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Available online at www.sciencedirect.com Diagnostic Microbiology and Infectious Disease 59 (2007) 415 – 419 www.elsevier.com/locate/diagmicrobio 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. 416 R.T. Alves et al. / Diagnostic Microbiology and Infectious Disease 59 (2007) 415–419 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 418 R.T. Alves et al. / Diagnostic Microbiology and Infectious Disease 59 (2007) 415–419 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. References Arruda ME, Aragaki C, Gagliardi F, Halle RW (1996) A seroprevalence and descriptive epidemiological study of malaria among Indian tribes of the Amazon basin of Brazil. Ann Trop Med Parasitol 90:135–143. Gonzales JM, Hurtado S, Arevalo-Herrera M, Herrera S (2001) Variants of the Plasmodium vivax circumsporozoite protein (VK210 and VK247) in Colombian isolates. Mem Inst Oswaldo Cruz 96:709–712. Gopinath R, Wongsrichanalai C, Cordón-rosales C, Mirabelli L, Kyle D, Kain KC (1994) Failure to detect a Plasmodium vivax-like malaria parasite in globally collected blood samples. 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