World Health Organization, 2009. Report. Available at: http://www.who.int/malaria/world_malaria_report_2009/en/index.html.
Roberts L, Enserink M, 2007. Did they really say eradication? Science 318: 1544–1545.
Tolia N, Enemark EJ, Sim BKL, Joshua-Tor L, 2005. Structural basis for the EBA-175 erythrocyte invasion pathway of the malaria parasite Plasmodium falciparum. Cell 122: 183–193.
Miller LH, Roberts T, Shahabuddin M, McCutchan TF, 1993. Analysis of sequence diversity in the Plasmodium falciparum merozoite surface protein-1 (MSP-1). Mol Biochem Parasitol 59: 1–14.
Takala SL, Plowe CV, 2009. Genetic diversity and malaria vaccine design, testing and efficacy: preventing and overcoming ‘vaccine resistant malaria'. Parasite Immunol 31: 560–573.
Ferreira M, da Silva Nunes M, Wunderlich G, 2004. Antigenic diversity and immune evasion by malaria parasites. Clin Diagn Lab Immunol 11: 987–995.
Luxemburger C, Thwai KL, White NJ, Webster HK, Kyle DE, Maelankirri L, Chongsuphajaisiddhi T, Nosten F, 1996. The epidemiology of malaria in a Karen population on the western border of Thailand. Trans R Soc Trop Med Hyg 90: 105–111.
Molineaux L, Gramiccia G, 1980. The Garki Project, Research on the Epidemiology and Control of Malaria in the Sudan Savanna of West Africa. Geneva: World Health Organization.
Theander T, 1998. Unstable malaria in Sudan. The influence of the dry season. Trans R Soc Trop Med Hyg 92: 589–592.
Anderson TJ, Haubold B, Williams JT, Estrada-Franco JG, Richardson L, Mollinedo R, Bockarie M, Mokili J, Mharakurwa S, French N, Whitworth J, Velez ID, Brockman AH, Nosten F, Ferreira MU, Day KP, 2000. Microsatellite markers reveal a spectrum of population structures in the malaria parasite Plasmodium falciparum. Mol Biol Evol 17: 1467–1482.
Chenet S, Branch OH, Escalante AA, Lucas CM, Bacon JD, 2007. Genetic diversity of vaccine candidate antigens in Plasmodium falciparum isolates from the Amazon basin of Peru. Malar J 7: 93.
Takala SL, Coulibaly D, Thera MA, Dicko A, Smith DL, Guindo AB, Kone AK, Traore K, Ouattara A, Djimde AA, Sehdev PS, Lyke KE, Diallo DA, Doumbo OK, Plowe CV, 2007. Dynamics of polymorphism in a malaria vaccine antigen at a vaccine-testing site in Mali. PLoS Med 4: e93.
Adams JH, Sim BK, Dolan SA, Fang X, Kaslow DC, Miller LH, 1992. A family of erythrocyte binding proteins of malaria parasites. Proc Natl Acad Sci USA 89: 7085–7089.
Sim BK, Orlandi PA, Haynes JD, Klotz FW, Carter JM, Camus D, Zegans ME, Chulay JD, 1990. Primary structure of the 175K Plasmodium falciparum erythrocyte binding antigen and identification of a peptide which elicits antibodies that inhibit malaria merozoite invasion. J Cell Biol 111: 1877–1884.
Toure F, Bisseye C, Mavoungou E, 2006. Imbalanced distribution of Plasmodium falciparum EBA-175 genotypes related to clinical status in children from Bakoumba, Gabon. Clin Med Res 4: 7–11.
Binks RH, Baum J, Oduola AM, Arnot DE, Babiker HA, Kremsner PG, Roper C, Greenwood BM, Conway DJ, 2001. Population genetic analysis of the Plasmodium falciparum erythrocyte binding antigen-175 (eba-175) gene. Mol Biochem Parasitol 114: 63–70.
Cramer JP, Mockenhaupt FP, Mohl I, Dittrich S, Dietz E, Otchwemah RN, Ehrhardt S, Bienzle U, Jelinek T, 2004. Allelic dimorphism of the erythrocyte binding antigen-175 (eba-175) gene of Plasmodium falciparum and severe malaria: significant association of the C-segment with fatal outcome in Ghanaian children. Malar J 3: 11.
Coley AM, Gupta A, Murphy VJ, Ba T, Kim H, Foley M, Anders RF, Batchelor AH, 208. Structure of the malaria antigen AMA1 in complex with a growth-inhibitory antibody. PLoS Pathog 3: 1308–1319.
Escalante A, Grebert HM, Chaiyaroj SC, Magris M, Biswas S, Nahlen BL, Lal AA, 2001. Polymorphism in the gene encoding the apical membrane antigen-1 (AMA-1) of Plasmodium falciparum. X. Asembo Bay Cohort Project. Mol Biochem Parasitol 113: 279–287.
Marshall V, Zhang L, Anders RF, Coppel RL, 1996. Diversity of the vaccine candidate AMA-1 of Plasmodium falciparum. Mol Biochem Parasitol 77: 109–113.
Kocken CH, Narum DL, Massougbodji A, Ayivi B, Dubbeld MA, van der Wel A, Conway DJ, Sanni A, Thomas AW, 2000. Molecular characterisation of Plasmodium reichenowi apical membrane antigen-1 (AMA-1), comparison with P. falciparum AMA-1, and antibody-mediated inhibition of red cell invasion. Mol Biochem Parasitol 109: 147–156.
Garg S, Alam MT, Das MK, Dev V, Kumar A, Dash AP, Sharma YD, 2007. Sequence diversity and natural selection at domain I of the apical membrane antigen 1 among Indian Plasmodium falciparum populations. Malar J 6: 154.
Oeuvray C, Bouharoun-Tayoun H, Gras-Masse H, Bottius E, Kaidoh T, Aikawa M, Filgueira MC, Tartar A, Druilhe P, 1994. Merozoite surface protein-3: a malaria protein inducing antibodies that promote Plasmodium falciparum killing by cooperation with blood monocytes. Blood 84: 1594–1602.
McColl D, Anders RF, 1997. Conservation of structural motifs and antigenic diversity in the Plasmodium falciparum merozoite surface protein-3 (MSP-3). Mol Biochem Parasitol 90: 21–31.
Oeuvray C, Bouharoun-Tayoun H, Grass-Masse H, Lepers JP, Ralamboranto L, Tartar A, Druilhe P, 1994. A novel merozoite surface antigen of Plasmodium falciparum (MSP-3) identified by cellular-antibody cooperative mechanism antigenicity and biological activity of antibodies. Mem Inst Oswaldo Cruz 89 (Suppl 2): 77–80.
Huber W, Felger I, Matile H, Lipps HJ, Steiger S, Beck HP, 1997. Limited sequence polymorphism in the Plasmodium falciparum merozoite surface protein 3. Mol Biochem Parasitol 87: 231–234.
Okenu DM, Thomas AW, Conway DJ, 2000. Allelic lineages of the merozoite surface protein 3 gene in Plasmodium reichenowi and Plasmodium falciparum. Mol Biochem Parasitol 109: 185–188.
Nebie I, Diarra A, Ouedraogo A, Soulama I, Bougouma EC, Tiono AB, Konate AT, Chilengi R, Theisen M, Dodoo D, Remarque E, Bosomprah S, Milligan P, Sirima SB, 2008. Humoral responses to Plasmodium falciparum blood-stage antigens and association with incidence of clinical malaria in children living in an area of seasonal malaria transmission in Burkina Faso, West Africa. Infect Immun 76: 759–766.
Samba-Kimbata J, 1978. Le Climat du bas Congo. Thèse Doctorat 3ème Cycle, Dijon.
Fondjo E, Robert V, Le Goff G, Toto JC, Carnevale P, 1992. Urban malaria in Yaounde (Cameroon). 2. Entomologic study in 2 suburban districts [in French]. Bull Soc Pathol Exot 85: 57–63.
Baird JK, Owusu Agyei S, Utz GC, Koram K, Barcus MJ, Jones TR, Fryauff DJ, Binka FN, Hoffman SL, Nkrumah FN, 2002. Seasonal malaria attack rates in infants and young children in northern Ghana. Am J Trop Med Hyg 66: 280–286.
Koram KA, Quaye L, Abuaku B, 2008. Efficacy of amodiaquine/artesunate combination therapy for uncomplicated malaria in children under five years in Ghana. Ghana Med J 42: 55–60.
Bereczky S, Martensson A, Gil JP, Farnert A, 2005. Short report: rapid DNA extraction from archive blood spots on filter paper for genotyping of Plasmodium falciparum. Am J Trop Med Hyg 72: 249–251.
Toure F, Mavoungou E, Ndong JM, Tshipamba P, Deloron P, 2001. Erythrocyte binding antigen (EBA-175) of Plasmodium falciparum: improved genotype determination by nested polymerase chain reaction. Trop Med Int Health 6: 767–769.
Peakall R, Smouse PE, 2005. Genalex 6: genetic analysis in Excel. Population genetic software for teaching and research. Mol Ecol Notes 6: 288–295.
Excoffier L, Estoup A, Cornuet JM, 2005. Bayesian analysis of an admixture model with mutations and arbitrarily linked markers. Genetics 169: 1727–1738.
Orloci L, 1978. Multivariate Analysis in Vegetation Research. The Hague, The Netherlands: Dr W. Junk b.v.
Conway DJ, 1997. Natural selection on polymorphic malaria antigens and the search for a vaccine. Parasitol Today 13: 26–29.
Soulama I, Bougouma EC, Diarra A, Nebié I, Sirima SB, 2010. Low-high season variation in Plasmodium falciparum erythrocyte binding antigen 175 (eba-175) in malaria endemic area of Burkina Faso. Trop Med Int Health 15: 51–59.
Ahouidi A, Bei AK, Neafsey DE, Sarr O, Volkman S, Milner D, Cox-Singh J, Ferreira MU, Ndir O, Premji Z, Mboup S, Duraisingh MT, 2010. Population genetic analysis of large sequence polymorphisms in Plasmodium falciparum blood-stage antigens. Infect Genet Evol 10: 200–206.
Polley SD, Tetteh KK, Lloyd JM, Akpogheneta OJ, Greenwood BM, Bojang KA, Conway DJ, 2007. Plasmodium falciparum merozoite surface protein 3 is a target of allele-specific immunity and alleles are maintained by natural selection. J Infect Dis 195: 279–287.
Umar F, Malla N, Dubey ML, 2009. Genetic polymorphism in msp-2, ama-1 and csp genes in Plasmodium falciparum field isolates from north and north-western India. J Vector Borne Dis 46: 109–116.
Oliveira DA, Udhayakumar V, Bloland P, Shi YP, Nahlen BL, Oloo AJ, Hawley WE, Lal AA, 1996. Genetic conservation of the Plasmodium falciparum apical membrane antigen-1 (AMA-1). Mol Biochem Parasitol 76: 333–336.
Wang J, Nguyen V, Glen J, Henderson B, Saul A, Miller LH, 2005. Improved yield of recombinant merozoite surface protein 3 (MSP3) from Pichia pastoris using chemically defined media. Biotechnol Bioeng 90: 838–847.
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The malaria vaccine candidate antigens erythrocyte binding antigen 175 (EBA-175), merozoite surface protein 3 (MSP-3), and apical membrane antigen (AMA-1) from Plasmodium falciparum isolates from countries in central and west Africa were assessed for allelic diversity. Samples were collected on filter paper from 600 P. falciparum-infected symptomatic patients in Cameroon, Republic of Congo, Burkina Faso, Ghana, and Senegal and screened for class-specific amplification fragments. Genetic diversity, assessed by mean heterozygosity, was comparable among countries. We detected a clinical increase in eba 175 F-allele frequency from west to east across the study region. No statistical difference in msp-3 allele distribution between countries was observed. The ama-1 3D7 alleles were present at a lower frequency in central Africa than in West Africa. We also detected little to no genetic differentiation among sampling locations. This finding indicates that, at least at the level of resolution offered by restriction fragment length polymorphism analysis, these antigens showed remarkable genetic homogeneity throughout the region sampled, perhaps caused by balancing selection to maintain a diverse array of antigen haplotyes.
Financial support: This study was supported by the Division of Microbiology and Infectious Disease, National Institutes of Allergy and Infectious Diseases, National Institutes of Health (Bethesda, MD) (Contract HHSN266200400016C) awarded to the Noguchi Memorial Institute for Medical Research, University of Ghana and subcontracted to the Centre National de Recherche et de Formation sur le Paludisme, Burkina Faso.
Authors' addresses: Issiaka Soulama and Edith C. Bougouma, Centre National de Recherche et de Formation sur le Paludisme, Ouagadougou, Burkina Faso, E-mails: a.soulama.cnrfp@fasonet.bf and eddy.cnrfp@fasonet.bf. Jude D. Bigoga, Department of Biochemistry and the Biotechnology Center, University of Yaounde I, Yaounde, Cameroon, E-mail: jbigoga@gmail.com. Magatte Ndiaye, Service de Parasitologie–Mycologie, Université Cheikh Anta Diop, Dakar, Senegal, E-mail: magou22000@yahoo.fr. Josephine Quagraine, Department of Parasitology Noguchi Memorial Institute for Medical Research, University of Ghana, Legon, Ghana, E-mail: Quagraine@noguchi.mimcom.org. Prisca N. Casimiro, Centre d'Etudes des Ressources Végétales, Unité de Pharmacologie, Brazzaville, Republic of Congo, E-mail: pncasimiro@yahoo.fr. Timothy T. Stedman, Malaria Research and Reference Reagent Resource Center, American Type Culture Collection, Manassas, VA, E-mail: TStedman@atcc.org. Sodiomon B. Sirima, Centre National de Recherche et de Formation sur le Paludisme, Ouagadougou, Burkina Faso and Groupe d'Action et de Recherche en Santé, Belgium, E-mail: s.sirima.cnlp@fasonet.bf.
World Health Organization, 2009. Report. Available at: http://www.who.int/malaria/world_malaria_report_2009/en/index.html.
Roberts L, Enserink M, 2007. Did they really say eradication? Science 318: 1544–1545.
Tolia N, Enemark EJ, Sim BKL, Joshua-Tor L, 2005. Structural basis for the EBA-175 erythrocyte invasion pathway of the malaria parasite Plasmodium falciparum. Cell 122: 183–193.
Miller LH, Roberts T, Shahabuddin M, McCutchan TF, 1993. Analysis of sequence diversity in the Plasmodium falciparum merozoite surface protein-1 (MSP-1). Mol Biochem Parasitol 59: 1–14.
Takala SL, Plowe CV, 2009. Genetic diversity and malaria vaccine design, testing and efficacy: preventing and overcoming ‘vaccine resistant malaria'. Parasite Immunol 31: 560–573.
Ferreira M, da Silva Nunes M, Wunderlich G, 2004. Antigenic diversity and immune evasion by malaria parasites. Clin Diagn Lab Immunol 11: 987–995.
Luxemburger C, Thwai KL, White NJ, Webster HK, Kyle DE, Maelankirri L, Chongsuphajaisiddhi T, Nosten F, 1996. The epidemiology of malaria in a Karen population on the western border of Thailand. Trans R Soc Trop Med Hyg 90: 105–111.
Molineaux L, Gramiccia G, 1980. The Garki Project, Research on the Epidemiology and Control of Malaria in the Sudan Savanna of West Africa. Geneva: World Health Organization.
Theander T, 1998. Unstable malaria in Sudan. The influence of the dry season. Trans R Soc Trop Med Hyg 92: 589–592.
Anderson TJ, Haubold B, Williams JT, Estrada-Franco JG, Richardson L, Mollinedo R, Bockarie M, Mokili J, Mharakurwa S, French N, Whitworth J, Velez ID, Brockman AH, Nosten F, Ferreira MU, Day KP, 2000. Microsatellite markers reveal a spectrum of population structures in the malaria parasite Plasmodium falciparum. Mol Biol Evol 17: 1467–1482.
Chenet S, Branch OH, Escalante AA, Lucas CM, Bacon JD, 2007. Genetic diversity of vaccine candidate antigens in Plasmodium falciparum isolates from the Amazon basin of Peru. Malar J 7: 93.
Takala SL, Coulibaly D, Thera MA, Dicko A, Smith DL, Guindo AB, Kone AK, Traore K, Ouattara A, Djimde AA, Sehdev PS, Lyke KE, Diallo DA, Doumbo OK, Plowe CV, 2007. Dynamics of polymorphism in a malaria vaccine antigen at a vaccine-testing site in Mali. PLoS Med 4: e93.
Adams JH, Sim BK, Dolan SA, Fang X, Kaslow DC, Miller LH, 1992. A family of erythrocyte binding proteins of malaria parasites. Proc Natl Acad Sci USA 89: 7085–7089.
Sim BK, Orlandi PA, Haynes JD, Klotz FW, Carter JM, Camus D, Zegans ME, Chulay JD, 1990. Primary structure of the 175K Plasmodium falciparum erythrocyte binding antigen and identification of a peptide which elicits antibodies that inhibit malaria merozoite invasion. J Cell Biol 111: 1877–1884.
Toure F, Bisseye C, Mavoungou E, 2006. Imbalanced distribution of Plasmodium falciparum EBA-175 genotypes related to clinical status in children from Bakoumba, Gabon. Clin Med Res 4: 7–11.
Binks RH, Baum J, Oduola AM, Arnot DE, Babiker HA, Kremsner PG, Roper C, Greenwood BM, Conway DJ, 2001. Population genetic analysis of the Plasmodium falciparum erythrocyte binding antigen-175 (eba-175) gene. Mol Biochem Parasitol 114: 63–70.
Cramer JP, Mockenhaupt FP, Mohl I, Dittrich S, Dietz E, Otchwemah RN, Ehrhardt S, Bienzle U, Jelinek T, 2004. Allelic dimorphism of the erythrocyte binding antigen-175 (eba-175) gene of Plasmodium falciparum and severe malaria: significant association of the C-segment with fatal outcome in Ghanaian children. Malar J 3: 11.
Coley AM, Gupta A, Murphy VJ, Ba T, Kim H, Foley M, Anders RF, Batchelor AH, 208. Structure of the malaria antigen AMA1 in complex with a growth-inhibitory antibody. PLoS Pathog 3: 1308–1319.
Escalante A, Grebert HM, Chaiyaroj SC, Magris M, Biswas S, Nahlen BL, Lal AA, 2001. Polymorphism in the gene encoding the apical membrane antigen-1 (AMA-1) of Plasmodium falciparum. X. Asembo Bay Cohort Project. Mol Biochem Parasitol 113: 279–287.
Marshall V, Zhang L, Anders RF, Coppel RL, 1996. Diversity of the vaccine candidate AMA-1 of Plasmodium falciparum. Mol Biochem Parasitol 77: 109–113.
Kocken CH, Narum DL, Massougbodji A, Ayivi B, Dubbeld MA, van der Wel A, Conway DJ, Sanni A, Thomas AW, 2000. Molecular characterisation of Plasmodium reichenowi apical membrane antigen-1 (AMA-1), comparison with P. falciparum AMA-1, and antibody-mediated inhibition of red cell invasion. Mol Biochem Parasitol 109: 147–156.
Garg S, Alam MT, Das MK, Dev V, Kumar A, Dash AP, Sharma YD, 2007. Sequence diversity and natural selection at domain I of the apical membrane antigen 1 among Indian Plasmodium falciparum populations. Malar J 6: 154.
Oeuvray C, Bouharoun-Tayoun H, Gras-Masse H, Bottius E, Kaidoh T, Aikawa M, Filgueira MC, Tartar A, Druilhe P, 1994. Merozoite surface protein-3: a malaria protein inducing antibodies that promote Plasmodium falciparum killing by cooperation with blood monocytes. Blood 84: 1594–1602.
McColl D, Anders RF, 1997. Conservation of structural motifs and antigenic diversity in the Plasmodium falciparum merozoite surface protein-3 (MSP-3). Mol Biochem Parasitol 90: 21–31.
Oeuvray C, Bouharoun-Tayoun H, Grass-Masse H, Lepers JP, Ralamboranto L, Tartar A, Druilhe P, 1994. A novel merozoite surface antigen of Plasmodium falciparum (MSP-3) identified by cellular-antibody cooperative mechanism antigenicity and biological activity of antibodies. Mem Inst Oswaldo Cruz 89 (Suppl 2): 77–80.
Huber W, Felger I, Matile H, Lipps HJ, Steiger S, Beck HP, 1997. Limited sequence polymorphism in the Plasmodium falciparum merozoite surface protein 3. Mol Biochem Parasitol 87: 231–234.
Okenu DM, Thomas AW, Conway DJ, 2000. Allelic lineages of the merozoite surface protein 3 gene in Plasmodium reichenowi and Plasmodium falciparum. Mol Biochem Parasitol 109: 185–188.
Nebie I, Diarra A, Ouedraogo A, Soulama I, Bougouma EC, Tiono AB, Konate AT, Chilengi R, Theisen M, Dodoo D, Remarque E, Bosomprah S, Milligan P, Sirima SB, 2008. Humoral responses to Plasmodium falciparum blood-stage antigens and association with incidence of clinical malaria in children living in an area of seasonal malaria transmission in Burkina Faso, West Africa. Infect Immun 76: 759–766.
Samba-Kimbata J, 1978. Le Climat du bas Congo. Thèse Doctorat 3ème Cycle, Dijon.
Fondjo E, Robert V, Le Goff G, Toto JC, Carnevale P, 1992. Urban malaria in Yaounde (Cameroon). 2. Entomologic study in 2 suburban districts [in French]. Bull Soc Pathol Exot 85: 57–63.
Baird JK, Owusu Agyei S, Utz GC, Koram K, Barcus MJ, Jones TR, Fryauff DJ, Binka FN, Hoffman SL, Nkrumah FN, 2002. Seasonal malaria attack rates in infants and young children in northern Ghana. Am J Trop Med Hyg 66: 280–286.
Koram KA, Quaye L, Abuaku B, 2008. Efficacy of amodiaquine/artesunate combination therapy for uncomplicated malaria in children under five years in Ghana. Ghana Med J 42: 55–60.
Bereczky S, Martensson A, Gil JP, Farnert A, 2005. Short report: rapid DNA extraction from archive blood spots on filter paper for genotyping of Plasmodium falciparum. Am J Trop Med Hyg 72: 249–251.
Toure F, Mavoungou E, Ndong JM, Tshipamba P, Deloron P, 2001. Erythrocyte binding antigen (EBA-175) of Plasmodium falciparum: improved genotype determination by nested polymerase chain reaction. Trop Med Int Health 6: 767–769.
Peakall R, Smouse PE, 2005. Genalex 6: genetic analysis in Excel. Population genetic software for teaching and research. Mol Ecol Notes 6: 288–295.
Excoffier L, Estoup A, Cornuet JM, 2005. Bayesian analysis of an admixture model with mutations and arbitrarily linked markers. Genetics 169: 1727–1738.
Orloci L, 1978. Multivariate Analysis in Vegetation Research. The Hague, The Netherlands: Dr W. Junk b.v.
Conway DJ, 1997. Natural selection on polymorphic malaria antigens and the search for a vaccine. Parasitol Today 13: 26–29.
Soulama I, Bougouma EC, Diarra A, Nebié I, Sirima SB, 2010. Low-high season variation in Plasmodium falciparum erythrocyte binding antigen 175 (eba-175) in malaria endemic area of Burkina Faso. Trop Med Int Health 15: 51–59.
Ahouidi A, Bei AK, Neafsey DE, Sarr O, Volkman S, Milner D, Cox-Singh J, Ferreira MU, Ndir O, Premji Z, Mboup S, Duraisingh MT, 2010. Population genetic analysis of large sequence polymorphisms in Plasmodium falciparum blood-stage antigens. Infect Genet Evol 10: 200–206.
Polley SD, Tetteh KK, Lloyd JM, Akpogheneta OJ, Greenwood BM, Bojang KA, Conway DJ, 2007. Plasmodium falciparum merozoite surface protein 3 is a target of allele-specific immunity and alleles are maintained by natural selection. J Infect Dis 195: 279–287.
Umar F, Malla N, Dubey ML, 2009. Genetic polymorphism in msp-2, ama-1 and csp genes in Plasmodium falciparum field isolates from north and north-western India. J Vector Borne Dis 46: 109–116.
Oliveira DA, Udhayakumar V, Bloland P, Shi YP, Nahlen BL, Oloo AJ, Hawley WE, Lal AA, 1996. Genetic conservation of the Plasmodium falciparum apical membrane antigen-1 (AMA-1). Mol Biochem Parasitol 76: 333–336.
Wang J, Nguyen V, Glen J, Henderson B, Saul A, Miller LH, 2005. Improved yield of recombinant merozoite surface protein 3 (MSP3) from Pichia pastoris using chemically defined media. Biotechnol Bioeng 90: 838–847.
Past two years | Past Year | Past 30 Days | |
---|---|---|---|
Abstract Views | 280 | 235 | 15 |
Full Text Views | 330 | 20 | 1 |
PDF Downloads | 105 | 16 | 1 |