is more useful in distinguishing P. vivax genotypes
than classifying them by using the CSP gene sequencing
technique (Kho et al., 1999). Finally, the simplicity,
specificity, and sensitivity of the PCR/RFLP system
described here should be sufficient to determine the
prevalence and the distribution of infections of these
P. vivax genotypes in endemic and nonendemic malaria
areas, enabling a better understanding their phylogeny.
Acknowledgments
The authors thank Ana Carolina Silva, Gustavo Capatti,
Valéria Fraga, and Luciana Moran for help in laboratory
work. Financial support was provided by FAPESP (Fundação de Amparo à Pesquisa do Estado de São Paulo, São
Paulo, Brazil, process number 04/15486-7) and CNPq
(Conselho Nacional de Desenvolvimento, Científico e
Tecnológico, Brasília, DE, Brazil, process number 475524/
2004-7). R.T.A. is a Masters student from the Genetic
Program Pos Graduation of the IBILCE/UNESP and has
received research studentship from CNPq.
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