Genetic diversity of Plasmodium vivax in a hyperendemic area predominated by Plasmodium falciparum; A preliminary study

Size: px

Start display at page:

Download "Genetic diversity of Plasmodium vivax in a hyperendemic area predominated by Plasmodium falciparum; A preliminary study"

Cleopatra Poole
5 years ago
Views:

1 Tropical Biomedicine 27(3): (2010) Genetic diversity of Plasmodium vivax in a hyperendemic area predominated by Plasmodium falciparum; A preliminary study Dhangadamajhi, G. 1, Rout, B.K. 2, Kar, S.K. 1 and Ranjit, M.R. 1* 1 Regional Medical Research Centre (ICMR), Bhubaneswar , Orissa, India 2 SUM Hospital, Bhubaneswar, Orissa, India *Corresponding author ranjit62@gmail.com Received 28 June 2010; received in revised form 2 August 2010; accepted 8 August 2010 Abstract. Understanding the genetic diversity, extent and distribution of variant forms of Plasmodium vivax parasites is crucial in the development of effective control measures and in Orissa, a hyperendemic state in the eastern part of India, the polymorphic nature of P. vivax isolates is largely lacking. The result of the study analyzing two highly polymorphic single copy genes for P. vivax circumsporozoite protein (pvcs) and P. vivax merozoite surface protein 3α (pvmsp3α) shows that the parasite population is highly heterogenous (33 distinct genotype from 35 isolates) in Orissa. However, the observation of the multiplicity of infection value of 1.34 and high frequency distribution of certain genotype with respect to individual marker (the VK247b allele with a frequency of 0.37; VK210e with 0.25 and VK210c with 0.14) suggests that the parasite population are likely to be under selective pressure and may either be due to preferential production of sporozoites carrying these variants in the available anopheline mosquito species of the state or selection of particular genotypes by host immune pressure. Moreover, although P. vivax in South-East Asia indicates an overall predominance of VK210 which is thought to be the best adapted variant of pvcs repeat type, the almost equal prevalence of both repeat type of pvcs; VK210 and VK247 in the present study is unexpected and needs further study for clarification. INTRODUCTION Malaria is one of the major public health problems in India and although, Plasmodium falciparum has shown an increased incidence during the last few decades, Plasmodium vivax continues to be a predominant species contributing around 60% of total cases of malaria in India (Maestre et al., 2004). According to the National Vector Borne Disease Control Programme (NVBDCP) 2009; Orissa, a hyperendemic state in the eastern part of the country contributes 24.5% of total malaria cases, 40.3% of P. falciparum cases and 18% of all reported deaths due to malaria, which is the highest in India. The climate of the state is favorable for the perennial transmission of malaria in many parts with a seasonal peak from July to October and all four Plasmodium species of human malaria have been reported from the state. Of which, P. falciparum is the major cause of malaria (>80%) followed by P. vivax (10-15%), Plasmodium malariae (up to 3%) in certain foothill areas and Plasmodium ovale being restricted to Koraput district of the state (Ranjit, 2006). In a recent study, we have shown the prevalence of P. malariae up to 44.6% and P.vivax up to 15% during the peak season of malaria transmission while the prevalence of P. falciparum remain unchanged by PCR method of diagnosis (Dhangadamajhi et al., 2009). 578

2 Genetic diversity study of Plasmodium parasite may help to predict the rate of spread of drug resistance mutations or escape from vaccine induced responses and has the practical importance of development and deployment of control strategies (Cui et al., 2003a). Majority of Plasmodium genetic diversity studies have been based on genes coding for antigenic determinants such as circumsporozoite surface protein (csp) and merozoite surface protein (msp) of P. falciparum, which is responsible for the most severe disease (Rich & Ayala, 2000). In recent years, although similar genetic diversity studies on P. vivax field isolates have been carried out in many geographic regions where P. vivax predominates, the study is limited in areas where it is less frequent. Several markers are available for molecular epidemiologic study of P. vivax and includes genes for circumsporozoite protein (pvcs), merozoite surface protein-1 (pvmsp1) merozoite surface protein-3α (pvmsp-3α) and 3β (pvmsp-3β), apical membrane antigen 1 (pvama1) and gametocyte antigen 1 (pvgam1) etc (de Souza-Neiras et al., 2007). However, the first 3 markers have been most widely used for population diversity study of P. vivax due to their highly polymorphic nature. The pvcs gene has a central repeat domain that varies in sequence (VK 210 has a nonapeptide repeat unit Gly-Asp- Arg-Ala- Asp/Ala-Gly-Gln-Pro-Ala and VK247 has Ala-Asn-Gly-Ala-Gly-Asn-Gln-Pro-Gly) and number of repeat units (Rosenberg et al., 1989; Arnot et al., 1985). The VK210 and VK247 have a worldwide distribution and four subtypes from VK210 and two subtypes from VK 247 can be differentiated by restricted enzyme digestion to show polymorphisms in both the pre- and postrepeat region (Imwong et al., 2005). Similarly, the pvmsp-3α gene encodes a merozoite surface protein with an alaninerich central domain that is predicted to form a coiled-coil tertiary structure and is a possible immunogens and vaccine candidate (Galinski et al., 1999). Use of the pvmsp-3α gene as genetic marker has been recently validated (Bruce et al., 1999; Rayner et al., 2002; Cui et al., 2003b; Zakeri et al., 2006). Thus, in the proposed study attempt has been made to study the genetic diversity of P. vivax in a region predominated by P. falciparum analyzing two highly polymorphic single copy genes the pvcs and the pvmsp-3α. MATERIALS AND METHODS The present study was a cross-sectional malaria survey carried out in Orissa during the peak season of malaria transmission in the year 2008 as described previously (Dhangadamajhi et al., 2009). Blood samples were collected from volunteered people complaining of malaria and were screened for the presence of P. vivax by both light microscopy and PCR diagnosis methods. The study was approved by the Ethics Committee of the Regional Medical Research Centre, Bhubaneswar. Genotyping of pvcs and pvmsp-3α Only those samples which were found positive for P. vivax by PCR diagnosis method were genotyped for the two highly polymorphic single copy genes pvcs and pvmsp-3α. Genotyping of pvcs was done by nested PCR followed by PCR-RFLP method as described by Imwong et al. (2005) and that of pvmsp-3α was done following the protocol of Bruce et al. (1999). The DNA fragments obtained for pvcs and pvmsp-3α gene following PCR were analyzed by 1.8% and 0.8% agarose gel electrophoresis respectively. For PCR-RFLP analysis, the digested products of both the genes were applied to 2% agarose gel. In order to see the cross-species amplification of genes, the primers for both of these genes were also used to amplify the DNA of P. falciparum and P. malariae monoinfected samples (five samples from each) and no amplifications were observed. RESULTS A total of 38 P. vivax isolates collected during 2008 (as described by 579

3 Dhangadamajhi et al., 2009) were genotyped for the two single copy genes pvcs and pvmsp 3α. Genotyping of pvcs gene was successful in all the 38 isolates, from which 15 (39.5%) isolates were only VK210 repeat type (with three size variants of approximately a; 680bp, b; 710bp, and c; 740bp), 10 (26.3%) were only VK247 repeat type (with two size variants b and c) and 13 (34.2%) isolates had both repeat types. Thus, 5 different allelic forms of pvcs were detected by simple analysis of repeat type and fragment size. The b variant dominated in 82.1% (23/28) of VK210 repeat type and the c variant 86.9% (20/23) in VK247 type. The pre- and post- repeat analysis by PCR- RFLP method further divided these 5 different pvcs variants into 11 different allelic types (Table 1), 8 for VK210 and 3 for VK 247.Of the 38 samples analyzed, a total of 51 bands were observed with mean multiplicity of infection (MOI) to be 1.34 and the frequency distribution of the pvcs allele showed the highest frequency of 0.25 and 0.14 for VK210e and VK210c of VK210 pvcs type respectively. For VK 247 repeat type, the VK247b allele represented the highest frequency of 0.37 and was the predominant allele of the samples analyzed (Table 1). Genotyping of pvmsp3α was successful in 37 of the isolates (represented by three size variants A; 1100bp B; 1500bp C; 1900bp), from which two isolates identified as mixed genotype infection by pvcs with MOI of 2 for each showed mixed infections by pvmsp3α as well. The frequency of A allele was 72.9% (27/37), B was 13.5% (5/37) and C was 18.9% (7/37). The PCR-RFLP analysis of the samples showed 3 different Alu I patterns and 4 different Hha I patterns (Figure 1). The combinational analysis of the size variants and RFLP patterns differentiated 16 different alleles from 35 isolates (2 samples with mixed infections were excluded since the sum of RFLP fragments sizes was less than that of uncut product), all of which were distributed randomly with allele A al1 h1 had a highest frequency of Combined genotype analysis of the two loci pvcs and pvmsp3α revealed that the P. vivax population of the state is genetically highly diverse and a total of 33 distinct genotypes were enumerated from 35 isolates and the most common two marker genotype were VK210e A a1 h1 and VK247b A a1 h1 which occurred at a frequency of (3/35) each. Table 1. Allele frequency of Pvcs variants classed by repeat type, size and presence of pre- and post repeat insertions Sl no. Allele Size variants Pre/Post repeat N (frequency) 1 VK 210a a N/Y 01 (0.02) 2 VK 210b a Y/N 01 (0.02) 3 VK 210c b N/Y 07 (0.14) 4 VK 210d b Y/Y 03 (0.06) 5 VK 210e b Y/N 13 (0.25) 6 VK 210f b N/N 01 (0.02) 7 VK 210g c N/Y 01 (0.02) 8 VK 210h c Y/N 01 (0.02) 9 VK 247a b Y 03 (0.06) 10 VK 247b c Y 19 (0.37) 11 VK 247c c N 01 (0.02) Note: Y; presence of pre/post repeat region, N; Absence of pre/post repeat region 580

4 Figure 1. PCR-RFLP patterns of P. vivax isolates based on pvmsp 3α. (A) The amplified products were digested with Hha I and the lane labeled with M represents 100bp ladder, L1 represents h1, L2 and L4 represents h4, L3 represents h2 and L5 represents h3 type of banding patterns. (B) The amplified products were digested with AluI and the lane M represents 100bp ladder, L1 and L4 represents a1, L2 and L5 represents a2 and L3 represents a3 type of banding patterns DISCUSSION The genetic diversity of Plasmodium species depend on complex interplay among themselves, between parasite-host and/or parasite-vector and varies considerably in different geographic regions. Although numerous genetic diversity studies on P. vivax field isolates have been carried out in many geographic regions where it predominates, its population structure and distribution is limited in areas where it is less frequent. The present study shows the genetic diversity of P. vivax in a region where its frequency distribution is less and mostly predominated by P. falciparum and P. malariae (Dhangadamajhi et al., 2009). The result of the study analyzing two highly polymorphic single copy genes pvcs and pvmsp3α shows that the parasite population is highly heterogenous (33 distinct genotype from 35 isolates). Earlier genetic analysis studies with respect to pvcs gene have demonstrated a global distribution of the two variant pvcs types; VK210 and VK247 with prevalence of individual genotype that varies geographically (Kain et al., 1992; Cui et al., 2003b; Zakeri et al., 2010 a & b). In general, P. vivax in South-East Asia indicates an overall predominance of VK210 (Kho et al., 1999; Kim et al., 2006; Moon et al., 2009). Further, VK210 type is prevalent in all geographical isolates and thought to be the best adapted variant (Machado & Póvoa, 2000) as supported by the observation of significant decrease over time in the proportions of VK247 in a two-year investigation performed in a malariahyperendemic area of Papua New Guinea in both the mosquito and human populations (Burkot et al., 1992). Contrast to this, in Mexico, a clear shift from VK210 to VK247 infections had been observed from 1996 to 1998 and the prevalence of pvcs phenotype appeared to be dependent on altitudes with VK210 being more prevalent in coastal region, VK247 in region above 170 meters from sea level and both phenotypes in region with intermediate altitudes (Rodriguez et al., 2000). However, the 581

5 result of the present study demonstrated that both VK210 and VK247 are equally prevalent in the state and is in contrast to the study in Kolkata (West Bengal, a neighbor state of Orissa), India where VK210 was the predominat pvcs type (99.3%).While the reasons for geographical variation of pvcs variants in India are unknown, they may reflect (i) differences in vector composition and variability in vector competence for the different genotypes of P. vivax as different anopheline mosquitoes were found to be differentially susceptible to infections by VK210 and VK247 variants (Gonzalez- Ceron et al., 1999) (ii) transmission intensity and variation in frequency distribution of Plasmodium species in these regions, (iii) ethnic differences of human host or (iv) temporal population dynamics of different mosquito vectors which are not equally susceptible to infections by VK210 or VK247 parasite isolates (Rodriguez et al., 2000). Moreover, although, the diversity of P. vivax parasite could be well differentiated by the combined genotype analysis, the parasite population appeared to be under selective pressure as evident by their high frequency distribution of certain genotype with respect to individual marker. Further, the observation of the multiplicity of infection value of 1.34 suggesting the transmission intensity to be considerably high, the frequency distribution results showed that of the 11 different pvcs allelic types, the VK247b allele was the predominant of all with a frequency of 0.37 followed by VK210e (0.25) and VK210c (0.14) of VK210 pvcs type respectively and may either be due to preferential production of sporozoites carrying these variants in the available mosquito species of the state or selection of particular genotypes by host immune pressure. In conclusion, the high levels of parasite population diversity and the high frequency observation of certain pvcs genotype belonging to both type of repeat units (VK210 and VK247) suggest that the parasite population are likely to be under selective pressure and may either be due to preferential production of sporozoites carrying these variants or selection of particular genotypes by host immune pressure. Thus, the development of recombinant malaria vaccines which includes the VK210, VK247 and any other variant CS epitopes may be effective in controlling P. vivax infections in this part and deserve further study for successful development and deployment of control strategies. Acknowledgements. We thank Mr. H.S. Nayak Lab Tech and Mr. Abhimanyu Das Lab Tech for their assistance in sample collection and microscopic examination of blood slides. We thankfully acknowledge National Vector Borne and Disease Control Programme, Government of Orissa for financial support and to the Director RMRC, Bhubaneswar for providing necessary laboratory facilities for the study. The authors also acknowledge Council of Scientific and Industrial Research, New Delhi for providing fellowship to Mr G Dhangadamajhi to carry out the research work. The authors are grateful to the patients who participated in the study. REFERENCES Arnot, D.E., Barnwell, J.W., Tam, J.P., Nussenzweig, V., Nussenzweig, R. & Enea, V. (1985). Circumsporozoite protein of Plasmodium vivax gene cloning and characterization of the immunodominant epitope. Science 230: Bruce, M.C., Galinski, M.R., Barnwell, J.W., Snounou, G. & Day, K.P. (1999). Polymorphism at the merozoite surface protein-3 alpha locus of Plasmodium vivax; global and local diversity. American Journal of Tropical Medicine and Hygiene 61: Burkot, T.R., Wirtz, R.A., Paru, R., Garner, P. & Alpers, M.P. (1992). The population dynamics in mosquitoes and humans of two Plasmodium vivax polymorphs distinguished by different circumsporozoite protein repeat regions 582

6 American Journal of Tropical Medicine and Hygiene 47: Cui, L., Escalante, A.A., Imwong, M. & Snounou, G. (2003a). The genetic diversity of Plasmodium vivax populations. Trends in Parasitology 19: Cui, L., Mascorro, C.N., Fan, Q., Rzomp, K.A., Khuntirat, B., Zhou, G., Chen, H., Yan, G. & Sattabongkot, J. (2003b). Genetic diversity and multiple infections of Plasmodium vivax malaria in Western Thailand. American Journal of Tropical Medicine and Hygiene 68: de Souza-Neiras, W.C., de Melo, L.M. & Machado, R.L. (2007). The genetic diversity of Plasmodium vivax-a review. Memórias do Instituto Oswaldo Cruz 102: Dhangadamajhi, G., Kar, S.K. & Ranjit, M.R. (2009). High prevalence and gender bias in distribution of Plasmodium malariae infection in central east-coast India. Tropical Biomedicine 26: Galinski, M.R., Corredor-Medina, C., Povoa, M., Crosby, J., Ingravallo, P. & Barnwell, J.W. (1999). Plasmodium vivax merozoite surface protein-3 contains coiled-coil motifs in an alanine-rich central domain. Molecular and Biochemical Parasitology 101: González-Cerón, L., Rodriguez, M.H., Nettel, J.A., Villarreal, C., Kain, K.C. & Hernández, J.E. (1999). Differential susceptibility of Anopheles albimanus and Anopheles pseudopunctipennis to infections with coindigenous Plasmodium vivax variants VK210 and VK247 in southern Mexico. Infection and Immunity 67: Imwong, M., Pukrittayakamee, S., Gruner, A.C., Renia, L., Letourneur, F., Looareesuwan, S., White, N.J. & Snounou, G. (2005). Practical PCR genotyping protocols for Plasmodium vivax using Pvcs and Pvmsp1. Malaria Journal 4: 20. Kain, K.C., Brown, A.E., Webster, H.K., Wirtz, R.A., Keystone, J.S., Rodriguez, M.H., Kinahan, J., Rowland, M. & Lanar, D.E. (1992). Circumsporozoite genotyping of global isolates of Plasmodium vivax from dried blood specimens. Journal of Clinical Microbiology 30: Kho, W.G., Park, Y.H., Chung, J.Y., Kim, J.P., Hong, S.T., Lee, W.J., Kim, T.S. & Lee, J.S. (1999). Two new genotypes of Plasmodium vivax circumsporozoite protein found in the republic of Korea. Korean Journal of Parasitology 37: Kim, J.R., Imwong, M., Nandy, A., Chotivanich, K., Nontprasert, A., Tonomsing, N., Maji, A., Addy, M., Day, N.P., White, N.J. & Pukrittayakamee, S. (2006). Genetic diversity of Plasmodium vivax in Kolkata, India. Malaria Journal 5: 71. Machado, R.L. & Póvoa, M.M. (2000). Distribution of Plasmodium vivax variants (VK210, VK247 and P. vivaxlike) in three endemic areas of the Amazon region of Brazil and their correlation with chloroquine treatment. Transaction of Royal Society of Tropical Medicine and Hygiene 94(4): Maestre, A., Sunil, S., Ahmad, G., Mohmmed, A., Echeverri, M., Corredor, M., Blair, S., Chauhan, V.S. & Malhotra, P. (2004). Inter-allelic recombination in the Plasmodium vivax merozoite surface protein 1 gene among Indian and Colombian isolates. Malaria Journal 3: 4. Moon, S.U., Lee, H.W., Kim, J.Y., Na, B.K., Cho, S.H., Lin, K., Sohn, W.M. & Kim, T.S. (2009). High frequency of genetic diversity of Plasmodium vivax field isolates in Myanmar. Acta Tropica 109(1): Ranjit, M.R. (2006). The epidemiology of malaria in Orissa. Indian Council of Medical Research Bulletin 36:

7 Rayner J.C., Corredor, V., Feldman, D., Ingravallo, P., Iderabdullah, F., Galinski, M.R. & Barnwell, J.W. (2002). Extensive polymorphism in the Plasmodium vivax merozoite surface coat protein MSP-3 alpha is limited to specific domains. Parasitology 125: Rich, S.M. & Ayala, F.J. (2000). Population structure and recent evolution of Plasmodium falciparum. Proceedings of the National Academy of Sciences USA 97: Rodriguez, M.H., Gonzalez-Ceron, L., Hernandez, J.E., Nettel, J.A., Villarreal, C., Kain, K.C. & Wirtz, R.A. (2000). Different prevalence of Plasmodium vivax phenotypes VK210 and VK247 associated with the distribution of Anopheles albimanus and Anopheles pseudopunctipennis in Mexico. American Journal of Tropical Medicine and Hygiene 62: Rosenberg, R., Wirtz, R.A., Lanar, D.E., Sattabongkot, J., Hall, T., Waters, A.P. & Prasittisuk, C. (1989). Circumsporozoite protein heterogeneity in the human malaria parasite Plasmodium vivax. Science 245: Zakeri, S., Barjesteh, H. & Djadid, N.D. (2006). Merozoite surface protein- 3alpha is a reliable marker for population genetic analysis of Plasmodium vivax. Malaria Journal 5: 53. Zakeri, S., Raeisi, A., Afsharpad, M., Kakar, Q., Ghasemi, F., Atta, H., Zamani, G., Memon, M.S., Salehi, M. & Djadid, N.D. (2010a). Molecular characterization of Plasmodium vivax clinical isolates in Pakistan and Iran using pvmsp-1, pvmsp-3α and pvcsp genes as molecular markers. Parasitology International 59: Zakeri, S., Safi, N., Afsharpad, M., Butt, W., Ghasemi, F., Abouie.Mehrizi, A., Atta, H., Zamani, G. & Djadid, N.D. (2010b). Genetic structure of Plasmodium vivax isolates from two malaria endemic areas in Afghanistan. Acta Tropica 113:

Partial Sequence Analysis of Merozoite Surface Proteine-3α Gene in Plasmodium vivax Isolates from Malarious Areas of Iran

Iranian J Parasitol: Vol. 3, No.4, 2008, pp. 1-8 Original Article Partial Sequence Analysis of Merozoite Surface Proteine-3α Gene in Plasmodium vivax Isolates from Malarious Areas of Iran A Shahbazi 1,2,