TRANSACTIONS THE ROYALSOCIETY TROPICAL OF OF MEDICINEAND HYGIENE(2000) 94,377-381Distribution of Plasmodium vivaxvariants (VK210, VK247 and P. vivax-like) inthree endemic areas of the Amazon region of Brazil and their correlation withchloroquine treatmentRicardo L. D. Machado and Marinete M. l%voa* Laboratdrio de Makiria, ServiGo de Parasitologia, Instituw EvandroChagaslFUNASA, Av. Almirante Barroso 492, 66 090-000 Be&n, Park, Brazil Abstract The present study evaluated the glassfibre membrane (GFM)-polymerase chain reaction (PCR) - enzyme- linked immunosorbent assay(ELISA) technique for genotyping the Plasmodium vivax variants, to verify the distribution of l? vivax variants (VK2 10, VK247 and Z? viva-like) in parts of Brazil and their correlation with levels of parasitaemia, previous malaria experience and clear&e-of parasitaemia linked to different treatment schedules. The samoleswere taken from individuals living in Macar& Port0 Velho and Belem. all ofwhich are endemic areasof&ax malaria in the Amazon region of?Brazil. B&d sampleswere collected on GFMs. The gene that codesfor the circumsporozoite proteins of l? vivaxvariants was amplified by PCRand the amplified fragments were hybridized to variant-specific, digoxigenin-labelled oligonucleotide probes by ELISA. The GFM-PCR-ELISA techniaue was shown to be accurate for eoidemiolonical survevs of the vivax complex. All variants were detected & all 3 areas,but only P. vivax VK2i 0 was fo&d as a sin&e agent of infection, while the other 2 occurred as mixed infections. The l? vivax-like variant was found to be associated with low parasitaemia and VK210 with the highest parasitaemia levels; none of the l? vivux variants was linked with a previous malaria experience. In all casesparasitaemia clearance was identical regarding the type of treatment and consequently it is not possible to confirm the previously reported correlation between I? vivax genotype and response to chloroquine. Keywords: malaria, Plasmodium vivax, variants,VK210, VK247, Plasmodiumv&x-like, epidemiology, chloroquine, glass fibre membranes, polymerase chain reaction, ELISA, Brazilian AmazonIntroduction to be absolute (COLLIGNON, 1994). Reduction in sus- Of the 4 known human malaria parasites, Plasmodium ceptibility to chloroquine was reported from Solomonvivax, I? falciparum, I? malariae, and l? ovale, only the Island (WHITBY et aZ., 1989), Papua New Guineafirst 3 species have been detected in Brazil (QARI et al., (SCHUURKAMPet aZ., 1992; MURPHY et aZ., 1993) and 1993a). During the past 5 years l? vivax has been India (GARG et al., 1995). Studies conducted by KAIN etresponsible for 77~2% of all the reported malaria cases al. (1993a) showed that the responseto chloroquine may (BRAZILIAN MINISTRY OF HEALTH, 1999). vary depending onthe type of Z?vivaxvariant. However, if Although the circumsporozoite protein (CSP) of the there is a relationship between CSP genotype and parasiteinfective sporozoite has been a major target in the clearance following treatment titi &ioroq&e, the development of recombinant malaria vaccines, this underlving mechanism is unknown UZAIN et al.. 1993b). approach has had to be re-evaluated because of the The-development of a sensitive and specific polymei- c&overy of sequence variation in the CSP gene (QARI ase chain reaction (PCR) method for detection of et al.. 1993a; GOPINATH et al.. 1994). Based on the CSP parasite DNA in blood samplesneeds to consider criteriagene; ROSE&BERG al. (1589) described a I? vivax et such as the selection of specific DNA, suitable DNAvariant form (VK247) in Thailand, and QAFU et al. extraction procedure and the conditions of this extrac- (1993a) reported the presence in Papua New Guinea of tion (WILSON et al., 199 1). As the PCR technique yields a human malaria parasite, referred to as ‘l? vivax-like’, amplification of only a single copy of the microiorga- morphologically resembling l? vivax but with the repe- nism’s genes (FOOTEet al., 1989; WILSON et al.. 1989) ittitive sequence of the central region of the CSP differing is regarded as-anexcellentmethdd to detect low levelsoffrom 2 described types of l? vivax. Several studies have DNA (KAIN et al., 1993b). Nevertheless, the majorbeen conducted to evaluate the global distribution of difficultv in the routine use of PCR amolification forvariant VK247: it was detected in indigenous popula- human blood samples is in obtaining an&purifying the tions of Brazil (COCHRANE et al., 199%), in endemic DNA. Furthermore, it is known that haemoglobin and areasof Thailand (WIRTZ et al.. 199 1: KPJN et al.. 1992. the other proteins can inhibit the PCR and that purified 1993a, 1993b), aid in South he&a, Africa (I&N e; DNA can contain traces of denaturing and inhibiting al., 1991), Mexico, Afghanistan and Papua New Guinea proteins (MERCIER et al., 1990; LONG et al., 1995). (KAIN et al., 1992). In addition, blood samples from hiACHAD0 et al. (1998) reported an adaptation of a Panua New Guinea. Indonesia. Brazil and Madagascar method for blood sample collection (WARHURSTet al., weie positive for I? &ax-like GNA (QARI et al., 1593b) 1991) for the extraction and amplification of Plasmodium Serological tests have detected all 3 P. vivax variants in DNA in the diagnosis of malaria infection (O-IRA et samplesfrom SPoPaula State (CUFZADO al., 1995) and et aZ., 1995). This new technique involving glass fibre indigenous communities of the Amazon region of Brazil membrane (GFM), GFM-PCR-ELISA, requires less (ARRUDAet al., 1996, 1998). expertise, saves time and reduces the cost for specific Overthepast30years,widespreadresistanceofmalaria malarial gene sequences. parasites to chloroquine has, so far, been restricted to P. The objective of the present study was to evaluate thefalciparum, and chloroquine still remains the drug of GFM-PCR-ELISA methodology for genotyping the P. choice for both prophylaxis and treatment of P. vivax vivax variants, to verify their distribution in 3 endemic infection (RIECM et al., 1989; BALDASSARRE al., et areas of the Amazon region of Brazil and to correlate 199 1). The first evidence that P. vivax is develoDinrr them with initial parasitaemia, previous malaria experi- resistance to chloroquine was reported in Papua hew ence and the time of parasitaemia clearance linked to Guinea by RIECKMANN et al. (1989). It is difficult to different treatment schedules. ascertain how common chloroquine resistance is in l? vivax infection, particularly asresistance does not appear Materials and Methods Study population Human blood was collected in Macapi (Amapi*Author for correspondence: e-mail email@example.com State), Beltm (Pa& State) and Port0 Velho (RondBnia
378 RICARDO L. D. MACHADO AND MARINETE M. POVOAState). Macapa and BelCm are in the eastern Amazon, amplification was carried out in 50 pL volume utilizingwhile Port0 Velho is in the western Amazon (Figure). prepared DNA (5 pL), with freshly prepared master-mixThe patients were randomly recruited to participate in [33.25 pL of double-distilled water, 0.25 pL (1.25U) ofthis studv at the National Health Foundation units in Taq polymerase (Bioloine, London, UK), 1 pL of eacheach study area, with the assistance of their physicians. dNTP to provide 200 p&i final concentration and 5 uL of 10Xreactionbuffer(10mMtris-HClpH8~3;0~01%(wiBlood sample collection v) gelatin; 1.5 mM MgCl,; 50 mM KCl)] . The biotiny- A questionnaire for information on malaria episodes, lated primer and the unlabelled primer designed frompatient’s identification, epidemiological details, and the specific terminal ends of the l? vivax CSP gene (QARI etmode of treatment was completed for each individual. al., 1993a) were used for amplification of the CSPgene.The samples from MacapP (n = 42) were collected in The reaction mixture was initiallv denatured at 94°C forSentember 1995, from Port0 Velho (n = 33) in Anril 5 min, followed by 30 cycles of amplification (94°C for1996 and from -BelCm (n = 40) during August and 1 min, 42°C for 50 s and 72°C for 90 s) and a finalSeptember 1996, with informed consent from all indivi- extension at 72°C for 5 min. The PCR products (6 @L)duals. The blood samples were obtained before therapy were electrophoresed on a 1% agarose gel and stainedwas initiated, and only from those individuals who had a with ethidium bromide. The PCR products (5 pL) werethick blood film and/or Quantitative Buffy Coat (QBC’) hybridized by liquid-phase non-isotopic method (OLI-microscopically positive for l? vivax parasites and who VERA et al., 1995) for homologous I? vivax variants-had agreed to participate in the study. Vacutainer tubes specific digoxigenin-labelled probes, designed from thecontaining EDTA (Becton Dickinson, UK) were used to CSP repetitive region (QARI et al., 1993a). Primer andcollect 10 mL of blood/individual for PCR typing of l? probe sequences and concentrations were describedvivax and cryopreservation; 100 pL of whole-blood were previously by QAFU et al. (1993a) and the hybridizationused for urenarine the GFM disc (Titertek. ICN Bio- conditions were described by OLIVEIRA et al. (1995). Formedicals &Limited,“UK) following the protocol described negative control we used 2 samples of non-infectedby WARHURST et al. (199 1). human blood and human DNA. Positive controls con- sisted of DNA from 3 different plasmids containing theTreatment CSP repetitive region of variants. Patients from Belem were treated as follows: chlor-oquine 10 mg/kg in a single dose, plus primaquine Statistical analysis0.50 mg/kg during 7 days (scheme A). Those from Port0 The data were organized in a database managerVelho and Macapa were treated with chloroquine 25 mg/ (Dbase III) and later on processed and analysed in ankg over 3 days (10 mg/kg on day 1 and 75 mg/kg on days epidemiology and statistical program (EpiInfo 6.0). For2 and 3), plus primaquine 0.25 mg/kg for 14 days testing the significance of the variables we used the(scheme B) . These were the standard treatments recom- Kruskal-Wallis and Mann-Whitney 2-tailed tests. Tomended by local physicians at each locality. obtain the independence among the proportions, the x2 test was applied with Yates’ correction or Fisher’s exactDNA template preparation, PCR amplification, test (Ztailed). The adopted significance level for statis-electrophoresis and hybridization tical inference was P < 0.05. We used the GFM-PCR-ELISA technique (h&XI-IA-DO et al., 1998). In brief, blood samples obtained directly Resultsfrom patients were spotted on GFM and prepared for All 115 samples collected were both microscopicallyPCR using the method of WARHuRsT et al. ( 199 1). PCR and QBC’ positive for I? vivax. The parasitaemia in theFigure. Study area for the I? &ax variants. Macape (00” 02’20”s; 51” 03’59”W), Belkm (-01” 27’21”s; 48” 30’16”W) andVelho (-08” 45’43”s; 63” 54’14”W).
BRAZILL4N I? VIVAXVARIANTS AND CHEMOTHERAPY 319MacapP patients ranged from 40 to 26 500 infected red previous malaria episodes and the infecting P. vivux typeblood cells/mm3 [geometric mean (GM) 12251; in those (P > 0.05) in all 3 study areas.from Port0 Velho it was from 375 to 6800 (GM 1445);and from Belem 350 to 23 000 (GM 3126). Discussion Samples with parasitaemia <500 infected red blood The existence of a new species or subspecies ofcells/mm3 (n = 30) did not produce a result using the Plasmodium causing human malaria would have impor-PCR-ELISA technique. However, using 5 l.tLofthe first tant implications for diagnosis and vaccine design.PCR products for re-amplification, it was possible to Furthermore, the P. vivax malaria variants may havedemonstrate the same fragment (1.2 Kb) as that ob- different characteristics in the intensity of symptoms, thetained from the samples with a parasitaemia >500 response to the drug treatment and to vector preference, (~1= 85) in agarose gel. which could cause drug resistance and failure of control The typing of l? vivax indicated that the prevalence measures (GOPINATH et al., 1994). It is important,and frequency of variants among the study areas were therefore, to determine the prevalence and distributionnot significantly different from one area to another of infection with these l? vivux genotypes. (P > 0.05), since all types were present in all areas (Table The results of this study have indicated that P. vivax 1). DNA can be detected and genotyped by GFM-PCR- Correlation of the l? r&ax genotype with the highest ELISA, even when parasitaemia is very low. This tech-initial parasitaemia was significant (P < 0.05) for l? nique offers advantages over microscopy for the study of&ax VK210 pure or in mixed infection with VK247, the P. vivax variants because they cannot be distin-whereas the P. vivax-like parasite was associated with the guished morphologically, and the technique is alsolowest initial parasitaemia (Table 2). suitable for epidemiological surveys on the vivax com- Table 2 shows that the time required for parasitaemia plex. clearance determined by thick smear for all l? vivux With regard to the distribution of P. vivax variants wegenotypes was similar, regardless of the treatment used have found the variant VK2 10 to be the most prevalent of (scheme A or B), and the differences did not reach the single variant infections. KAIN et al. (1991) had statistical significance. Table 2 also demonstrates that similar results when analysing samples from South there is no correlation between the mean number of America, West Africa and the Indian subcontinent. ThisTable 1. Distribution of P. vivax genotypes in the three study areas in the Amazon region of Brazil Pure types” Mixed typesStudy area 1 2 3 1+2 1+3 2+3 1+2+3Macapa 1; y;;; 0 0 1 (2.4%) 1 (2.4%) 6 (14.3%) 23 (54.8%)Port0 Velho 0 0 4 (12.1%) 1 (3.0%) 5 (15.2%) 14 (42.4%)Belem 15 (37.5;) 0 0 8 (20.0%) 1 (2.5%) 5 (12.5%) 11 (27.5%)P = 0.23bValues are numbers (%) in each category.;T,ype 1, VK2 10; type 2, VK247; type 3, I? v&xx-like. x. Table 2. P. vivax genotypes and their correlation with initial parasitaemia, past history of malaria and the number of days for the parasitaemia clearance Parasitaemia Previous Total clearance after malaria number of chloroquine and experience’ l? vivux P. vivux Parasitaemiab primaquine (number of genotypes genotype” (parasites/mm3) treatmentc (days) episodes) found 1 6122 f 5999 2.2 f 1.35 1.4 zt 1.98 35 1+2 5880 f 5740 2.2 f 1.09 0.8 3~ 1.42 13 1+2+3 1840 f 1597 2.0 f 1.30 1.9 f 1.95 48 1+3 900 f 540 2.7 dr 1.15 1.7 f 2.88 03 2+3 1506 f 1708 2.0 It 1.15 1.7 f 2.88 16 pi 0.01 0.86 0.39 “Type l,VK210; type 2, VK247; type 3, P. vivux-like. b,cValues are bgeometric means f standard deviation (SD), or cmeans f SD. dKruskal-Wallis test. Mann-Whitney 2-tailed test comparing P. &ax genotype with time for parasite clearance after treatment P value Typelvstypes1+2 0.56 Typelvstypes1+2+3 0.25 Typelvstypes1+3 0.69 Typelvstypes2+3 0.52 Types1+2vstypes1+2+3 0.60 Types1+2vstypes1+3 0.82 Types1+2vstypes2+3 0.93 Types1+2+3vstypes1+3 0.95 Types1+2+3vstypes2+3 0.48 Types1+3vstypes2+3 0.81
380 RICARDO L. D. MACHADO AND MARINETE M. POVOA fact suggests that this variant is well adapted worldwide. tients, but also found no correlation with the genotypesThe VK247 variant has a worldwide distribution and is of I? vivax. As immunity in malaria is directly related tofrequently found as a mixed infection with the VK210 the number of infections, it is likely that our patients hadgenotype (COCHRANE et aZ., 1990; WIRTZ et aZ., 1990; not developed immunity and consequently there was noCURADOetal., lg%?);&RUDAeta~., 1996,1998).While selection of genotypes.this genotype has also been detected as the only kfecting In order to differentiate better the 3 P. vivax geno-agent of malaria in Brazil. Afhanistan. Thailand. Panua types, studies focusing on other genes might providegew Guinea and Mexicd(Q&u etal., i991,199i; FIN further genetic information, enabling a better under-et al., 1992, 1993a, 1993b), we did not find it as a pure standing of their phylogeny and relationship with otherinfection. Studies have shown that pure infection is more human and non-human malaria parasites.frequent in Asia than in South America, suggesting abetter adaptation of this genotype in this continent Acknowledgements (KAIN et al., 1992, 1993a, 1993b; QARI et al., 1992). We thank Josi Maria de Souza Nascimento for technical QARI et al. (1993a, 1993b) drew attention to the l? support and Professor Ralph Lainson, Dr Alexandre Linharesv&x-like parasite and demonstrated the global occur- and Dr Jan E. Conn for critical reading of the manuscript. We thank Dr Shoukat Qari and Altaf La1 (CDC, Atlanta, USA) forrence of this variant in mixed infection, including in the technical support and suggestions. Financial support wasBrazilian Amazon Region. ARRUDA et al. (1996, 1998) provided by the British Council (BRAS/881/161)/CNPq (Pro-and CURADO et al. (1997) had demonstrated by serolo- cess 9101191953), CNPq (Process 830381/95.3), PICD/gical methods the occurrence of this variant co-circulat- CAPES, CDC Atlanta, Wellcome Trust and the Institutoing with other types, but do not indicate whether the Evandro Chagas/FUNASA. R.L.D.M. is a PhD student frominfections were mixed or the antibodies were raised from Universidade Federal do Pari supported by CNPq. The proto-separate single infections. Similar results were obtained col for this study was reviewed and approved by the Researchin the present study. If our results are compared with Board of the Evandro Chagas Institute.those of D. A. Oliveira (personal communication) (39% Referencesof the P. z&z-like variant in mixed infections), we note Arruda,M., Dutra, L., Veiga, E., Souza, R. & Clavijo, P. 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Wide distribution of the 593-595. variant form of the human malaria parasite PZasmodium vivax. Journal of Biological Chemistry, 266,16297-16300.Qari, S. H., Goldman, I. F., Povoa, M. M., Sand, S., Alpers, Received 9 August 1999; revised 19January 2000; accepted MI’. & Lal, A. A. (1992). Polymorphism in the circumspor- for publication 26 January 20001 Announcement ] The Tropical Health and Education Trust (THET) Since THET was established in 1988, it has actively supported training for health care in tropical countries, where resources are limited. THET works with those who are responsible for such training so that their goals can be reached, as they prepare their staff and students for the tasks that they will be called on to do, relevant to the needs of their local community. THET achieves this strategy through collaborative links between a hospital or medical/nursing school in the United Kingdom and its counterpart overseas. 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Trustees: Professor Eldryd Parry (chairman), Professor Roy Anderson, FRS, Professor Brian Greenwood, FRS, Miss Thelma Holland, Mrs Helen Parry, Mr John Rennie FRCS, Mr Richard Southwell, QC and Professor David Warrell. For further information please contact THET, Euston House, 24 Eversholt Street, London NW1 lAD, UK; phone +44 (0)20 7611 870516, fax t-44 (0)20 7611 068314, e-mail firstname.lastname@example.org