416 R.T. Alves et al. / Diagnostic Microbiology and Infectious Disease 59 (2007) 415–419The proportion of positive sera, specific for the VK210 and fresh blood samples collected in different endemic areas ofVK247 variants, ranged from 28% to 66% in Thailand the Brazilian Amazon region, all with positive results for(Wirtz et al., 1990). Nevertheless, VK247 genotype was P. vivax thick blood films (TBFs). TBFs were examined byidentified in 58% of all patients infected with both genotypes independent experienced microscopists who were unaware of(Kain et al., 1992, 1993). In Brazil, all variants were each result as recommended by the World Health Organiza-genotyped, but only VK210 was found as a single agent of tion. Furthermore, molecular confirmation of P. vivax wasinfection, whereas the other 2 occurred as mixed infections performed for all samples according to the method described(Machado and Póvoa, 2000; Silva et al., 2006). On the other by Kimura et al. (1997). The protocol for this study washand, serological approaches had shown higher levels of reviewed and approved by the Ethics Research Board of thepositivity for antibodies against the 3 variants in Brazilian Medicine School in São José do Rio Preto, Brazil.endemic and nonendemic areas (Arruda et al., 1996;Oliveira-Ferreira et al., 2004). VK247 variant was mainly 2.2. Target DNA sequences and design of syntheticfound as a single infection in West Africa and the Indian oligonucleotidessubcontinent. In addition, the majority of the studied DNA was extracted from blood samples by the phenol–individuals had mixed infections with both variants, the chloroform method (Pena et al., 1991). To amplify the CSPpredominant and VK210 (Kain et al., 1991; Gonzales et al., gene, 2 sets of forward and reverse primers were designed2001). In Southern Mexico, it was observed that all patients based on the conserved central portion of the CSP gene.were infected with VK210 and most of them also had VK247 The CSP sequences are available in the GenBank database(Rodriguez et al., 2000). All variants were detected in field (VK210, accession number 11926; VK247, accessionisolates from malarious regions of Papua New Guinea, number M69061; P. vivax-like, accession numberIndonesia, and Madagascar, although no pure P. vivax-like L13724). The sequences were amplified using the follow-isolate was verified (Qari et al., 1991, 1993). ing set of primers: PR1 (5′-ACT TTT ATT CGA CTT TGT Kain et al. (1992) developed a genotype-specific poly- TGG TC-3′) and PR2 (5′-ATG GAC TCC ATG CAG TGTmerase chain reaction (PCR) technique by 32P-end–labeled AAC C-3′). The optimal specificity was achieved using theoligoprobes to detect the VK210 and VK247 variants, BLAST program (http://www.ncbi.nlm.nih.gov/BLAST/).whereas Kho et al. in 1999 investigated the polymorphisms A conformational analysis was made to investigate theof the CSP gene in isolates from Korea by the PCR/ possibility of secondary structure formations (primerrestriction fragment length polymorphism (RFLP) technique. dimer). All oligonucleotide primers were synthesized byThe first methodology developed to identify P. vivax the Integrated DNA Technologies (Coralville, IA).genotypes was PCR/hybridization, which also uses radi-olabeled oligoprobes, but the technique is expensive and 2.3. PCR standardizationtime-consuming, and also requires an adequate laboratorialstructure for elimination of the oligoprobes proper disposal Different PCR conditions were tested, varying PCR mixer(Qari et al., 1993). Six years ago, Machado and Póvoa (2000) concentrations, primer annealing, and number of cycles.optimized the Glass Fiber Membrane (GFM)/PCR/enzyme- After optimization, DNA (1.5 μL) was amplified in a totallinked immunosorbent assay (ELISA) method; however, it reaction volume of 25 μL consisting of 1× PCR bufferneeds much time, as well as uses initiating biotinylated (10 mmol/L Tris–HCl, pH 8.3, 50 mmol/L KCl), 1.5 mmol/Lprimers and digoxigenin-labeled probes, raising the cost of of MgCl2, 1.0 μmol/L of each primer, 200 μmol/Lthe procedure. In 2006, a protocol of nested-PCR/RFLP was deoxyribonucleotide triphospate (dNTPs), 2.5 U ampli-Taqstandardized for the diagnosis of 2 of the 3 genotypes: DNA polymerase, 1% betaine, and water (25 μL). Twenty-VK210 and VK247 (Zakeri et al., 2006). five cycles of amplification were performed in a thermo- The analysis of RFLPs of PCR products is a fast and cycler (DNA MasterCycler, Eppendorf, Hamburg, Germany)simple technique (Trost et al., 2004) normally used in after initial denaturation of DNA at 94 °C for 5 min. Eachmolecular biology laboratories in malaria endemic countries, cycle consisted of a denaturation step at 93 °C for 60 s, anrequiring only basic equipment (Tahar et al., 1998). Here, we annealing step at 41 °C for 90 s, and an extension step atreport on the standardization of a new PCR/RFLP for the 72 °C for 2 min, with a final extension at 72 °C for 10 minidentification of the 3 described P. vivax CSP gene variants. after the last cycle. The PCR products were analyzed by electrophoresis using 1.5% agarose gels and stained with ethidium bromide.2. Materials and methods 2.4. Restriction digests of PCR products2.1. Samples The selected enzymes were required to have at least 1 For PCR standardization, we used 3 different plasmids cleavage site in the amplification of each variant, resulting in(BlueScript, Stratagene, La Jolla, CA), one for the DNA fragments that are easily visible in polyacrylamide gel.characteristic CSP repetitive region of each variant Restriction digests were set up with 10 μL of PCR product(VK210, VK247, and P. vivax-like), and 45 frozen plus 10 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 417San Diego, CA), incubated for 1 h at 37 °C. Restrictionfragments were separated by electrophoresis in 12.5%polyacrylamide gels. The gels were stained with ethidiumbromide and analyzed with a Gel Doc 2000 illuminator (Bio-Rad, Hercules, CA).2.5. PCR sensitivity threshold Three P. vivax blood samples from patients withparasitemia ranging from 300 to 12,500 parasites permicroliter were used. These samples were serially dilutedin blood from an uninfected donor to a final level ofparasitemia corresponding to 10−6 and further processedfor PCR amplification. After that, a parasitologic evalua-tion was performed to compare the sensitivity among the 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 ladderPCR products. (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.2.6. PCR specificity As a negative control, blood samples obtained from 20molecularly diagnosed Plasmodium falciparum-infected with human DNA alone or with samples containing onlypatients and from 10 blood donors living in the same areas P. falciparum parasites. The sensitivity of PCR was determinedwith negative molecular results for Plasmodium were used. by serial dilutions of P. vivax blood samples with known parasitemia. PCR of the P. vivax CSP gene detected levels of2.7. P. vivax CSP gene amplification control parasitemia corresponding to 0.0069 parasites per microliter. A single amplification of a CSP gene fragment using a set 3.2. RFLP analysisof previously described oligonucleotide primers (AL60 5′-GTC GGA ATT CAT GAA GAA CTT CAT TCT C-3′and To distinguish among the 3 P. vivax genotypes, RFLPAL61 5′-CAG CGG ATC CTT AAT TGA ATA ATG CTA using AluI identified fragments of 10, 27, 38, 54, 106, and 135GG-3′) was performed for all DNA samples (Machado and bp for VK210 and 10, 38, and 673 bp for VK247, whereasPóvoa, 2000). 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), whereas3. Results P. vivax-like presented as 2 fragments (39 and 697 bp). The3.1. Amplification of the P. vivax CSP gene fragment second enzyme has no restriction site for VK210 (Fig. 2). Other fragments below 38 bp, not considered for variant As shown in Fig. 1, DNA from all samples of P. vivax determination, were also formed after the RFLP procedure.included in this study were amplified with the PR1 and PR2primers. PCR products had lengths of 694 bp (VK210), 4. Discussion721 bp (VK247), to 736 bp (P. vivax-like), as expected fromthe BLAST program analysis. No amplifications were observed 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 toFig. 1. P. vivax CSP gene PCR products. Lane 1, 100-bp DNA ladder achieve easily distinguishable restriction fragments. The(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; choice of restriction enzymes was also influenced by ourlanes 2–6, P. vivax CSP gene amplified with PR1 and PR2 primers; lanes objective of creating an efficient test with optimal resolution7–8, P. vivax CSP gene amplified with AL60 and AL61. of restriction profiles. Based on the sequence analysis of
418 R.T. Alves et al. / Diagnostic Microbiology and Infectious Disease 59 (2007) 415–419P. vivax variants available in GenBank, the AluI and DpnI parasite in globally collected blood samples. J Infect Dis 170:endonucleases were found to be the most suitable enzymes 1630–1633. Herrera S, Corradim G, Arévalo-Herrera M (2007) An update on the searchfor this purpose. AluI showed optimal discriminatory power for a Plasmodium vivax vaccine. Trends Parasitol :122–127.to distinguish VK210 and P. vivax-like, but it was not adequate Imwong M, Pukrittayakamee S, Gruner AC, Renia L, Letourneur F,to identify VK247 in mixed infections with the P. vivax-like Looareesuwan S, White NJ, Snounou G (2005) Practical PCRvariant. We solved this problem by adding a second genotyping protocols for Plasmodium vivax using Pvcs and Pvmsp1.restriction step of the PCR product using DpnI, thereby Malar J 27:20. Kain KC, Keystone J, Franke ED, Lanar DE (1991) Global distribution of aunequivocally separating the VK247 and P. vivax-like variant of the circumsporozoite gene of Plasmodium vivax. J Infect Disvariants, which allowed us the detection of mixed infections. 164:208–210. The high cost and the need for adequate laboratory Kain KC, Brown AE, Webster HK, Wirtz RA, Keystone JS, Rodriguez MH,conditions are the most frequently used arguments against Kinahan J, Rowland M, Lanar DE (1992) Circumsporozoite genotypingusing PCR in developing countries (Torres et al., 2006). of global isolates of Plasmodium vivax from dried blood specimens. J Clin Microbiol 30:1863–1866.However, PCR-based assays have advantages over micro- Kain KC, Brown AE, Lanar DE, Ballou WR, Webster HK (1993) Responsescopic tests because of their great capacity to distinguish P. of Plasmodium vivax variants to chloroquine as determined byvivax genotypes, as all 3 variants are morphologically similar microscopy and quantitative polymerase chain reaction. Am J Trop(Qari et al., 1993). Our method is sensitive and specific to Med Hyg 49:478–484. Kho WG, Park YH, Chung JY, Kim JP, Hong ST, Lee WJ, Kim TS, Leedetect P. vivax variants in both fresh and frozen samples. A JS (1999) Two new genotypes Plasmodium vivax circumsporozoiteprevious PCR/RFLP assay described by Zakeri et al. (2006) protein found in the Republic of Korea. Korean J Parasitol 37:can only detect VK210 and VK247 and is inadequate for 265–270.complete large-scale studies. In addition, our methodology Kimura M, Kaneko O, Liu Q, Zhou M, Kawamoto F, Wataya Y,saves time and reduces the cost by more than one-half of the Otani S, Yamaguchi Y, Tanabe K (1997) Identification of the fourprice of the GFM/PCR/ELISA method developed by species of human malaria parasites by nested PCR that targets variant sequences in the small subunit rRNA gene. Parasitol IntMachado and Póvoa (2000). Moreover, it does not use 46:91–95.oligonucleotide primers labeled with radioisotopes as Machado RLD, Póvoa MM (2000) Distribution of Plasmodium vivaxdescribed by Qari et al. (1993). variants (VK210, VK247 and P. vivax-like) in three endemic areas of RFLP is more useful in distinguishing P. vivax genotypes Amazonian Brazil and their correlation with chloroquine-treatment.than classifying them by using the CSP gene sequencing Trans R Soc Trop Med Hyg 94:377–381. Machado RLD, Figueriredo-Filho AF, Calvosa VSP, Figueredo MC,technique (Kho et al., 1999). Finally, the simplicity, Nascimento JM, Póvoa MM (2003) Correlation between Plasmodiumspecificity, and sensitivity of the PCR/RFLP system vivax variants in Belém, Pará State, Brazil and symptoms and clearancedescribed here should be sufficient to determine the of parasitemia. Braz J Infect Dis 7:175–177.prevalence and the distribution of infections of these Oliveira-Ferreira J, Pratt-Riccio LR, Santos M, Ribeiro F, Goldberg CT, Banic AC (2004) HLA class II and antibody responses toP. vivax genotypes in endemic and nonendemic malaria circumsporozoite protein repeats of P. vivax (VK210, VK247 and P.areas, enabling a better understanding their phylogeny. vivax-like) in individuals naturally exposed to malaria. Acta Trop 92: 63–69.Acknowledgments Pena SDJ, Macedo AM, Gontijo NF (1991) DNA bioprints: simple non- isotopic DNA fingerprints with biotinylated probes. Electrophoresis 12: The authors thank Ana Carolina Silva, Gustavo Capatti, 14–52. Qari SH, Goldman IF, Povoa MM, Oliveira S, Alpers MP, Lal AA (1991)Valéria Fraga, and Luciana Moran for help in laboratory Wide distribution of the variant form of the human malaria parasitework. Financial support was provided by FAPESP (Funda- Plasmodium vivax. J Biol Chem 266:16297–16300.ção de Amparo à Pesquisa do Estado de São Paulo, São Qari SH, Shi YP, Goldman IF, Udhayakumar V, Alpers MP, Collins WE, LalPaulo, Brazil, process number 04/15486-7) and CNPq AA (1993) Identification of Plasmodium vivax-like human malaria(Conselho Nacional de Desenvolvimento, Científico e parasite. Lancet 341:780–783. Rieckmann KH, Davis DR, Hutton DC (1989) Plasmodium vivax resistanceTecnológico, Brasília, DE, Brazil, process number 475524/ to chloroquine? Lancet 18:1183–1184.2004-7). 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