Microsatellite Genotyping of Plasmodium vivax Isolates from Pregnant Women in Four Malaria Endemic Countries

RESEARCH ARTICLE Microsatellite Genotyping of Plasmodium vivax Isolates from Pregnant Women in Four Malaria Endemic Countries Michela Menegon1‡, Azucena Bardají2‡, Flor Martínez-Espinosa3, Camila BôttoMenezes3,4, Maria Ome-Kaius5, Ivo Mueller2,5,6, Inoni Betuela5, Myriam Arévalo-Herrera7, Swati Kochar8, Sanjay K. Kochar8, Puneet Jaju9, Dhiraj Hans9, Chetan Chitnis9, Norma Padilla10, María Eugenia Castellanos10, Lucía Ortiz10, Sergi Sanz2, Mireia Piqueras2, Meghna Desai11, Alfredo Mayor2, Hernando del Portillo2,12‡, Clara Menéndez2‡, Carlo Severini1‡* OPEN ACCESS Citation: Menegon M, Bardají A, Martínez-Espinosa F, Bôtto-Menezes C, Ome-Kaius M, Mueller I, et al. (2016) Microsatellite Genotyping of Plasmodium vivax Isolates from Pregnant Women in Four Malaria Endemic Countries. PLoS ONE 11(3): e0152447. doi:10.1371/journal.pone.0152447 Editor: Luzia Helena Carvalho, Centro de Pesquisa Rene Rachou/Fundação Oswaldo Cruz (FiocruzMinas), BRAZIL Received: July 27, 2015 Accepted: March 14, 2016 Published: March 24, 2016 Copyright: This is an open access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication. Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Funding: The PregVax collaborative project was an EU-FP7 funded programme (FP7-HEALTH-201588). This research was also supported by the Malaria in Pregnancy Consortium, which is funded through a grant from the Bill and Melinda Gates Foundation to the Liverpool School of Tropical Medicine. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. 1 Department of Infectious, Parasitic and Immunomediated Diseases, Istituto Superiore di Sanità, Rome, Italy, 2 Barcelona Centre for International Health Research (CRESIB, Hospital Clínic-Universitat de Barcelona), Barcelona, Spain, 3 Gerência de Malária, Fundação de Medicina Tropical do Amazonas Dr. Heitor Vieira Dourado, Manaus, Brazil, 4 Universidade do Estado do Amazonas, Manaus, Amazonas, Brazil, 5 Papua New Guinea Institute of Medical Research, Goroka, Papua New Guinea, 6 Walter and Eliza Hall Institute, Parkville, Australia, 7 Centro Internacional de Vacunas / Faculty of Health, Universidad del Valle, Cali, Colombia, 8 Sardar Patel Medical College, Bikaner, India, 9 Malaria Group, International Centre for Genetic Engineering and Biotechnology, New Delhi, India, 10 Centro de Estudios en Salud, Universidad del Valle de Guatemala, Guatemala City, Guatemala, 11 Centers for Disease Control and Prevention, Center for Global Health, Division of Parasitic Diseases and Malaria, Atlanta, United States of America, 12 Institució Catalana de Recerca i Estudis Avançats, Barcelona, Spain ‡ MM and AB are shared first authors on this work. HdP, CM and CS are shared senior authors on this work. * Abstract Plasmodium vivax is the most widely distributed human parasite and the main cause of human malaria outside the African continent. However, the knowledge about the genetic variability of P. vivax is limited when compared to the information available for P. falciparum. We present the results of a study aimed at characterizing the genetic structure of P. vivax populations obtained from pregnant women from different malaria endemic settings. Between June 2008 and October 2011 nearly 2000 pregnant women were recruited during routine antenatal care at each site and followed up until delivery. A capillary blood sample from the study participants was collected for genotyping at different time points. Seven P. vivax microsatellite markers were used for genotypic characterization on a total of 229 P. vivax isolates obtained from Brazil, Colombia, India and Papua New Guinea. In each population, the number of alleles per locus, the expected heterozygosity and the levels of multilocus linkage disequilibrium were assessed. The extent of genetic differentiation among populations was also estimated. Six microsatellite loci on 137 P. falciparum isolates from three countries were screened for comparison. The mean value of expected heterozygosity per country ranged from 0.839 to 0.874 for P. vivax and from 0.578 to 0.758 for P. falciparum. P. vivax populations were more diverse than those of P. falciparum. In some of the studied countries, the diversity of P. vivax population was very high compared to the PLOS ONE | DOI:10.1371/journal.pone.0152447 March 24, 2016 1 / 13 P. vivax Genotyping in Different Endemic Settings Competing Interests: The authors have declared that no competing interests exist. respective level of endemicity. The level of inter-population differentiation was moderate to high in all P. vivax and P. falciparum populations studied. Introduction In endemic areas where Plasmodium vivax predominates, malaria in pregnancy is associated with detrimental effects on the health of the affected mothers and their infants [1–8]. Yet, there are still many gaps in the understanding of the mechanisms involved in the pathology of P. vivax infection in pregnancy. Genetic diversity in the Plasmodium populations is reported to be associated with the intensity of transmission. In P. falciparum, the genetic diversity is often, but not always, directly associated with transmission intensity [9–12]; in P. vivax the situation is more complicated and a number of studies reported high genetic diversity in parasite populations from low transmission settings [9,11,13–16]. As with Plasmodium falciparum, to understand the epidemiology, diversity, distribution and transmission dynamics of natural P. vivax populations in different epidemiological regions is crucial to develop specific control tools that target the distinctive biology of this neglected parasite. With malaria elimination back on the global agenda, mapping of global and local P. vivax population structure is essential prior to establishing goals for elimination and the roll out of interventions [17]. In recent years, reliable methods to genotype populations of P. falciparum and P. vivax have been developed. Genotyping methods based on the analysis of the polymorphic genes encoding antigens under immune selective pressure [18–20] might lead to a misunderstanding of the effective process of transmission [21]. Microsatellite (MS) markers, which are neutral or nearly neutral genetic markers, showed a high degree of allelic variation [22, 23] and are efficiently used for studies on genetic diversity and structure of both P. falciparum [24,25] and P. vivax populations [26]. Moreover, the use of MS markers can improve the capability to distinguish recrudescences/relapses from new infections in clinical trials [27]. The knowledge about the genetic variability of P. vivax is limited when compared to the information available for P. falciparum. Rece (...truncated)