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Genetic structure of Plasmodium vivax in Nicaragua, a country in the control phase, based on the carboxyl terminal region of the merozoite surface protein-1
Genetic structure of Plasmodium vivax in Nicaragua, a country in the control phase, based on the carboxyl terminal region of the merozoite surface protein-1 Sleidher Guti´errez, Lilia Gonz´alez-Cer´on, Alberto Montoya, Marco A. Sandoval, Maritza E. T´orres, Rene Cerritos PII: DOI: Reference:
S1567-1348(15)00363-9 doi: 10.1016/j.meegid.2015.08.040 MEEGID 2469
To appear in: Received date: Revised date: Accepted date:
1 June 2015 26 August 2015 27 August 2015
Please cite this article as: Guti´errez, Sleidher, Gonz´alez-Cer´ on, Lilia, Montoya, Alberto, Sandoval, Marco A., T´ orres, Maritza E., Cerritos, Rene, Genetic structure of Plasmodium vivax in Nicaragua, a country in the control phase, based on the carboxyl terminal region of the merozoite surface protein-1, (2015), doi: 10.1016/j.meegid.2015.08.040
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Title
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Genetic structure of Plasmodium vivax in Nicaragua, a country in the control phase, based on the carboxyl terminal region of the merozoite surface protein-1
Authors
Sleidher Gutiérrez1†, Lilia González-Cerón1†, Alberto Montoya2, Marco A Sandoval1, Maritza E
contributed with similar effort to this work
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*corresponding author
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†
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Tórres2, Rene Cerritos*3
Affiliations 1
Mexico 30700. 2
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Regional Center for Research in Public Health, National Institute of Public Health, Tapachula, Chiapas,
Nicaragua. 3
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Parasitology Department, National Center for Diagnosis and Reference (CNDR), Ministry of Health,
Departamento de Medicina Experimental, Facultad de Medicina, Universidad Nacional Autónoma de
México, México D.F. 04510, México. Email: [email protected]
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Abstract
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Malaria is still a grave public health problem in tropical areas of the world. The greater genetic
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diversity of Plasmodium vivax at geographic sites with less control over infection evidences the
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importance of genetic studies of these parasites. The present genetic study compares P. vivax in Nicaragua, which is still in the control phase, with this species in several other countries. In Nicaragua, P. vivax causes over 80% of malaria cases, most occurring in two remote northern
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regions. Plasmodium asexual blood-stage antigens, implicated in reticulocyte invasion, are possible
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molecular markers for analyzing parasite population genetics and for developing vaccines. The aim of this work was to investigate the genetic structure of P. vivax based on the 42 kD merozoite surface
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protein-1 (PvMSP-142), which may represent a sensitive marker for evaluating malaria transmission
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control. From blood samples of patients with P. vivax, we amplified PvMSP-142, obtained the nucleotide sequences, and compared them to homologous sequences of parasites from other
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geographic sites, retrieved from the GenBank. The 92 nucleotide sequences of P. vivax resulted in the resolution of eight haplotypes, six exclusive to Nicaragua. The great nucleotide diversity
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(π=0.020), the minimal recombination events (Rm = 11), and the dN-dS values were similar to other control phase countries. FST values between parasites were low (0.069) for Nicaragua versus Brazil but higher for Nicaragua versus other regions (0.134-0.482). The haplotype network revealed five lineages: two are very frequent in Nicaragua and closely related to American parasites; three have been detected in multiple geographic sites around the world. These results suggest that P. vivax in Nicaragua is a differentiated and genetically diverse population (mainly due to mutation, positive balancing selection and recombination) and that PvMSP-142 may be a sensitive marker for evaluating sustained reduction in malaria transmission and for developing vaccines. 2
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Keywords: Plasmodium vivax, merozoite surface protein-1, Nicaragua, genetic diversity, natural
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selection, genetic differentiation, genealogy.
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1. Introduction
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Plasmodium vivax is widely distributed in Latin-America, Asia, the Middle East and Oceania
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(Guerra et al., 2010){FormattingCitation}{Formatting Citation}. An estimated 25 million people in
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the Americas are considered at high risk. Thirteen countries, including Nicaragua, reported a decline of over 75% in laboratory-confirmed malaria cases between 2000 and 2012 (PAHO, 2012). In 2012,
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the American continent reported 469,380 confirmed malaria cases and 108 malaria deaths (WHO, 2013).
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Currently, several Central American countries are in the control phase, while El Salvador, Costa Rica and Belize are in the pre-elimination phase. In the North Atlantic Autonomous Region
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(RAAN) in Nicaragua, P. vivax and P. falciparum are transmitted throughout the year, and on the
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North Pacific coast transmission is sporadic with periodic outbreaks. The RAAN is adjacent to
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Honduras, and there is a large territorial extension covering both sides of the border where most malaria transmission takes place. In 2012, Honduras and Nicaragua reported 6,434 and 1,235 malaria
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cases, respectively (PAHO, 2012). The current strategy and Plan of Action for Malaria (2011-2025) prioritizes different actions including malaria prevention, surveillance, early case detection, outbreak containment, monitoring and evaluation (Martinelli, 2014). To support these actions, genetic population studies are necessary to identify the haplotypes circulating at each geographic site and determine how they originate, persist and disperse in the region. The degree of genetic diversity and multiple clone infections might determine the transmission intensity and complexity of Plasmodium species (Arnott et al., 2012).
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Although the Mesoamerican region (from central Mexico to northern Costa Rica) shares
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ecological and socioeconomical conditions, there might be a restriction on parasite circulation. For
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instance, the circumsporozoite repeat genotype vk247 highly prevalent in southern Mexico
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(Rodriguez et al., 2000) and Colombia (Hernandez-Martinez et al., 2011) has not been reported in Guatemala (Mendizábal-Cabrera, 2006), Nicaragua (Gonzalez-Ceron et al., 2013) or Honduras (Lopez et al., 2012). In the southern part of Mexico bordering with Guatemala, the different P. vivax
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lineages identified show several hybridization events followed by diversification (Cerritos et al.,
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2014).
Gene markers with high evolutionary dynamics may be useful for explaining how P. vivax
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populations evolve and produce new haplotypes, and for elucidating the changes in haplotype
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frequency (Barry et al., 2015). The gene encoding for the merozoite surface protein-1 (MSP-1) plays an important role in the invasion of the host blood reticulocyte and is an important vaccine candidate
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(Ahlborg et al., 2002; Han et al., 2011; Thakur et al., 2008). It is synthesized as a precursor of the 200 kDa protein during schizogony, and its processing produces four polypeptides of approximately
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83, 30, 38 and 42 kDa (Kang et al., 2010; Miahipour et al., 2012; Putaporntip et al., 2002). During the invasion process, the C-terminal 42 kDa is further processed into two fragments of 33 kDa (MSP-133) and 19 kDa (MSP-119), although only the 19 kDa fragment remains on the merozoite surface (Holder et al., 1992; Kang et al., 2010). In P. vivax, it has been reported that PvMSP-133 is highly polymorphic, while PvMSP-119 is better conserved. Both fragments are capable of inducing blocking antibodies, but PvMSP-142 is more immunogenic than PvMSP-119 (Bastos et al., 2007; Wickramarachchi et al., 2007; Zeyrek et al., 2010).
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The aim of the present study was to analyze the genetic structure of P. vivax in Nicaraguan
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parasites based on the gene fragment pvmsp-142. The corresponding nucleotide sequences from
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parasites of other geographic sites, retrieved from the GenBank data base, were used to compare
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differentiation and the genealogical relationships.
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genetic diversity, natural selection and recombination, and to determine the degree of genetic
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2. Methods
This study was approved by the Ethical Committee of the National Center for Diagnosis and
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National Institute of Public Health.
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Reference (CNDR) of the Health Ministry in Nicaragua, and the Ethical Committee of the Mexican
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2.1. Blood samples and geographic origin Following informed consent, infected blood samples were obtained from symptomatic patients
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seeking malaria diagnosis during 2012 and 2013 in the sentinel laboratory network established by the Nicaraguan Health Ministry at head municipalities: Waspam (50 msnm, 14° 43.994´ N, 83° 56.458´ W), Rosita (66 msnm, 13° 52.004´ N, 84° 23.467´ W), Puerto Cabezas (16msnm, 14° 1.828´ N, 83° 23.089´ W), Bonanza (220 msnm, 14° 1.800´ N, 84° 35.715´ W), Siuna (200 msnm, 13° 44.114´ N, 84° 46.653´ W) and Prinzapolka (22 msnm, 13° 30.589´ N, 84° 13.200´ W) in the North Atlantic Autonomous Region (RAAN); Chinandega (65 msnm, 12° 37.474´ N, 87° 7.802´ W) and El Viejo (50 msnm, 12° 39.769´ N, 87° 9.958´ W) on the North Pacific side. The diagnosis of P. vivax was carried out by microscopic examination of stained thick blood smears, and then patients were asked 6
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to donate capillary blood to impregnated filter paper (Whatman #2). After P. vivax infected blood
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samples were dried in silica gel, they were preserved in that state and protected from light until
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analyzed. Whole genomic DNA was extracted from dried blood spots using a commercially available
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Qiagen Qiamp blood Minikit (Qiagen, USA) following the manufacturer’s instructions. DNA obtained from three punches (0.5 cm in diameter) was eluted in 50 μL of water and stored at -20 °C
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until use. 2.2. PCR amplification and sequencing
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From total genomic DNA, pvmsp-142 was amplified by polymerase chain reaction (PCR) using oligonucleotides: For 5´- GCC GAG GAC TAC GAC AAA G - 3´ and Rev 5´- CCT CCA GCT
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TCC TAA GCT TG - 3´. The PCR reaction was prepared as follows: 2.6μl μL of each primer
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(10pM), 2.6μL of the dNTP mixture (1.25mM), 5.1μL of 10× PCR buffer, 1.3μL of MgSO4 (50mM),
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0.2μL Taq Platinum DNA polymerase (Invitrogen Corporation, Carlsbad, CA), and 4 μL of extracted DNA for a final volume of 50 μL. The PCR reaction conditions were as follows: 5 min at 95 °C, 1 min at 95 °C, 1 min at 60 °C, and 75 sec at 72 °C for 35 cycles; afterwards, there was a final
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extension of 72 °C for 10 min, all run in a MyCycler (BioRad, Hercules, CA, USA). The amplified products were examined in agarose gels at 1%, and stained with 0.2µg/ml ethidium bromide using an electrophoresis chamber Midicell primo (Thermo EC330, New York, USA). The 100bp ladder (Invitrogen Corporation, Carlsbad, CA, USA) was used as molecular marker. The amplified PCR product was purified using a MiniElute PCR Purification Kit (Qiagen, Valencia, CA, USA) following the manufacturer’s instructions. The purified products were Sanger sequenced using forward and reverse primers at the High Throughput Genomics Unit, Department of 7
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Genome Sciences, University of Washington, Seattle, WA, USA. The quality of pherograms with the
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forward and reverse nucleotide sequences was verified manually and by using Bioedit v7.1.3
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software. The consensus sequences obtained for each gene fragment were submitted to the NCBI-
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Gen Bank [accession numbers: KR871926-KR872017]. 2.3. Data analysis
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The nucleotide sequence of pvmsp-142 for the Sal 1 strain (XM_001614792.1) was used as a reference. For genetic analysis, nucleotide sequences from other geographic sites were extracted
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from the NBCI, GenBank. For South Korea (SK), n=200: JQ446312 – JQ446322 (Kang et al., 2012), HQ171934– HQ171941 (Han et al., 2011), AF435635-AF435638. For Vanuatu (VAN), n=2:
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AF435632, AF435634 (Putaporntip et al., 2002). For Turkey (TUK), n=30: AB564559 - AB564588
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(Zeyrek et al., 2010). For Thailand (THL), n=93: AF435595, AF435615 (Putaporntip et al., 2002), GQ890917 – GQ890974 (Jongwutiwes et al., 2010), AF199393- AF199404, AF199408- AF199410
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(Putaporntip et al., 2000). For Singapore (SNG), n=50: GU971656 – GU971705 (Ng et al., 2010). For India-Bangladesh (IND-BG), n= 35: EU430452- EU430479, KF612323; AF435639 (Putaporntip
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et al., 2000), AF435616-AF435620 (Putaporntip et al., 2002). For Brazil (BRZ), n=11: AF43562225,27,29,30,31, AF199405,6,7 (Putaporntip et al., 2000). For Sri Lanka (SLK), n=106: AJ292349AJ292359, GU175174-GU175268 (Dias et al., 2011). For Myanmar (MYN), n= 28: JX490129 JX490156. For China (CHN), n=2: JX993754-JX993755 (Zhou and Chen, unpublished). For Cambodia (CAM), n=27 (Parobek et al., 2014). For Mexico (MEX), n=35 (Gonzalez-Ceron et al., unpublished). 2.4. Population genetic analysis 8
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The genetic diversity was measured by calculating π and θ indexes for the complete DNA sequence
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and by using 100 pb windows assuming different levels of variation across the pvmsp-142 fragment.
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The mean of pairwise nucleotide changes detected along the sequences (using the Jukes-Cantor
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correction) is represented by π (Nei and Li, 1979), while θ is the number of segregating sites in a group of sequences (Watterson, 1975). The number of polymorphic sites (S) and haplotypes (H) and the minimal number of recombination events (Rm) were calculated. These analyses were computed
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using DnaSP software (Librado and Rozas, 2009).
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To test whether positive selection shaped the evolution of the pvmsp-142 subfragment in Nicaraguan parasites and to compare them to those from other geographic sites, the number of
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synonymous (s) and non-synonymous (ns) nucleotide changes and the rate of synonymous versus
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non-synonymous changes (dS-dN) were determined by using the Nei-Gojobori proportion method
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with 1000 bootstrap replicates in MEGA 6.0 software (Tamura et al., 2013). The genetic differentiation of the pvmsp-142 was calculated between parasites of different countries by using Wright’s fixation index (FST) (Wright, 1951). Pairwise fixation values between
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each gene population were calculated using the Kimura 2P model in DnaSPv5.1 software (Rozas et al., 2003). FST values from 0 to 0.05 evidence little or no genetic differentiation; values between 0.05 and 0.15 indicate moderate genetic differentiation, while those between 0.15 and 0.25 represent high genetic differentiation. Finally, values above 0.25 point to very high genetic differentiation (Hartl and Clark, 1989). In order to reconstruct the evolutionary history and establish the genealogical relationships of haplotypes from Nicaraguan with those from other geographic sites, a haplotype network was 9
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constructed using TCS 1.21 (Clement et al., 2000). In this way the connection between haplotypes is
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represented by mutational steps, and the non-sampled or extinct haplotypes are shown by empty
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the size of the circle represents the frequency of each haplotype.
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squares. The color of the circles represents the geographic origins (country) of each haplotype, while
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3. Results
Of 119 samples, 92 were amplified and the consensus sequences were obtained for pvmsp-142.
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Patients from the North Atlantic Autonomous Region (RAAN) provided 85 samples, including those from Rosita (n=31), Bonanza (n=22), Waspam (n=16), Siuna (n=11), Puerto Cabezas (n=3) and
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Prinzapolka (n=2). The other 7 samples were obtained from the North Pacific (NP) coast towns of
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from RAAN and 4 from NP.
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Chinandega (n=2) and El Viejo (n=5). Of the 27 samples that were not amplified by PCR, 23 were
The 92 Nicaraguan pvmsp-142 sequences were comprised of 981 nucleotides had 59
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polymorphic sites (51 non-synonymous and 9 synonymous). All polymorphisms were detected in the pvmsp-133 subfragment between nucleotides 1384 and 2080, while the pvmsp-119 subfragment (nucleotides 2081- 2365) did not present any change (Supplementary table 1). In this group of parasites, there was no sign of mixed genotype infections. 3.1. Genetic diversity and geographic distribution of haplotypes The nucleotide changes evidence the resolution of eight haplotypes, named N1-N8 based on their frequency. Six of them were exclusive to Nicaragua (haplotypes N2, N3, N5, N6, N7 and N8). The 10
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most frequent haplotype (N1) and another (N4) were previously detected in other countries. N1 was
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detected in all municipalities from RAAN as well as in parasites from a nearby country (MEX),
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while N4 was identified in parasites from South America (BRZ) and outside the Americas (IND,
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SNG, THL, CAM and MYN). Haplotype N2 was detected in all Nicaraguan locations, while N1, N3 and N5 were found exclusively in the RAAN. There were three single haplotypes (N6, N7, and N8).
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N6 and N7 were detected in Rosita and N8 in Chinandega (Figure 1).
The nucleotide diversity of the pvmsp-142 in P. vivax of Nicaragua (π = 0.020) was lesser than
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that of P. vivax in BRZ, CAM, IND-BNG, SLK, THL and MYN and greater than that of this species in MEX, SK and SNG. The lowest diversity was reported in TUR. At a global level, 99 haplotypes
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have been found with a total of 711 nucleotide sequences, representing great diversity (π = 0.024 and
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θ = 0.017) (Table 1). Sliding window analysis, used to compare genetic diversity across pvmsp-142,
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detected the highest values for π and θ in the window comprising nucleotides 1735 to 1834, followed by the adjacent windows. Only in Nicaraguan isolates (not at other geographic sites) did the window
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comprising nucleotides 1935-2034 show great diversity (Supplementary figure 1). 3.2. Natural selection and recombination In Nicaraguan isolates, non-synonymous substitutions were more numerous than synonymous ones in subfragment pvmsp-133, as found in parasites from other countries. Accordingly, the dN-dS value was positive but not significant (0.779; p=0.426). In other geographic locations this value varied from 0.403 in TUR to 4.087 in SK, being significantly positive for parasites found in SK, CAM, THL, SNG and MYN (Table 1).
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The N8 haplotype presented eight of the nine total synonymous changes detected in the
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nucleotide sequences, including codons 1476, 1509, 1528, 1530, 1533, 1538, 1564 and 1570
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(Supplementary table 1). The minimal number of recombination events for pvmsp-142 in Nicaraguan
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parasites was similar (Rm = 11) to that calculated for some other geographic sites such as SNG, IND, THL and MYN (Rm = 11-12), followed by MEX (Rm = 9) > SK (Rm = 4) > TUR (Rm = 0) (Table
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1). 3.3. Genetic differentiation (FST)
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Based on pvmsp-142, FST values ranged from 0.069 to 0.481 between parasites of Nicaragua and other countries (Table 2). The lowest values were exhibited with NIC versus BRZ, followed by NIC
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versus SLK or IND-BNG. Between NIC and its nearest neighbor (MEX) the differentiation value
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was high (0.231). The highest values (0.363-0.481) were found when comparing NIC and SNG, SK
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or TUR. Also, TUR (0.14-0.502) and SK (0.11-502) had high values when compared to other geographic sites. Finally, the highest differentiation value was between TUR and SK.
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3.4. Haplotype network
Genealogical reconstruction revealed five lineages among the eight haplotypes found in Nicaragua (Figure 2). Three of them were more frequent, while the other two comprised many parasites of distinct geographic origins. Haplotype N1, also detected in MEX (two isolates), was at one mutational step from other MEX haplotype and four mutational steps from N6 and likely comprised one lineage. Haplotypes N2, N3 and N8 constituted a different lineage and were separated by four mutational steps from a Mexican parasite (see also supplementary Table 1). Haplotype N3 was separated from N2 by two mutational step and haplotype N8 from N2 by three mutational 12
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steps. Haplotype N5 was separated by five mutational steps from IND, and by 13 from a very
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frequent haplotype shared by American (MEX, BRZ) and non-American (MYN and TUR) parasites.
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The Nicaraguan haplotype N4, also detected in BRZ, CAM, IND-BNG, SNG, THL and MYN,
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was at 28 mutational steps from N7, which in turn was related to parasites from BRZ, CAM, THL and MYN. N4 and N7 represented different genealogical clusters. There were detected signs of local diversification in SNG and THL, and at less degree in NIC. Also, several loops apparent in the
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network were observed, likely due to recombination events.
4. Discussion
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The results suggest that in Nicaragua P. vivax showed a differentiated population with moderate genetic diversity. Patterns of genetic diversity and the intensity of selection for pvmsp-142 were
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similar to what was found in other countries under the control phase. The five genealogical groups represent different relationships, likely the result of recombination events, parasite migration, and the
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long lasting transmission in the region. Historically, malaria has been an endemic disease in Nicaragua. Both P. falciparum and P. vivax are transmitted in the affected regions, with at least 80% of the confirmed cases being caused by P. vivax (PAHO, 2012). Currently, Nicaragua is under the control phase, and it is expected to advance towards the pre-elimination phase shortly. However, in the North Atlantic Autonomous Region (RAAN) malaria transmission has been more persistent than at other sites. In fact, more than 90% of samples herein analyzed were from this region.
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RAAN is the largest department in Nicaragua, comprising about 33,000 km² and having a
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population of about 250,000 (2005 census) representing different indigenous groups (INIDE, 2015).
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In addition, this region borders with Honduras along the Coco River. In fact, both countries share a
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large area affected by malaria transmission (PAHO, 2012). Nicaragua has marked migration movements of its population, mainly across the border region of RAAN. Undocumented people traffic by land and air (OIM, 2013). The number of malaria cases in Nicaragua decreased between
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1996 and 2012 from 70,000 to 999, while in Honduras the decrease was slightly less prominent
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(from 73,313 to 5,000 during the same period). Lower incidence and sporadic transmission have been reported on the Pacific coast, bordering with El Salvador (PAHO, 2012).
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Furthermore, in Nicaragua malaria transmission occurs in remote communities in which
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social and economic deficiencies cause difficulties for disease diagnosis and treatment. This dynamic of transmission contributes to the maintenance of the genetic diversity found with P. vivax in the
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region. Honduras, the neighbor to the north, is also under the control phase and has reported high diversity in different gene markers, including pvama-1, pvcsp and pvmsp-1 (Lopez et al., 2012).
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When considering geographic sites in the pre-elimination phase (e.g., MEX), P. vivax showed lower diversity than in NIC. TUR had the lowest diversity in P. vivax samples, which were obtained during 2007-2008 (Zeyrek et al., 2010). Since 2000, in this country, the number of malaria cases has undergone a gradual decrease, and in 2010 reached the elimination phase (Özbilgin et al., 2011; WHO, 2010). This suggests that if low numbers of malaria cases are sustained for several years, parasite diversity may decrease significantly. The Mexican region bordering Guatemala shares a haplotype (N1) with Nicaragua. Whereas the frequency of N1 is high in Nicaragua (42%), it is low in MEX (5.7%). At present, the probability 14
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that this parasite will increase in Mexican territory due to migration is quite low, as migrants spend
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few days in this border region. Indeed, only asymptomatic P. vivax seropositive individuals were
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detected (Betanzos-Reyes et al., 2012).
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Currently, the P. falciparum vaccine based on PfMSP-142 is under clinical trials (Ellis et al., 2010; Huaman et al., 2008; Malkin et al., 2007; Ogutu et al., 2009). Similarly, PvMSP-142 is also a
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good vaccine candidate against blood stages. However, its application might be limited by global and local diversity. Six of the eight haplotypes herein detected were exclusive to Nicaragua, and local
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diversification might have occurred as in other regions under the control phase. For instance, there was an exclusive and divergent haplotype (N8) in the municipality of Chinandega, with unique
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characteristics given by the high diversity between codons 1476 and 1570 as well as by the presence
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of eight synonymous changes. There is a possibility that N8 diverged from N2 on the Central American region, supported by the fact that N2, N3 and N8 formed a group unique to Nicaragua.
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Since haplotype N2 is widespread in all affected regions of the present study, this high frequency might be the result of its successful transmission. The accelerated and inherent evolutionary
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dynamics of P. vivax merozoite genes mainly due to positive balancing selection and recombination might require periodic monitoring of the ongoing antigenic diversity of the parasite and vaccine modifications (Kang et al., 2012). Genealogical reconstruction based on the haplotype network suggests the presence of five P. vivax lineages in Nicaragua, separated by many mutational steps and closely related to haplotypes reported in different regions inside and outside the Americas. However, there are still gaps in our knowledge about genealogical reconstruction. For example, the importance of recombination versus mutation in the generation of the divergent haplotypes is still unknown. In order to understand the 15
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transmission process, pathogenesis and drug resistance of P. vivax, it is necessary to gain insights
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into its origin, way of dispersion and genealogical relationships. The mitochondrial DNA analysis
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shows that parasites from Central America and South America are closely related (FST = 0.016; not
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significant above zero). Indeed, the recent parasite introduction was predicted for Central America along this and other old migration routes between continents (Taylor et al., 2013). Accordingly, in this study, the FST value between parasites from NIC and BRZ (using pvmsp-142) was much lower
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(0.069) than that between NIC and other geographic sites. Further studies using mitochondrial DNA
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are required to clarify the higher FST values obtained between NIC and MEX. It is important to consider that pvmsp-1 is highly recombinant (Putaporntip et al., 2002), and
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that some haplotypes detected herein could have been generated by recombination. The pvmsp-142
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fragment showed 11 minimal recombination events in the Nicaraguan sample. Pvmsp-142 was recently classified into 12 global groups without geographic correlation, evidenced by the
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heterogeneity shown by the phylogenetic analysis (Dias et al., 2011). The previous finding was confirmed by the genealogical reconstruction of the present study: the most abundant haplotypes
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from Nicaragua (N1, N2 and N3) were genetically related to haplotypes from Mexico and are located in groups separated by few mutation steps (from none to four steps). On the other hand, N7 was closely related to a haplotype from Brazil (along with other countries). Since malaria control campaigns can drastically modify haplotype frequency within the region herein studied, genetic parameters should be incorporated into monitoring with the aim of facilitating the advance to the upcoming pre-elimination or elimination phase in Nicaragua. Besides its importance as a vaccine candidate, pvmsp142 offers a good and sensitive genetic marker that is 16
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under different evolutionary forces, including mutation, positive selection and high levels of
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recombination.
Acknowledgments
This work was supported by CONACyT-Mexico (project CB-2009-01-131247), AMI/RAVEDRA-
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OPS/OMS-Nicaragua, and global fund-malaria component-Nicaragua (2012-2013). We are grateful
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to the lab personnel at the sentinel sites in Nicaragua that collaborated to prepare the P. vivax blood samples on filter paper. We give special thanks to Valentina Manzanares in Siuna, Martha Lopez in
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Rosita, and Dexter Manzanares in Waspam, as well as the local authorities for the use of the
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facilities. Thanks to Valeria Zermeño for assistance in manuscript revision, and to Frida Santillán
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assistance.
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and Olga L. Paloqueme at the Regional Center for Research in Public Health in Mexico for technical
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5. References
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Ahlborg, N., Ling, I.T., Howard, W., Holder, A.A., Riley, E.M., 2002. Protective Immune Responses to the 42-Kilodalton (kDa) Region of Plasmodium yoelii Merozoite Surface Protein 1 Are Induced by the C-Terminal 19-kDa Region but Not by the Adjacent 33-kDa Region. Infect. Immun. 70, 820825.
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Arnott, A., Barry, A.E., Reeder, J.C., 2012. Understanding the population genetics of Plasmodium vivax is essential for malaria control and elimination. Malar. J. 11, 14.
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Barry, A.E., Waltmann, A., Koepfli, C., Barnadas, C., Mueller, I., 2015. Uncovering the transmission dynamics of Plasmodium vivax using population genetics. Pathog. Glob. Health 109, 142-152.
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Bastos, M.S., da Silva-Nunes, M., Malafronte, R.S., Hoffmann, E.H., Wunderlich, G., Moraes, S.L., Ferreira, M.U., 2007. Antigenic polymorphism and naturally acquired antibodies to Plasmodium vivax merozoite surface protein 1 in rural Amazonians. Clin. Vaccine Immunol. 14, 1249-1259. Betanzos-Reyes, A.F., González-Cerón, L., Rodríguez, M.H., Torres-Monzón, J.A., 2012. Malaria's seroepidemiology in a group of migrants in transit (Chiapas, 2008). Salud Publica Mex. 54, 523-529.
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Cerritos, R., González-Cerón, L., Nettel, J.A., Wegier, A., 2014. Genetic structure of Plasmodium vivax using the merozoite surface protein 1 icb5-6 fragment reveals new hybrid haplotypes in southern Mexico. Malar. J. 13, 35-35.
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Clement, M., Posada, D., Crandall, K.A., 2000. TCS: a computer program to estimate gene genealogies. Mol. Ecol. 9, 1657-1659.
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Dias, S., Longacre, S., Escalante, A.A., Udagama-Randeniya, P.V., 2011. Genetic diversity and recombination at the C-terminal fragment of the merozoite surface protein-1 of Plasmodium vivax (PvMSP-1) in Sri Lanka. Infect. Genet. Evol. 11, 145-156. Ellis, R.D., Sagara, I., Doumbo, O., Wu, Y., 2010. Blood stage vaccies for Plasmodium falciparum. Current status and the way forward. Hum. Vaccin. 6, 627-634. Gonzalez-Ceron, L., Martinez-Barnetche, J., Montero-Solis, C., Santillan, F., Soto, A.M., Rodriguez, M.H., Espinosa, B.J., Chavez, O.A., 2013. Molecular epidemiology of Plasmodium vivax in Latin America: polymorphism and evolutionary relationships of the circumsporozoite gene. Malar. J. 12, 243. Guerra, C.A., Howes, R.E., Patil, A.P., Gething, P.W., Van Boeckel, T.P., Temperley, W.H., Kabaria, C.W., Tatem, A.J., Manh, B.H., Elyazar, I.R., Baird, J.K., Snow, R.W., Hay, S.I., 2010. The international limits and population at risk of Plasmodium vivax transmission in 2009. PLoS Negl. Trop. Dis. 4, e774. 18
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Han, E.-T., Wang, Y., Lim, C.S., Cho, J.H., Chai, J.-Y., 2011. Genetic diversity of the malaria vacine candidate merozoite surface protein 1 gene of Plasmodium vivax field isolates in Republic of Korea. Parasitol. Res. 109, 1571-1576.
IP
Hartl, D.L., Clark, A.G., 1989. Principles of Population Genetics in: Associates, S. (Ed.), Sutherland, Massachusetts, pp. 118-119.
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Hernandez-Martinez, M.A., Escalante, A.A., Arevalo-Herrera, M., Herrera, S., 2011. Antigenic diversity of the Plasmodium vivax circumsporozoite protein in parasite isolates of Western Colombia. Am. J. Trop. Med. Hyg. 84, 51-57.
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Holder, A.A., Blackman, M.J., Burghaus, P.A., Chappel, J.A., Ling, I.T., McCallum-Deighton, N., Shai, S., 1992. A malaria merozoite surface protein (MSP1) structure, processing and function. Mem Inst Oswaldo Cruz 87 (3), 37-42.
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Huaman, M.C., Martin, L.B., Malkin, E., Narum, D.L., Miller, L.H., Mahanty, S., Long, C.A., 2008. Ex Vivo Cytokine and Memory T Cell Responses to The 42-kDa Fragment of Plasmodium falciparum Merozoite Surface Protein-1 in Vaccinated Volunteers. The Journal of Immunology 180, 1451-1461.
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Instituto Nacional de información de Desarrollo de Nicaragua, INIDE; census 2015. http://www.inide.gob.ni
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Jongwutiwes, S., Putaporntip, C., Hughes, A.L., 2010. Bottleneck Effects on Vaccine-Candidate Antigen Diversity of Malaria Parasites in Thailand. Vaccine 28, 3112-3117.
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Kang, J.-M., Ju, H.-L., Kang, Y.-M., Lee, D.-H., Moon, S.-U., Sohn, W.-M., Park, J.-W., Kim, T.-S., Na, B.-K., 2012. Genetic polymorphism and natural selection in the C-terminal 42 kDa region of merozoite surface protein-1 among Plasmodium vivax Korean isolates. Malar. J. 11, 1-9. Kang, J.M., Moon, S.U., Kim, J.Y., Cho, S.H., Lin, K., Sohn, W.M., Kim, T.S., Na, B.K., 2010. Genetic polymorphism of merozoite surface protein-1 and merozoite surface protein-2 in Plasmodium falciparum field isolates from Myanmar. Malar. J. 9, 131. Librado, P., Rozas, J., 2009. DnaSP v5: a software for comprehensive analysis of DNA polymorphism data. Bioinformatics 25, 1451-1452. Lopez, A.C., Ortiz, A., Coello, J., Sosa-Ochoa, W., Torres, R.E., Banegas, E.I., Jovel, I., Fontecha, G.A., 2012. Genetic diversity of Plasmodium vivax and Plasmodium falciparum in Honduras. Malar. J. 11, 391. Malkin, E., Long, C.A., Stowers, A.W., Zou, L., Singh, S., MacDonald, N.J., Narum, D.L., Miles, A.P., Orcutt, A.C., Muratova, O., Moretz, S.E., Zhou, H., Diouf, A., Fay, M., Tierney, E., Leese, P., 19
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Mahanty, S., Miller, L.H., Saul, A., Martin, L.B., 2007. Phase 1 Study of Two Merozoite Surface Protein 1 (MSP1(42)) Vaccines for Plasmodium falciparum Malaria. PLoS Clin. Trials 2, e12.
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Martinelli, S., 2014. Malaria Free by 2025: A Global Fund Regional Initiative. Friends of the Global Fight Washington, DC. link: http://theglobalfight.org/malaria-free-by-2025-mesoamerica-hispaniolaregional-initiative last accessed August 25, 2015.
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Mendizábal-Cabrera, R., and Padilla, N., 2006. Diversidad genética de Plasmodium vivax en regiones de alto riesgo de malaria en Guatemala [Genetic Diversity of Plasmodium vivax in areas of high risk of malaria in Guatemala], Revista de la Universidad del Valle de Guatemala, Guatemala, pp. 62-79.
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Miahipour, A., Keshavarz, H., Heidari, A., Raeisi, A., Rezaeian, M., Rezaie, S., 2012. Genetic Variation of MSP-1 Gene in Plasmodium vivax Isolated from Patients in Hormozgan Province, Iran using SSCP-PCR. Iran J. Parasitol. 7, 1-7. Nei, M., Li, W.H., 1979. Mathematical model for studying genetic variation in terms of restriction endonucleases. Proc. Natl. Acad. Sci. U. S. A. 76, 5269-5273.
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Ng, L.C., Lee, K.S., Tan, C.H., Ooi, P.L., Lam-Phua, S.G., Lin, R., Pang, S.C., Lai, Y.L., Solhan, S., Chan, P.P., Wong, K.Y., Ho, S.T., Vythilingam, I., 2010. Entomologic and molecular investigation into Plasmodium vivax transmission in Singapore, 2009. Malar. J. 9, 305.
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Ogutu, B.R., Apollo, O.J., McKinney, D., Okoth, W., Siangla, J., Dubovsky, F., Tucker, K., Waitumbi, J.N., Diggs, C., Wittes, J., Malkin, E., Leach, A., Soisson, L.A., Milman, J.B., Otieno, L., Holland, C.A., Polhemus, M., Remich, S.A., Ockenhouse, C.F., Cohen, J., Ballou, W.R., Martin, S.K., Angov, E., Stewart, V.A., Lyon, J.A., Heppner, D.G., Withers, M.R., for the, M.S.P.M.V.W.G., 2009. Blood Stage Malaria Vaccine Eliciting High Antigen-Specific Antibody Concentrations Confers No Protection to Young Children in Western Kenya. PLoS ONE 4, e4708. Organización Internacional para las Migraciones OIM, 2013. Perfil Migratorio de Nicaragua 2012. http://migracion-ue- alc.eu/documents/keydocs /ES/Perfil_ Migratorio_Nicaragua_2012.pdf Özbilgin, A., Topluoglu, S., Es, S., Islek, E., Mollahaliloglu, S., Erkoc, Y., 2011. Malaria in Turkey: Successful control and strategies for achieving elimination. Acta Trop. 120, 15-23. Pan American Health Organization PAHO, 2012. Interactive Malaria Statistics. http://www.paho.org/hq/index.php?option=com_content&view=article&id=2632&Itemid=2130&lan g=en. Parobek, C.M., Bailey, J.A., Hathaway, N.J., Socheat, D., Rogers, W.O., Juliano, J.J., 2014. Differing Patterns of Selection and Geospatial Genetic Diversity within Two Leading Plasmodium vivax Candidate Vaccine Antigens. PLoS Negl. Trop. Dis. 8, e2796. 20
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Putaporntip, C., Jongwutiwes, S., Sakihama, N., Ferreira, M.U., Kho, W.G., Kaneko, A., Kanbara, H., Hattori, T., Tanabe, K., 2002. Mosaic organization and heterogeneity in frequency of allelic recombination of the Plasmodium vivax merozoite surface protein-1 locus. Proc. Natl. Acad. Sci. U. S. A. 99, 16348-16353.
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Putaporntip, C., Jongwutiwes, S., Seethamchai, S., Kanbara, H., Tanabe, K., 2000. Intragenic recombination in the 3′ portion of the merozoite surface protein 1 gene of Plasmodium vivax. Mol. Biochem. Parasitol. 109, 111-119.
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Rodriguez, M.H., Gonzalez-Ceron, L., Hernandez, J.E., Nettel, J.A., Villarreal, C., Kain, K.C., Wirtz, R.A., 2000. Different prevalences of Plasmodium vivax phenotypes VK210 and VK247 associated with the distribution of Anopheles albimanus and Anopheles pseudopunctipennis in Mexico. Am. J. Trop. Med. Hyg. 62, 122-127.
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Rozas, J., Sanchez-del Barrio, J.C., Messeguer, X., Rozas, R., 2003. DnaSP, DNA polymorphism analyses by the coalescent and other methods. Bioinformatics 19, 2496-2497. Tamura, K., Stecher, G., Peterson, D., Filipski, A., Kumar, S., 2013. MEGA6: Molecular Evolutionary Genetics Analysis Version 6.0. Mol. Biol. Evol. 30, 2725-2729.
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Taylor, J.E., Pacheco, M.A., Bacon, D.J., Beg, M.A., Machado, R.L., Fairhurst, R.M., Herrera, S., Kim, J.-Y., Menard, D., Póvoa, M.M., Villegas, L., Mulyanto, Snounou, G., Cui, L., Zeyrek, F.Y., Escalante, A.A., 2013. The Evolutionary History of Plasmodium vivax as Inferred from Mitochondrial Genomes: Parasite Genetic Diversity in the Americas. Mol. Biol. Evol. 30, 20502064.
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Thakur, A., Alam, M.T., Sharma, Y.D., 2008. Genetic diversity in the C-terminal 42 kDa region of merozoite surface protein-1 of Plasmodium vivax (PvMSP-1(42)) among Indian isolates. Acta Trop. 108, 58-63. Watterson, G.A., 1975. On the number of segregating sites in genetical models without recombination. Theor. Popul. Biol. 7, 256-276. Wickramarachchi, T., Illeperuma, R.J., Perera, L., Bandara, S., Holm, I., Longacre, S., Handunnetti, S.M., Udagama-Randeniya, P.V., 2007. Comparison of naturally acquired antibody responses against the C-terminal processing products of Plasmodium vivax Merozoite Surface Protein-1 under low transmission and unstable malaria conditions in Sri Lanka. Int. J. Parasitol. 37, 199-208. World Health Organizatio WHO, 2010. World Malaria report 2010. Geneva, Switzerland. http://www.who.int/malaria/world_malaria_report_2010/en/ World Health Organization WHO, 2013. World Malaria Report 2013. Geneva, Switzerland. http://www.who.int/malaria/world_malaria_report_2013/en/ 21
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Wright, S., 1951. The genetical structure of populations. Ann. Eugenics 15, 323-354.
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Zeyrek, F.Y., Tachibana, S.-I., Yuksel, F., Doni, N., Palacpac, N., Arisue, N., Horii, T., Coban, C., Tanabe, K., 2010. Limited Polymorphism of the Plasmodium vivax Merozoite Surface Protein 1 Gene in Isolates from Turkey. Am. J. Trop. Med. Hyg. 83, 1230-1237.
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Figure legends
Figure 1. Geographic distribution of the pvmsp-142 haplotypes in Nicaragua. Each haplotype is
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distinguished by grayscale/grid/stripe. The haplotype frequency is indicated by the circles. The RAAN municipalities include: R) Rosita, B) Bonanza, W) Waspam, S) Siuna, C) Puerto Cabezas, and P) Prinzapolka. The North Pacific coast is represented by G) Chinandega. n= the number of isolates. Figure 2. Haplotype network of pvmsp142. Each cycle corresponds to one haplotype and countries are indicated by different colors. Related genotypes are connected by lines, and squares and
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additional mutational steps between haplotypes are indicated by small diamonds. Nicaraguan
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haplotypes, named from N1 to N8, represent five different lineages.
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Fig. 1
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Fig. 2
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N
H
Diversity indexes π Ө
Recombination Rm
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Country
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8 0.020 0.013 11 NIC 92 8 0.018 0.014 9 MEX 35 8 0.023 0.020 10 BRZ 11 10 0.017 0.009 4 SK 196 22 0.023 0.016 11 CAM 27 30 0.024 0.017 14 IND-BNG 35 47 0.024 0.014 23 SLK 106 43 0.023 0.012 12 THL 93 3 0.007 0.007 0 TUR 30 27 0.019 0.022 12 SNG 50 26 0.024 0.017 13 MYN 28 188 0.025 0.019 24 Total 702 *Significance at 95% IC; N, number of isolates; H, number of haplotypes
Selection dN-dS P* 0.779 0.426 1.275 0.205 0.972 0.333 4.087 0 2.251 0.026 1.931 0.056 1.523 0.130 2.155 0.033 0.403 0.688 2.638 0.008 2.218 0.028 2.061 0.041
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Table 1. The genetic diversity, recombination and natural selection of Pvmsp-142 from Nicaragua compared to the same parameters found at other geographic sites. Similar indexes were shown in almost all countries.
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SK
INDBNG
BRZ
0.275 0.093 0.099 0.234 0.087 0.174 0.140 0.202 0.170
0.111 0.218 0.320 0.206 0.272 0.502 0.167 0.216
0.043 0.127 0.012 0.048 0.307 0.022 0.019
0.275 0.038 0.113 0.355 0.070 0.097
SNG
SLK
THL
TUR
CAM
0.380 0.049 0.009
0.420 0.360
0.021
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MEX
NU
0.153 0.101 0.406 0.180 0.097
0.037 0.298 0.061 0.027
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MEX SK IND-BNG BRZ SNG SLK THL TUR CAM MYN
NIC 0.231 0.384 0.183 0.069 0.363 0.134 0.202 0.481 0.225 0.190
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Table 2. FST indexes when comparing the pvmsp-142 of parasites in Nicaraguan versus those at other geographic sites.
NIC, Nicaragua; MEX, Mexico; BRZ, Brazil; SK, South Korea; IND-BNG, India-Bangladesh; SNG, Singapore; SLK, SriLanka; THL, Thailand; TUR, Turkey; CAM, Cambodia; MYN, Myanmar. The lowest
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value was between NIC and BRZ and the highest values were between NIC and Asian-Middle East parasites (SK, TUR and SNG).
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Highlights
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1. The pvmsp142 genetic diversity in Nicaragua was lower than other countries in control phase.
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2. Pvmsp133 exposed strong positive and negative selection, and pvmsp119 was conserved. 3. Nicaraguan haplotypes reveals processes of migration and local diversification.
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4. P. vivax in Nicaragua shows a differentiated population and micro heterogeneity.
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5. The haplotype network exposed five Nicaraguan lineages.
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S1567-1348(15)00363-9 doi: 10.1016/j.meegid.2015.08.040 MEEGID 2469
To appear in: Received date: Revised date: Accepted date:
1 June 2015 26 August 2015 27 August 2015
Please cite this article as: Guti´errez, Sleidher, Gonz´alez-Cer´ on, Lilia, Montoya, Alberto, Sandoval, Marco A., T´ orres, Maritza E., Cerritos, Rene, Genetic structure of Plasmodium vivax in Nicaragua, a country in the control phase, based on the carboxyl terminal region of the merozoite surface protein-1, (2015), doi: 10.1016/j.meegid.2015.08.040
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Title
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Genetic structure of Plasmodium vivax in Nicaragua, a country in the control phase, based on the carboxyl terminal region of the merozoite surface protein-1
Authors
Sleidher Gutiérrez1†, Lilia González-Cerón1†, Alberto Montoya2, Marco A Sandoval1, Maritza E
contributed with similar effort to this work
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*corresponding author
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†
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Tórres2, Rene Cerritos*3
Affiliations 1
Mexico 30700. 2
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Regional Center for Research in Public Health, National Institute of Public Health, Tapachula, Chiapas,
Nicaragua. 3
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Parasitology Department, National Center for Diagnosis and Reference (CNDR), Ministry of Health,
Departamento de Medicina Experimental, Facultad de Medicina, Universidad Nacional Autónoma de
México, México D.F. 04510, México. Email: [email protected]
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Abstract
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Malaria is still a grave public health problem in tropical areas of the world. The greater genetic
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diversity of Plasmodium vivax at geographic sites with less control over infection evidences the
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importance of genetic studies of these parasites. The present genetic study compares P. vivax in Nicaragua, which is still in the control phase, with this species in several other countries. In Nicaragua, P. vivax causes over 80% of malaria cases, most occurring in two remote northern
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regions. Plasmodium asexual blood-stage antigens, implicated in reticulocyte invasion, are possible
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molecular markers for analyzing parasite population genetics and for developing vaccines. The aim of this work was to investigate the genetic structure of P. vivax based on the 42 kD merozoite surface
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protein-1 (PvMSP-142), which may represent a sensitive marker for evaluating malaria transmission
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control. From blood samples of patients with P. vivax, we amplified PvMSP-142, obtained the nucleotide sequences, and compared them to homologous sequences of parasites from other
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geographic sites, retrieved from the GenBank. The 92 nucleotide sequences of P. vivax resulted in the resolution of eight haplotypes, six exclusive to Nicaragua. The great nucleotide diversity
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(π=0.020), the minimal recombination events (Rm = 11), and the dN-dS values were similar to other control phase countries. FST values between parasites were low (0.069) for Nicaragua versus Brazil but higher for Nicaragua versus other regions (0.134-0.482). The haplotype network revealed five lineages: two are very frequent in Nicaragua and closely related to American parasites; three have been detected in multiple geographic sites around the world. These results suggest that P. vivax in Nicaragua is a differentiated and genetically diverse population (mainly due to mutation, positive balancing selection and recombination) and that PvMSP-142 may be a sensitive marker for evaluating sustained reduction in malaria transmission and for developing vaccines. 2
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Keywords: Plasmodium vivax, merozoite surface protein-1, Nicaragua, genetic diversity, natural
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selection, genetic differentiation, genealogy.
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1. Introduction
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Plasmodium vivax is widely distributed in Latin-America, Asia, the Middle East and Oceania
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(Guerra et al., 2010){FormattingCitation}{Formatting Citation}. An estimated 25 million people in
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the Americas are considered at high risk. Thirteen countries, including Nicaragua, reported a decline of over 75% in laboratory-confirmed malaria cases between 2000 and 2012 (PAHO, 2012). In 2012,
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the American continent reported 469,380 confirmed malaria cases and 108 malaria deaths (WHO, 2013).
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Currently, several Central American countries are in the control phase, while El Salvador, Costa Rica and Belize are in the pre-elimination phase. In the North Atlantic Autonomous Region
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(RAAN) in Nicaragua, P. vivax and P. falciparum are transmitted throughout the year, and on the
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North Pacific coast transmission is sporadic with periodic outbreaks. The RAAN is adjacent to
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Honduras, and there is a large territorial extension covering both sides of the border where most malaria transmission takes place. In 2012, Honduras and Nicaragua reported 6,434 and 1,235 malaria
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cases, respectively (PAHO, 2012). The current strategy and Plan of Action for Malaria (2011-2025) prioritizes different actions including malaria prevention, surveillance, early case detection, outbreak containment, monitoring and evaluation (Martinelli, 2014). To support these actions, genetic population studies are necessary to identify the haplotypes circulating at each geographic site and determine how they originate, persist and disperse in the region. The degree of genetic diversity and multiple clone infections might determine the transmission intensity and complexity of Plasmodium species (Arnott et al., 2012).
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Although the Mesoamerican region (from central Mexico to northern Costa Rica) shares
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ecological and socioeconomical conditions, there might be a restriction on parasite circulation. For
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instance, the circumsporozoite repeat genotype vk247 highly prevalent in southern Mexico
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(Rodriguez et al., 2000) and Colombia (Hernandez-Martinez et al., 2011) has not been reported in Guatemala (Mendizábal-Cabrera, 2006), Nicaragua (Gonzalez-Ceron et al., 2013) or Honduras (Lopez et al., 2012). In the southern part of Mexico bordering with Guatemala, the different P. vivax
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lineages identified show several hybridization events followed by diversification (Cerritos et al.,
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2014).
Gene markers with high evolutionary dynamics may be useful for explaining how P. vivax
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populations evolve and produce new haplotypes, and for elucidating the changes in haplotype
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frequency (Barry et al., 2015). The gene encoding for the merozoite surface protein-1 (MSP-1) plays an important role in the invasion of the host blood reticulocyte and is an important vaccine candidate
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(Ahlborg et al., 2002; Han et al., 2011; Thakur et al., 2008). It is synthesized as a precursor of the 200 kDa protein during schizogony, and its processing produces four polypeptides of approximately
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83, 30, 38 and 42 kDa (Kang et al., 2010; Miahipour et al., 2012; Putaporntip et al., 2002). During the invasion process, the C-terminal 42 kDa is further processed into two fragments of 33 kDa (MSP-133) and 19 kDa (MSP-119), although only the 19 kDa fragment remains on the merozoite surface (Holder et al., 1992; Kang et al., 2010). In P. vivax, it has been reported that PvMSP-133 is highly polymorphic, while PvMSP-119 is better conserved. Both fragments are capable of inducing blocking antibodies, but PvMSP-142 is more immunogenic than PvMSP-119 (Bastos et al., 2007; Wickramarachchi et al., 2007; Zeyrek et al., 2010).
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The aim of the present study was to analyze the genetic structure of P. vivax in Nicaraguan
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parasites based on the gene fragment pvmsp-142. The corresponding nucleotide sequences from
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parasites of other geographic sites, retrieved from the GenBank data base, were used to compare
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differentiation and the genealogical relationships.
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genetic diversity, natural selection and recombination, and to determine the degree of genetic
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2. Methods
This study was approved by the Ethical Committee of the National Center for Diagnosis and
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National Institute of Public Health.
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Reference (CNDR) of the Health Ministry in Nicaragua, and the Ethical Committee of the Mexican
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2.1. Blood samples and geographic origin Following informed consent, infected blood samples were obtained from symptomatic patients
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seeking malaria diagnosis during 2012 and 2013 in the sentinel laboratory network established by the Nicaraguan Health Ministry at head municipalities: Waspam (50 msnm, 14° 43.994´ N, 83° 56.458´ W), Rosita (66 msnm, 13° 52.004´ N, 84° 23.467´ W), Puerto Cabezas (16msnm, 14° 1.828´ N, 83° 23.089´ W), Bonanza (220 msnm, 14° 1.800´ N, 84° 35.715´ W), Siuna (200 msnm, 13° 44.114´ N, 84° 46.653´ W) and Prinzapolka (22 msnm, 13° 30.589´ N, 84° 13.200´ W) in the North Atlantic Autonomous Region (RAAN); Chinandega (65 msnm, 12° 37.474´ N, 87° 7.802´ W) and El Viejo (50 msnm, 12° 39.769´ N, 87° 9.958´ W) on the North Pacific side. The diagnosis of P. vivax was carried out by microscopic examination of stained thick blood smears, and then patients were asked 6
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to donate capillary blood to impregnated filter paper (Whatman #2). After P. vivax infected blood
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samples were dried in silica gel, they were preserved in that state and protected from light until
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analyzed. Whole genomic DNA was extracted from dried blood spots using a commercially available
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Qiagen Qiamp blood Minikit (Qiagen, USA) following the manufacturer’s instructions. DNA obtained from three punches (0.5 cm in diameter) was eluted in 50 μL of water and stored at -20 °C
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until use. 2.2. PCR amplification and sequencing
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From total genomic DNA, pvmsp-142 was amplified by polymerase chain reaction (PCR) using oligonucleotides: For 5´- GCC GAG GAC TAC GAC AAA G - 3´ and Rev 5´- CCT CCA GCT
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TCC TAA GCT TG - 3´. The PCR reaction was prepared as follows: 2.6μl μL of each primer
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(10pM), 2.6μL of the dNTP mixture (1.25mM), 5.1μL of 10× PCR buffer, 1.3μL of MgSO4 (50mM),
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0.2μL Taq Platinum DNA polymerase (Invitrogen Corporation, Carlsbad, CA), and 4 μL of extracted DNA for a final volume of 50 μL. The PCR reaction conditions were as follows: 5 min at 95 °C, 1 min at 95 °C, 1 min at 60 °C, and 75 sec at 72 °C for 35 cycles; afterwards, there was a final
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extension of 72 °C for 10 min, all run in a MyCycler (BioRad, Hercules, CA, USA). The amplified products were examined in agarose gels at 1%, and stained with 0.2µg/ml ethidium bromide using an electrophoresis chamber Midicell primo (Thermo EC330, New York, USA). The 100bp ladder (Invitrogen Corporation, Carlsbad, CA, USA) was used as molecular marker. The amplified PCR product was purified using a MiniElute PCR Purification Kit (Qiagen, Valencia, CA, USA) following the manufacturer’s instructions. The purified products were Sanger sequenced using forward and reverse primers at the High Throughput Genomics Unit, Department of 7
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Genome Sciences, University of Washington, Seattle, WA, USA. The quality of pherograms with the
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forward and reverse nucleotide sequences was verified manually and by using Bioedit v7.1.3
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software. The consensus sequences obtained for each gene fragment were submitted to the NCBI-
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Gen Bank [accession numbers: KR871926-KR872017]. 2.3. Data analysis
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The nucleotide sequence of pvmsp-142 for the Sal 1 strain (XM_001614792.1) was used as a reference. For genetic analysis, nucleotide sequences from other geographic sites were extracted
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from the NBCI, GenBank. For South Korea (SK), n=200: JQ446312 – JQ446322 (Kang et al., 2012), HQ171934– HQ171941 (Han et al., 2011), AF435635-AF435638. For Vanuatu (VAN), n=2:
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AF435632, AF435634 (Putaporntip et al., 2002). For Turkey (TUK), n=30: AB564559 - AB564588
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(Zeyrek et al., 2010). For Thailand (THL), n=93: AF435595, AF435615 (Putaporntip et al., 2002), GQ890917 – GQ890974 (Jongwutiwes et al., 2010), AF199393- AF199404, AF199408- AF199410
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(Putaporntip et al., 2000). For Singapore (SNG), n=50: GU971656 – GU971705 (Ng et al., 2010). For India-Bangladesh (IND-BG), n= 35: EU430452- EU430479, KF612323; AF435639 (Putaporntip
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et al., 2000), AF435616-AF435620 (Putaporntip et al., 2002). For Brazil (BRZ), n=11: AF43562225,27,29,30,31, AF199405,6,7 (Putaporntip et al., 2000). For Sri Lanka (SLK), n=106: AJ292349AJ292359, GU175174-GU175268 (Dias et al., 2011). For Myanmar (MYN), n= 28: JX490129 JX490156. For China (CHN), n=2: JX993754-JX993755 (Zhou and Chen, unpublished). For Cambodia (CAM), n=27 (Parobek et al., 2014). For Mexico (MEX), n=35 (Gonzalez-Ceron et al., unpublished). 2.4. Population genetic analysis 8
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The genetic diversity was measured by calculating π and θ indexes for the complete DNA sequence
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and by using 100 pb windows assuming different levels of variation across the pvmsp-142 fragment.
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The mean of pairwise nucleotide changes detected along the sequences (using the Jukes-Cantor
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correction) is represented by π (Nei and Li, 1979), while θ is the number of segregating sites in a group of sequences (Watterson, 1975). The number of polymorphic sites (S) and haplotypes (H) and the minimal number of recombination events (Rm) were calculated. These analyses were computed
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using DnaSP software (Librado and Rozas, 2009).
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To test whether positive selection shaped the evolution of the pvmsp-142 subfragment in Nicaraguan parasites and to compare them to those from other geographic sites, the number of
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synonymous (s) and non-synonymous (ns) nucleotide changes and the rate of synonymous versus
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non-synonymous changes (dS-dN) were determined by using the Nei-Gojobori proportion method
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with 1000 bootstrap replicates in MEGA 6.0 software (Tamura et al., 2013). The genetic differentiation of the pvmsp-142 was calculated between parasites of different countries by using Wright’s fixation index (FST) (Wright, 1951). Pairwise fixation values between
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each gene population were calculated using the Kimura 2P model in DnaSPv5.1 software (Rozas et al., 2003). FST values from 0 to 0.05 evidence little or no genetic differentiation; values between 0.05 and 0.15 indicate moderate genetic differentiation, while those between 0.15 and 0.25 represent high genetic differentiation. Finally, values above 0.25 point to very high genetic differentiation (Hartl and Clark, 1989). In order to reconstruct the evolutionary history and establish the genealogical relationships of haplotypes from Nicaraguan with those from other geographic sites, a haplotype network was 9
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constructed using TCS 1.21 (Clement et al., 2000). In this way the connection between haplotypes is
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represented by mutational steps, and the non-sampled or extinct haplotypes are shown by empty
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the size of the circle represents the frequency of each haplotype.
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squares. The color of the circles represents the geographic origins (country) of each haplotype, while
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3. Results
Of 119 samples, 92 were amplified and the consensus sequences were obtained for pvmsp-142.
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Patients from the North Atlantic Autonomous Region (RAAN) provided 85 samples, including those from Rosita (n=31), Bonanza (n=22), Waspam (n=16), Siuna (n=11), Puerto Cabezas (n=3) and
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Prinzapolka (n=2). The other 7 samples were obtained from the North Pacific (NP) coast towns of
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from RAAN and 4 from NP.
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Chinandega (n=2) and El Viejo (n=5). Of the 27 samples that were not amplified by PCR, 23 were
The 92 Nicaraguan pvmsp-142 sequences were comprised of 981 nucleotides had 59
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polymorphic sites (51 non-synonymous and 9 synonymous). All polymorphisms were detected in the pvmsp-133 subfragment between nucleotides 1384 and 2080, while the pvmsp-119 subfragment (nucleotides 2081- 2365) did not present any change (Supplementary table 1). In this group of parasites, there was no sign of mixed genotype infections. 3.1. Genetic diversity and geographic distribution of haplotypes The nucleotide changes evidence the resolution of eight haplotypes, named N1-N8 based on their frequency. Six of them were exclusive to Nicaragua (haplotypes N2, N3, N5, N6, N7 and N8). The 10
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most frequent haplotype (N1) and another (N4) were previously detected in other countries. N1 was
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detected in all municipalities from RAAN as well as in parasites from a nearby country (MEX),
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while N4 was identified in parasites from South America (BRZ) and outside the Americas (IND,
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SNG, THL, CAM and MYN). Haplotype N2 was detected in all Nicaraguan locations, while N1, N3 and N5 were found exclusively in the RAAN. There were three single haplotypes (N6, N7, and N8).
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N6 and N7 were detected in Rosita and N8 in Chinandega (Figure 1).
The nucleotide diversity of the pvmsp-142 in P. vivax of Nicaragua (π = 0.020) was lesser than
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that of P. vivax in BRZ, CAM, IND-BNG, SLK, THL and MYN and greater than that of this species in MEX, SK and SNG. The lowest diversity was reported in TUR. At a global level, 99 haplotypes
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have been found with a total of 711 nucleotide sequences, representing great diversity (π = 0.024 and
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θ = 0.017) (Table 1). Sliding window analysis, used to compare genetic diversity across pvmsp-142,
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detected the highest values for π and θ in the window comprising nucleotides 1735 to 1834, followed by the adjacent windows. Only in Nicaraguan isolates (not at other geographic sites) did the window
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comprising nucleotides 1935-2034 show great diversity (Supplementary figure 1). 3.2. Natural selection and recombination In Nicaraguan isolates, non-synonymous substitutions were more numerous than synonymous ones in subfragment pvmsp-133, as found in parasites from other countries. Accordingly, the dN-dS value was positive but not significant (0.779; p=0.426). In other geographic locations this value varied from 0.403 in TUR to 4.087 in SK, being significantly positive for parasites found in SK, CAM, THL, SNG and MYN (Table 1).
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The N8 haplotype presented eight of the nine total synonymous changes detected in the
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nucleotide sequences, including codons 1476, 1509, 1528, 1530, 1533, 1538, 1564 and 1570
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(Supplementary table 1). The minimal number of recombination events for pvmsp-142 in Nicaraguan
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parasites was similar (Rm = 11) to that calculated for some other geographic sites such as SNG, IND, THL and MYN (Rm = 11-12), followed by MEX (Rm = 9) > SK (Rm = 4) > TUR (Rm = 0) (Table
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1). 3.3. Genetic differentiation (FST)
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Based on pvmsp-142, FST values ranged from 0.069 to 0.481 between parasites of Nicaragua and other countries (Table 2). The lowest values were exhibited with NIC versus BRZ, followed by NIC
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versus SLK or IND-BNG. Between NIC and its nearest neighbor (MEX) the differentiation value
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was high (0.231). The highest values (0.363-0.481) were found when comparing NIC and SNG, SK
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or TUR. Also, TUR (0.14-0.502) and SK (0.11-502) had high values when compared to other geographic sites. Finally, the highest differentiation value was between TUR and SK.
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3.4. Haplotype network
Genealogical reconstruction revealed five lineages among the eight haplotypes found in Nicaragua (Figure 2). Three of them were more frequent, while the other two comprised many parasites of distinct geographic origins. Haplotype N1, also detected in MEX (two isolates), was at one mutational step from other MEX haplotype and four mutational steps from N6 and likely comprised one lineage. Haplotypes N2, N3 and N8 constituted a different lineage and were separated by four mutational steps from a Mexican parasite (see also supplementary Table 1). Haplotype N3 was separated from N2 by two mutational step and haplotype N8 from N2 by three mutational 12
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steps. Haplotype N5 was separated by five mutational steps from IND, and by 13 from a very
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frequent haplotype shared by American (MEX, BRZ) and non-American (MYN and TUR) parasites.
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The Nicaraguan haplotype N4, also detected in BRZ, CAM, IND-BNG, SNG, THL and MYN,
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was at 28 mutational steps from N7, which in turn was related to parasites from BRZ, CAM, THL and MYN. N4 and N7 represented different genealogical clusters. There were detected signs of local diversification in SNG and THL, and at less degree in NIC. Also, several loops apparent in the
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network were observed, likely due to recombination events.
4. Discussion
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The results suggest that in Nicaragua P. vivax showed a differentiated population with moderate genetic diversity. Patterns of genetic diversity and the intensity of selection for pvmsp-142 were
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similar to what was found in other countries under the control phase. The five genealogical groups represent different relationships, likely the result of recombination events, parasite migration, and the
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long lasting transmission in the region. Historically, malaria has been an endemic disease in Nicaragua. Both P. falciparum and P. vivax are transmitted in the affected regions, with at least 80% of the confirmed cases being caused by P. vivax (PAHO, 2012). Currently, Nicaragua is under the control phase, and it is expected to advance towards the pre-elimination phase shortly. However, in the North Atlantic Autonomous Region (RAAN) malaria transmission has been more persistent than at other sites. In fact, more than 90% of samples herein analyzed were from this region.
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RAAN is the largest department in Nicaragua, comprising about 33,000 km² and having a
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population of about 250,000 (2005 census) representing different indigenous groups (INIDE, 2015).
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In addition, this region borders with Honduras along the Coco River. In fact, both countries share a
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large area affected by malaria transmission (PAHO, 2012). Nicaragua has marked migration movements of its population, mainly across the border region of RAAN. Undocumented people traffic by land and air (OIM, 2013). The number of malaria cases in Nicaragua decreased between
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1996 and 2012 from 70,000 to 999, while in Honduras the decrease was slightly less prominent
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(from 73,313 to 5,000 during the same period). Lower incidence and sporadic transmission have been reported on the Pacific coast, bordering with El Salvador (PAHO, 2012).
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Furthermore, in Nicaragua malaria transmission occurs in remote communities in which
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social and economic deficiencies cause difficulties for disease diagnosis and treatment. This dynamic of transmission contributes to the maintenance of the genetic diversity found with P. vivax in the
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region. Honduras, the neighbor to the north, is also under the control phase and has reported high diversity in different gene markers, including pvama-1, pvcsp and pvmsp-1 (Lopez et al., 2012).
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When considering geographic sites in the pre-elimination phase (e.g., MEX), P. vivax showed lower diversity than in NIC. TUR had the lowest diversity in P. vivax samples, which were obtained during 2007-2008 (Zeyrek et al., 2010). Since 2000, in this country, the number of malaria cases has undergone a gradual decrease, and in 2010 reached the elimination phase (Özbilgin et al., 2011; WHO, 2010). This suggests that if low numbers of malaria cases are sustained for several years, parasite diversity may decrease significantly. The Mexican region bordering Guatemala shares a haplotype (N1) with Nicaragua. Whereas the frequency of N1 is high in Nicaragua (42%), it is low in MEX (5.7%). At present, the probability 14
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that this parasite will increase in Mexican territory due to migration is quite low, as migrants spend
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few days in this border region. Indeed, only asymptomatic P. vivax seropositive individuals were
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detected (Betanzos-Reyes et al., 2012).
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Currently, the P. falciparum vaccine based on PfMSP-142 is under clinical trials (Ellis et al., 2010; Huaman et al., 2008; Malkin et al., 2007; Ogutu et al., 2009). Similarly, PvMSP-142 is also a
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good vaccine candidate against blood stages. However, its application might be limited by global and local diversity. Six of the eight haplotypes herein detected were exclusive to Nicaragua, and local
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diversification might have occurred as in other regions under the control phase. For instance, there was an exclusive and divergent haplotype (N8) in the municipality of Chinandega, with unique
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characteristics given by the high diversity between codons 1476 and 1570 as well as by the presence
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of eight synonymous changes. There is a possibility that N8 diverged from N2 on the Central American region, supported by the fact that N2, N3 and N8 formed a group unique to Nicaragua.
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Since haplotype N2 is widespread in all affected regions of the present study, this high frequency might be the result of its successful transmission. The accelerated and inherent evolutionary
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dynamics of P. vivax merozoite genes mainly due to positive balancing selection and recombination might require periodic monitoring of the ongoing antigenic diversity of the parasite and vaccine modifications (Kang et al., 2012). Genealogical reconstruction based on the haplotype network suggests the presence of five P. vivax lineages in Nicaragua, separated by many mutational steps and closely related to haplotypes reported in different regions inside and outside the Americas. However, there are still gaps in our knowledge about genealogical reconstruction. For example, the importance of recombination versus mutation in the generation of the divergent haplotypes is still unknown. In order to understand the 15
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transmission process, pathogenesis and drug resistance of P. vivax, it is necessary to gain insights
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into its origin, way of dispersion and genealogical relationships. The mitochondrial DNA analysis
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shows that parasites from Central America and South America are closely related (FST = 0.016; not
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significant above zero). Indeed, the recent parasite introduction was predicted for Central America along this and other old migration routes between continents (Taylor et al., 2013). Accordingly, in this study, the FST value between parasites from NIC and BRZ (using pvmsp-142) was much lower
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(0.069) than that between NIC and other geographic sites. Further studies using mitochondrial DNA
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are required to clarify the higher FST values obtained between NIC and MEX. It is important to consider that pvmsp-1 is highly recombinant (Putaporntip et al., 2002), and
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that some haplotypes detected herein could have been generated by recombination. The pvmsp-142
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fragment showed 11 minimal recombination events in the Nicaraguan sample. Pvmsp-142 was recently classified into 12 global groups without geographic correlation, evidenced by the
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heterogeneity shown by the phylogenetic analysis (Dias et al., 2011). The previous finding was confirmed by the genealogical reconstruction of the present study: the most abundant haplotypes
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from Nicaragua (N1, N2 and N3) were genetically related to haplotypes from Mexico and are located in groups separated by few mutation steps (from none to four steps). On the other hand, N7 was closely related to a haplotype from Brazil (along with other countries). Since malaria control campaigns can drastically modify haplotype frequency within the region herein studied, genetic parameters should be incorporated into monitoring with the aim of facilitating the advance to the upcoming pre-elimination or elimination phase in Nicaragua. Besides its importance as a vaccine candidate, pvmsp142 offers a good and sensitive genetic marker that is 16
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under different evolutionary forces, including mutation, positive selection and high levels of
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recombination.
Acknowledgments
This work was supported by CONACyT-Mexico (project CB-2009-01-131247), AMI/RAVEDRA-
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OPS/OMS-Nicaragua, and global fund-malaria component-Nicaragua (2012-2013). We are grateful
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to the lab personnel at the sentinel sites in Nicaragua that collaborated to prepare the P. vivax blood samples on filter paper. We give special thanks to Valentina Manzanares in Siuna, Martha Lopez in
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Rosita, and Dexter Manzanares in Waspam, as well as the local authorities for the use of the
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facilities. Thanks to Valeria Zermeño for assistance in manuscript revision, and to Frida Santillán
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assistance.
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and Olga L. Paloqueme at the Regional Center for Research in Public Health in Mexico for technical
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5. References
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Ahlborg, N., Ling, I.T., Howard, W., Holder, A.A., Riley, E.M., 2002. Protective Immune Responses to the 42-Kilodalton (kDa) Region of Plasmodium yoelii Merozoite Surface Protein 1 Are Induced by the C-Terminal 19-kDa Region but Not by the Adjacent 33-kDa Region. Infect. Immun. 70, 820825.
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Arnott, A., Barry, A.E., Reeder, J.C., 2012. Understanding the population genetics of Plasmodium vivax is essential for malaria control and elimination. Malar. J. 11, 14.
NU
Barry, A.E., Waltmann, A., Koepfli, C., Barnadas, C., Mueller, I., 2015. Uncovering the transmission dynamics of Plasmodium vivax using population genetics. Pathog. Glob. Health 109, 142-152.
MA
Bastos, M.S., da Silva-Nunes, M., Malafronte, R.S., Hoffmann, E.H., Wunderlich, G., Moraes, S.L., Ferreira, M.U., 2007. Antigenic polymorphism and naturally acquired antibodies to Plasmodium vivax merozoite surface protein 1 in rural Amazonians. Clin. Vaccine Immunol. 14, 1249-1259. Betanzos-Reyes, A.F., González-Cerón, L., Rodríguez, M.H., Torres-Monzón, J.A., 2012. Malaria's seroepidemiology in a group of migrants in transit (Chiapas, 2008). Salud Publica Mex. 54, 523-529.
TE
D
Cerritos, R., González-Cerón, L., Nettel, J.A., Wegier, A., 2014. Genetic structure of Plasmodium vivax using the merozoite surface protein 1 icb5-6 fragment reveals new hybrid haplotypes in southern Mexico. Malar. J. 13, 35-35.
CE P
Clement, M., Posada, D., Crandall, K.A., 2000. TCS: a computer program to estimate gene genealogies. Mol. Ecol. 9, 1657-1659.
AC
Dias, S., Longacre, S., Escalante, A.A., Udagama-Randeniya, P.V., 2011. Genetic diversity and recombination at the C-terminal fragment of the merozoite surface protein-1 of Plasmodium vivax (PvMSP-1) in Sri Lanka. Infect. Genet. Evol. 11, 145-156. Ellis, R.D., Sagara, I., Doumbo, O., Wu, Y., 2010. Blood stage vaccies for Plasmodium falciparum. Current status and the way forward. Hum. Vaccin. 6, 627-634. Gonzalez-Ceron, L., Martinez-Barnetche, J., Montero-Solis, C., Santillan, F., Soto, A.M., Rodriguez, M.H., Espinosa, B.J., Chavez, O.A., 2013. Molecular epidemiology of Plasmodium vivax in Latin America: polymorphism and evolutionary relationships of the circumsporozoite gene. Malar. J. 12, 243. Guerra, C.A., Howes, R.E., Patil, A.P., Gething, P.W., Van Boeckel, T.P., Temperley, W.H., Kabaria, C.W., Tatem, A.J., Manh, B.H., Elyazar, I.R., Baird, J.K., Snow, R.W., Hay, S.I., 2010. The international limits and population at risk of Plasmodium vivax transmission in 2009. PLoS Negl. Trop. Dis. 4, e774. 18
ACCEPTED MANUSCRIPT
T
Han, E.-T., Wang, Y., Lim, C.S., Cho, J.H., Chai, J.-Y., 2011. Genetic diversity of the malaria vacine candidate merozoite surface protein 1 gene of Plasmodium vivax field isolates in Republic of Korea. Parasitol. Res. 109, 1571-1576.
IP
Hartl, D.L., Clark, A.G., 1989. Principles of Population Genetics in: Associates, S. (Ed.), Sutherland, Massachusetts, pp. 118-119.
SC R
Hernandez-Martinez, M.A., Escalante, A.A., Arevalo-Herrera, M., Herrera, S., 2011. Antigenic diversity of the Plasmodium vivax circumsporozoite protein in parasite isolates of Western Colombia. Am. J. Trop. Med. Hyg. 84, 51-57.
NU
Holder, A.A., Blackman, M.J., Burghaus, P.A., Chappel, J.A., Ling, I.T., McCallum-Deighton, N., Shai, S., 1992. A malaria merozoite surface protein (MSP1) structure, processing and function. Mem Inst Oswaldo Cruz 87 (3), 37-42.
MA
Huaman, M.C., Martin, L.B., Malkin, E., Narum, D.L., Miller, L.H., Mahanty, S., Long, C.A., 2008. Ex Vivo Cytokine and Memory T Cell Responses to The 42-kDa Fragment of Plasmodium falciparum Merozoite Surface Protein-1 in Vaccinated Volunteers. The Journal of Immunology 180, 1451-1461.
TE
D
Instituto Nacional de información de Desarrollo de Nicaragua, INIDE; census 2015. http://www.inide.gob.ni
CE P
Jongwutiwes, S., Putaporntip, C., Hughes, A.L., 2010. Bottleneck Effects on Vaccine-Candidate Antigen Diversity of Malaria Parasites in Thailand. Vaccine 28, 3112-3117.
AC
Kang, J.-M., Ju, H.-L., Kang, Y.-M., Lee, D.-H., Moon, S.-U., Sohn, W.-M., Park, J.-W., Kim, T.-S., Na, B.-K., 2012. Genetic polymorphism and natural selection in the C-terminal 42 kDa region of merozoite surface protein-1 among Plasmodium vivax Korean isolates. Malar. J. 11, 1-9. Kang, J.M., Moon, S.U., Kim, J.Y., Cho, S.H., Lin, K., Sohn, W.M., Kim, T.S., Na, B.K., 2010. Genetic polymorphism of merozoite surface protein-1 and merozoite surface protein-2 in Plasmodium falciparum field isolates from Myanmar. Malar. J. 9, 131. Librado, P., Rozas, J., 2009. DnaSP v5: a software for comprehensive analysis of DNA polymorphism data. Bioinformatics 25, 1451-1452. Lopez, A.C., Ortiz, A., Coello, J., Sosa-Ochoa, W., Torres, R.E., Banegas, E.I., Jovel, I., Fontecha, G.A., 2012. Genetic diversity of Plasmodium vivax and Plasmodium falciparum in Honduras. Malar. J. 11, 391. Malkin, E., Long, C.A., Stowers, A.W., Zou, L., Singh, S., MacDonald, N.J., Narum, D.L., Miles, A.P., Orcutt, A.C., Muratova, O., Moretz, S.E., Zhou, H., Diouf, A., Fay, M., Tierney, E., Leese, P., 19
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Mahanty, S., Miller, L.H., Saul, A., Martin, L.B., 2007. Phase 1 Study of Two Merozoite Surface Protein 1 (MSP1(42)) Vaccines for Plasmodium falciparum Malaria. PLoS Clin. Trials 2, e12.
IP
T
Martinelli, S., 2014. Malaria Free by 2025: A Global Fund Regional Initiative. Friends of the Global Fight Washington, DC. link: http://theglobalfight.org/malaria-free-by-2025-mesoamerica-hispaniolaregional-initiative last accessed August 25, 2015.
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Mendizábal-Cabrera, R., and Padilla, N., 2006. Diversidad genética de Plasmodium vivax en regiones de alto riesgo de malaria en Guatemala [Genetic Diversity of Plasmodium vivax in areas of high risk of malaria in Guatemala], Revista de la Universidad del Valle de Guatemala, Guatemala, pp. 62-79.
MA
NU
Miahipour, A., Keshavarz, H., Heidari, A., Raeisi, A., Rezaeian, M., Rezaie, S., 2012. Genetic Variation of MSP-1 Gene in Plasmodium vivax Isolated from Patients in Hormozgan Province, Iran using SSCP-PCR. Iran J. Parasitol. 7, 1-7. Nei, M., Li, W.H., 1979. Mathematical model for studying genetic variation in terms of restriction endonucleases. Proc. Natl. Acad. Sci. U. S. A. 76, 5269-5273.
TE
D
Ng, L.C., Lee, K.S., Tan, C.H., Ooi, P.L., Lam-Phua, S.G., Lin, R., Pang, S.C., Lai, Y.L., Solhan, S., Chan, P.P., Wong, K.Y., Ho, S.T., Vythilingam, I., 2010. Entomologic and molecular investigation into Plasmodium vivax transmission in Singapore, 2009. Malar. J. 9, 305.
AC
CE P
Ogutu, B.R., Apollo, O.J., McKinney, D., Okoth, W., Siangla, J., Dubovsky, F., Tucker, K., Waitumbi, J.N., Diggs, C., Wittes, J., Malkin, E., Leach, A., Soisson, L.A., Milman, J.B., Otieno, L., Holland, C.A., Polhemus, M., Remich, S.A., Ockenhouse, C.F., Cohen, J., Ballou, W.R., Martin, S.K., Angov, E., Stewart, V.A., Lyon, J.A., Heppner, D.G., Withers, M.R., for the, M.S.P.M.V.W.G., 2009. Blood Stage Malaria Vaccine Eliciting High Antigen-Specific Antibody Concentrations Confers No Protection to Young Children in Western Kenya. PLoS ONE 4, e4708. Organización Internacional para las Migraciones OIM, 2013. Perfil Migratorio de Nicaragua 2012. http://migracion-ue- alc.eu/documents/keydocs /ES/Perfil_ Migratorio_Nicaragua_2012.pdf Özbilgin, A., Topluoglu, S., Es, S., Islek, E., Mollahaliloglu, S., Erkoc, Y., 2011. Malaria in Turkey: Successful control and strategies for achieving elimination. Acta Trop. 120, 15-23. Pan American Health Organization PAHO, 2012. Interactive Malaria Statistics. http://www.paho.org/hq/index.php?option=com_content&view=article&id=2632&Itemid=2130&lan g=en. Parobek, C.M., Bailey, J.A., Hathaway, N.J., Socheat, D., Rogers, W.O., Juliano, J.J., 2014. Differing Patterns of Selection and Geospatial Genetic Diversity within Two Leading Plasmodium vivax Candidate Vaccine Antigens. PLoS Negl. Trop. Dis. 8, e2796. 20
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Putaporntip, C., Jongwutiwes, S., Sakihama, N., Ferreira, M.U., Kho, W.G., Kaneko, A., Kanbara, H., Hattori, T., Tanabe, K., 2002. Mosaic organization and heterogeneity in frequency of allelic recombination of the Plasmodium vivax merozoite surface protein-1 locus. Proc. Natl. Acad. Sci. U. S. A. 99, 16348-16353.
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IP
Putaporntip, C., Jongwutiwes, S., Seethamchai, S., Kanbara, H., Tanabe, K., 2000. Intragenic recombination in the 3′ portion of the merozoite surface protein 1 gene of Plasmodium vivax. Mol. Biochem. Parasitol. 109, 111-119.
NU
Rodriguez, M.H., Gonzalez-Ceron, L., Hernandez, J.E., Nettel, J.A., Villarreal, C., Kain, K.C., Wirtz, R.A., 2000. Different prevalences of Plasmodium vivax phenotypes VK210 and VK247 associated with the distribution of Anopheles albimanus and Anopheles pseudopunctipennis in Mexico. Am. J. Trop. Med. Hyg. 62, 122-127.
MA
Rozas, J., Sanchez-del Barrio, J.C., Messeguer, X., Rozas, R., 2003. DnaSP, DNA polymorphism analyses by the coalescent and other methods. Bioinformatics 19, 2496-2497. Tamura, K., Stecher, G., Peterson, D., Filipski, A., Kumar, S., 2013. MEGA6: Molecular Evolutionary Genetics Analysis Version 6.0. Mol. Biol. Evol. 30, 2725-2729.
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D
Taylor, J.E., Pacheco, M.A., Bacon, D.J., Beg, M.A., Machado, R.L., Fairhurst, R.M., Herrera, S., Kim, J.-Y., Menard, D., Póvoa, M.M., Villegas, L., Mulyanto, Snounou, G., Cui, L., Zeyrek, F.Y., Escalante, A.A., 2013. The Evolutionary History of Plasmodium vivax as Inferred from Mitochondrial Genomes: Parasite Genetic Diversity in the Americas. Mol. Biol. Evol. 30, 20502064.
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Thakur, A., Alam, M.T., Sharma, Y.D., 2008. Genetic diversity in the C-terminal 42 kDa region of merozoite surface protein-1 of Plasmodium vivax (PvMSP-1(42)) among Indian isolates. Acta Trop. 108, 58-63. Watterson, G.A., 1975. On the number of segregating sites in genetical models without recombination. Theor. Popul. Biol. 7, 256-276. Wickramarachchi, T., Illeperuma, R.J., Perera, L., Bandara, S., Holm, I., Longacre, S., Handunnetti, S.M., Udagama-Randeniya, P.V., 2007. Comparison of naturally acquired antibody responses against the C-terminal processing products of Plasmodium vivax Merozoite Surface Protein-1 under low transmission and unstable malaria conditions in Sri Lanka. Int. J. Parasitol. 37, 199-208. World Health Organizatio WHO, 2010. World Malaria report 2010. Geneva, Switzerland. http://www.who.int/malaria/world_malaria_report_2010/en/ World Health Organization WHO, 2013. World Malaria Report 2013. Geneva, Switzerland. http://www.who.int/malaria/world_malaria_report_2013/en/ 21
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Wright, S., 1951. The genetical structure of populations. Ann. Eugenics 15, 323-354.
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Zeyrek, F.Y., Tachibana, S.-I., Yuksel, F., Doni, N., Palacpac, N., Arisue, N., Horii, T., Coban, C., Tanabe, K., 2010. Limited Polymorphism of the Plasmodium vivax Merozoite Surface Protein 1 Gene in Isolates from Turkey. Am. J. Trop. Med. Hyg. 83, 1230-1237.
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Figure legends
Figure 1. Geographic distribution of the pvmsp-142 haplotypes in Nicaragua. Each haplotype is
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distinguished by grayscale/grid/stripe. The haplotype frequency is indicated by the circles. The RAAN municipalities include: R) Rosita, B) Bonanza, W) Waspam, S) Siuna, C) Puerto Cabezas, and P) Prinzapolka. The North Pacific coast is represented by G) Chinandega. n= the number of isolates. Figure 2. Haplotype network of pvmsp142. Each cycle corresponds to one haplotype and countries are indicated by different colors. Related genotypes are connected by lines, and squares and
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additional mutational steps between haplotypes are indicated by small diamonds. Nicaraguan
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haplotypes, named from N1 to N8, represent five different lineages.
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Fig. 1
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Fig. 2
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Diversity indexes π Ө
Recombination Rm
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8 0.020 0.013 11 NIC 92 8 0.018 0.014 9 MEX 35 8 0.023 0.020 10 BRZ 11 10 0.017 0.009 4 SK 196 22 0.023 0.016 11 CAM 27 30 0.024 0.017 14 IND-BNG 35 47 0.024 0.014 23 SLK 106 43 0.023 0.012 12 THL 93 3 0.007 0.007 0 TUR 30 27 0.019 0.022 12 SNG 50 26 0.024 0.017 13 MYN 28 188 0.025 0.019 24 Total 702 *Significance at 95% IC; N, number of isolates; H, number of haplotypes
Selection dN-dS P* 0.779 0.426 1.275 0.205 0.972 0.333 4.087 0 2.251 0.026 1.931 0.056 1.523 0.130 2.155 0.033 0.403 0.688 2.638 0.008 2.218 0.028 2.061 0.041
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Table 1. The genetic diversity, recombination and natural selection of Pvmsp-142 from Nicaragua compared to the same parameters found at other geographic sites. Similar indexes were shown in almost all countries.
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0.275 0.093 0.099 0.234 0.087 0.174 0.140 0.202 0.170
0.111 0.218 0.320 0.206 0.272 0.502 0.167 0.216
0.043 0.127 0.012 0.048 0.307 0.022 0.019
0.275 0.038 0.113 0.355 0.070 0.097
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0.380 0.049 0.009
0.420 0.360
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0.153 0.101 0.406 0.180 0.097
0.037 0.298 0.061 0.027
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NIC 0.231 0.384 0.183 0.069 0.363 0.134 0.202 0.481 0.225 0.190
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Table 2. FST indexes when comparing the pvmsp-142 of parasites in Nicaraguan versus those at other geographic sites.
NIC, Nicaragua; MEX, Mexico; BRZ, Brazil; SK, South Korea; IND-BNG, India-Bangladesh; SNG, Singapore; SLK, SriLanka; THL, Thailand; TUR, Turkey; CAM, Cambodia; MYN, Myanmar. The lowest
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Highlights
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1. The pvmsp142 genetic diversity in Nicaragua was lower than other countries in control phase.
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2. Pvmsp133 exposed strong positive and negative selection, and pvmsp119 was conserved. 3. Nicaraguan haplotypes reveals processes of migration and local diversification.
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4. P. vivax in Nicaragua shows a differentiated population and micro heterogeneity.
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5. The haplotype network exposed five Nicaraguan lineages.
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