Spotlights
Tracking malaria parasites in the eradication era Marcelo U. Ferreira and Priscila T. Rodrigues Department of Parasitology, Institute of Biomedical Sciences, University of Sa˜o Paulo, Av. Prof. Lineu Prestes 1374, 05508-900 Sa˜o Paulo (SP), Brazil
As more endemic countries enter the elimination phase, the detection of sporadic malaria infections or outbreaks elicits many questions: (i) are the infections locally acquired or imported? (ii) If imported, where do they come from? (iii) Do outbreak strains have a single or multiple geographic origins? New molecular barcoding methods provide ways to analyze clinical malaria parasite samples and answer these and other crucial questions. Conservation biology of endangered animals and malaria eradication programs entail similar analytical strategies, despite their diametrically opposite aims [1]. For example, DNA analysis of illegal African elephant ivory shipments seized on the way to the Far East has now became the standard method to locate the original sites of elephant poaching, helping to elucidate ivory smuggling routes in Africa [2]. To guide public health interventions, malariologists are often interested in understanding how new parasite phenotypes (such as drug resistance, increased virulence, or novel antigenic variants) originate and spread in human populations. Furthermore, accurate and reproducible methods to characterize parasites and assign strains to specific regions and countries are required to design strategies to prevent malaria reintroduction in areas approaching elimination that remain vulnerable to malaria resurgences. Preston and colleagues [3] have recently developed a novel molecular genotyping strategy to identify the geographic origin of Plasmodium falciparum strains. They started by analyzing the full-length genomes of two organelles, the mitochondrion (6 kb) and the apicoplast (29.4 kb), from over 700 P. falciparum isolates originating from 14 countries, mostly from Africa. They next used an interactive haplotype search algorithm to select the 23 most informative single nucleotide polymorphisms (SNPs), five from the mitochondrial and 18 from the apicoplast genome, with the aim of developing a molecular barcode. Because mitochondrial and apicoplast lineages are uniparentally inherited, they do not recombine with each other and can thus provide more stable region-specific genotypes than nuclear genome SNPs [3]. The 23-SNP barcoding strategy was 92% accurate in identifying the continental origin of P. falciparum samples from West and East Africa, Southeast Asia, Oceania, and South America. Most
Corresponding author: Ferreira, M.U. ([email protected], [email protected]) Keywords: malaria; genotyping; eradication; single-nucleotide polymorphism; microsatellites. 1471-4922/ ß 2014 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.pt.2014.08.003
(26 of 34) distinct haplotypes characterized by genotyping were unique to a single region [3]. A global map of sequence diversity is crucial for assigning P. falciparum organellar genomes to specific regions of malaria endemicity. One of the limitations of the current 23-SNP barcode is the lack of reference DNA sequences from sites such as South Asia, Central America and the Caribbean, and southern Africa [3]. South Asia is a particularly challenging region, due to the lack of mitochondrial, and especially of apicoplast, reference genomes in open access databases. Moreover, the existence of two divergent genetic clusters (‘southern’ and ‘northern’) of P. falciparum isolates in South America, and putatively also in Central America and the Caribbean, further complicates the geographic assignment of samples from this region. These clusters may have originated from separate colonization events associated with the slave trade [4]. Similar issues will arise when applying the molecular barcoding approach to other human malaria parasites, such as Plasmodium vivax. Although many P. vivax mitochondrial genomes cluster according to the geographic origin of the isolates, those from South Asia, Central Asia, Africa and the Middle East are closely related to each other and fail to provide population-specific molecular barcodes. Therefore, mitochondrial genome analysis lacks geographic resolution for assigning many imported P. vivax infections to their regions of origin [5]. As only three P. vivax apicoplast genomes have been sequenced, no comprehensive organellar SNP panel is currently available for this species. The next crucial step for the further development and large-scale use of malaria barcoding approaches consists of creating and maintaining a comprehensive and freely accessible database of geo-referenced sequences. The ever-growing multilocus sequencing typing (MLST) research community offers a success story in this field. Microbiologists created curated DNA sequence databases for molecular epidemiologic surveillance of several human pathogens. MLST databases comprise thousands of 450 to 500 bp DNA sequences of known origin derived from six or seven housekeeping genes per organism. Databases are currently available online for 75 bacteria, three fungi, and one protozoon [6]. Showing the potential utility of molecular barcoding in academic research and epidemiologic surveillance will hopefully further stimulate contributions of reference DNA sequences to the open access databases, resulting in improved accuracy for this technique in tracing the origin of strains. Conventional phylogeny-based tracing techniques can help to determine which reference site a malaria sample matches best [3,5]. However, while a global database of Trends in Parasitology, October 2014, Vol. 30, No. 10
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Spotlights Box 1. Molecular characterization of malaria outbreaks For an epidemiologist, it is crucial to determine whether all infections diagnosed during an outbreak had a single or multiple origins in order to design proper containment strategies. DNA analysis can help in this task. The short-term spread of a single or a few strains in a community can originate a malaria outbreak, as recently documented in an area of declining malaria transmission in rural Amazonia [7]. The characterization of P. vivax isolates collected in a single farming settlement over three years, with 14 highly polymorphic microsatellite markers, revealed the lowest parasite diversity, with the smallest proportion of multiple-clone infections and the strongest linkage disequilibrium at the time of the outbreak. All features are consistent with a clonal expansion of outbreak strain(s). Alternatively, outbreaks can involve several genetically divergent strains, with putatively multiple geographic origins. The first example comes from the 2011 P. vivax outbreak in Evrotas, Greece; 27 samples were genotyped with three repetitive DNA markers, revealing at least six different haplotypes co-circulating in the area [8]. The second example of multiple-strain P. vivax outbreak comes from Trincomalee, Eastern Province of Sri Lanka; both microsatellite [9] and nuclear SNP genotyping [10] revealed surprisingly high levels of diversity, with no single haplotype being shared between samples. These findings argue for multiple independent introductions of P. vivax strains into these receptive areas, with major implications for malaria surveillance and prevention of re-emergence. In fact, P. vivax populations in Sri Lanka remained highly diverse over the past decade, despite the dramatic decline in malaria transmission countrywide [9]. These data are consistent with strains of various origins being continuously introduced in the country or, alternatively, with genetically diverse strains being maintained in a large asymptomatic reservoir within the country.
malaria parasite variation can comprise a large number of reference sites, not all areas of malaria endemicity in more than 100 countries and territories are included. Consequently, geo-statistical models are needed to assign individual infections to locations where no reference DNA sequences are available. Conservation biologists apply spatial smoothing methods to infer allele frequencies across the entire area of distribution of African elephants, using genotypes of reference samples from a limited number of geo-referenced sites [2]. Validating analogous geo-statistical approaches for tracing malaria parasites worldwide would be crucial for further advancing the field. Genetic analyses can help to elucidate the origin and routes of dissemination of parasite strains and to define the target area for public health interventions during malaria outbreaks (Box 1). However, the 23-SNP barcode is unlikely to reveal subtle differences between local and imported P. falciparum strains. In other words, it may lack genetic resolution to distinguish between ongoing autochthonous transmission and malaria infections imported from one or
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more nearby locations. Highly polymorphic microsatellites (tandem repeats of motifs of one to six nucleotides) potentially offer a greater genetic resolution for outbreak investigation. These are fast-evolving markers, with 10 3 to 10 4 mutations per locus per generation mostly caused by strandslippage events during DNA replication. Microsatellites are useful to characterize community-, country- or region-level genetic diversity over relatively short periods of time, as often required in outbreak investigations, but they do not necessarily match everyone’s needs. More stable markers, such as nuclear and organellar SNPs, are better suited for tracing the origin and dispersal of parasite strains across and between continents. It is now clear that no single barcoding strategy will provide all answers. Public health specialists must be familiar with the wide range of parasite typing methods and their potential applications in different settings. The 23-SNP barcode developed by Preston and colleagues [3] represents a welcome complement to our arsenal of genotyping tools for malaria parasites. Acknowledgments The authors received scholarships from the Conselho Nacional de Desenvolvimento Cientı´fico e Tecnolo´gico (CNPq), Brazil.
References 1 Nkhoma, S.C. et al. (2013) Population genetic correlates of declining transmission in a human pathogen. Mol. Ecol. 22, 273–285 2 Wasser, S.K. et al. (2007) Using DNA to track the origin of the largest ivory seizure since the 1989 trade ban. Proc. Natl. Acad. Sci. U.S.A. 104, 4228–4233 3 Preston, M.D. et al. (2014) A barcode of organellar genome polymorphisms identifies the geographic origin of Plasmodium falciparum strains. Nat. Commun. 5, 4052 4 Yalcindag, E. et al. (2012) Multiple independent introductions of Plasmodium falciparum in South America. Proc. Natl. Acad. Sci. U.S.A. 109, 511–516 5 Rodrigues, P.T. et al. (2014) Using mitochondrial genome sequences to track the origin of imported Plasmodium vivax infections diagnosed in the United States. Am. J. Trop. Med. Hyg. 90, 1102–1108 6 Pe´rez-Losada, M. et al. (2013) Pathogen typing in the genomics era: MLST and the future of molecular epidemiology. Infect. Genet. Evol. 16, 38–53 7 Batista, C.L. et al. (2014) Genetic diversity of Plasmodium vivax over time and space: a community-based study in rural Amazonia. Parasitology http://dx.doi.org/10.1017/S0031182014001176 8 Spanakos, G. et al. (2013) Genotyping Plasmodium vivax isolates from the 2011 outbreak in Greece. Malar. J. 12, 463 9 Gunawardena, S. et al. (2014) The Sri Lankan paradox: high genetic diversity in Plasmodium vivax populations despite decreasing levels of malaria transmission. Parasitology 141, 880–890 10 Orjuela-Sa´nchez, P. et al. (2010) Single-nucleotide polymorphism, linkage disequilibrium and geographic structure in the malaria parasite Plasmodium vivax: prospects for genome-wide association studies. BMC Genet. 11, 65
Tracking malaria parasites in the eradication era Marcelo U. Ferreira and Priscila T. Rodrigues Department of Parasitology, Institute of Biomedical Sciences, University of Sa˜o Paulo, Av. Prof. Lineu Prestes 1374, 05508-900 Sa˜o Paulo (SP), Brazil
As more endemic countries enter the elimination phase, the detection of sporadic malaria infections or outbreaks elicits many questions: (i) are the infections locally acquired or imported? (ii) If imported, where do they come from? (iii) Do outbreak strains have a single or multiple geographic origins? New molecular barcoding methods provide ways to analyze clinical malaria parasite samples and answer these and other crucial questions. Conservation biology of endangered animals and malaria eradication programs entail similar analytical strategies, despite their diametrically opposite aims [1]. For example, DNA analysis of illegal African elephant ivory shipments seized on the way to the Far East has now became the standard method to locate the original sites of elephant poaching, helping to elucidate ivory smuggling routes in Africa [2]. To guide public health interventions, malariologists are often interested in understanding how new parasite phenotypes (such as drug resistance, increased virulence, or novel antigenic variants) originate and spread in human populations. Furthermore, accurate and reproducible methods to characterize parasites and assign strains to specific regions and countries are required to design strategies to prevent malaria reintroduction in areas approaching elimination that remain vulnerable to malaria resurgences. Preston and colleagues [3] have recently developed a novel molecular genotyping strategy to identify the geographic origin of Plasmodium falciparum strains. They started by analyzing the full-length genomes of two organelles, the mitochondrion (6 kb) and the apicoplast (29.4 kb), from over 700 P. falciparum isolates originating from 14 countries, mostly from Africa. They next used an interactive haplotype search algorithm to select the 23 most informative single nucleotide polymorphisms (SNPs), five from the mitochondrial and 18 from the apicoplast genome, with the aim of developing a molecular barcode. Because mitochondrial and apicoplast lineages are uniparentally inherited, they do not recombine with each other and can thus provide more stable region-specific genotypes than nuclear genome SNPs [3]. The 23-SNP barcoding strategy was 92% accurate in identifying the continental origin of P. falciparum samples from West and East Africa, Southeast Asia, Oceania, and South America. Most
Corresponding author: Ferreira, M.U. ([email protected], [email protected]) Keywords: malaria; genotyping; eradication; single-nucleotide polymorphism; microsatellites. 1471-4922/ ß 2014 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.pt.2014.08.003
(26 of 34) distinct haplotypes characterized by genotyping were unique to a single region [3]. A global map of sequence diversity is crucial for assigning P. falciparum organellar genomes to specific regions of malaria endemicity. One of the limitations of the current 23-SNP barcode is the lack of reference DNA sequences from sites such as South Asia, Central America and the Caribbean, and southern Africa [3]. South Asia is a particularly challenging region, due to the lack of mitochondrial, and especially of apicoplast, reference genomes in open access databases. Moreover, the existence of two divergent genetic clusters (‘southern’ and ‘northern’) of P. falciparum isolates in South America, and putatively also in Central America and the Caribbean, further complicates the geographic assignment of samples from this region. These clusters may have originated from separate colonization events associated with the slave trade [4]. Similar issues will arise when applying the molecular barcoding approach to other human malaria parasites, such as Plasmodium vivax. Although many P. vivax mitochondrial genomes cluster according to the geographic origin of the isolates, those from South Asia, Central Asia, Africa and the Middle East are closely related to each other and fail to provide population-specific molecular barcodes. Therefore, mitochondrial genome analysis lacks geographic resolution for assigning many imported P. vivax infections to their regions of origin [5]. As only three P. vivax apicoplast genomes have been sequenced, no comprehensive organellar SNP panel is currently available for this species. The next crucial step for the further development and large-scale use of malaria barcoding approaches consists of creating and maintaining a comprehensive and freely accessible database of geo-referenced sequences. The ever-growing multilocus sequencing typing (MLST) research community offers a success story in this field. Microbiologists created curated DNA sequence databases for molecular epidemiologic surveillance of several human pathogens. MLST databases comprise thousands of 450 to 500 bp DNA sequences of known origin derived from six or seven housekeeping genes per organism. Databases are currently available online for 75 bacteria, three fungi, and one protozoon [6]. Showing the potential utility of molecular barcoding in academic research and epidemiologic surveillance will hopefully further stimulate contributions of reference DNA sequences to the open access databases, resulting in improved accuracy for this technique in tracing the origin of strains. Conventional phylogeny-based tracing techniques can help to determine which reference site a malaria sample matches best [3,5]. However, while a global database of Trends in Parasitology, October 2014, Vol. 30, No. 10
465
Spotlights Box 1. Molecular characterization of malaria outbreaks For an epidemiologist, it is crucial to determine whether all infections diagnosed during an outbreak had a single or multiple origins in order to design proper containment strategies. DNA analysis can help in this task. The short-term spread of a single or a few strains in a community can originate a malaria outbreak, as recently documented in an area of declining malaria transmission in rural Amazonia [7]. The characterization of P. vivax isolates collected in a single farming settlement over three years, with 14 highly polymorphic microsatellite markers, revealed the lowest parasite diversity, with the smallest proportion of multiple-clone infections and the strongest linkage disequilibrium at the time of the outbreak. All features are consistent with a clonal expansion of outbreak strain(s). Alternatively, outbreaks can involve several genetically divergent strains, with putatively multiple geographic origins. The first example comes from the 2011 P. vivax outbreak in Evrotas, Greece; 27 samples were genotyped with three repetitive DNA markers, revealing at least six different haplotypes co-circulating in the area [8]. The second example of multiple-strain P. vivax outbreak comes from Trincomalee, Eastern Province of Sri Lanka; both microsatellite [9] and nuclear SNP genotyping [10] revealed surprisingly high levels of diversity, with no single haplotype being shared between samples. These findings argue for multiple independent introductions of P. vivax strains into these receptive areas, with major implications for malaria surveillance and prevention of re-emergence. In fact, P. vivax populations in Sri Lanka remained highly diverse over the past decade, despite the dramatic decline in malaria transmission countrywide [9]. These data are consistent with strains of various origins being continuously introduced in the country or, alternatively, with genetically diverse strains being maintained in a large asymptomatic reservoir within the country.
malaria parasite variation can comprise a large number of reference sites, not all areas of malaria endemicity in more than 100 countries and territories are included. Consequently, geo-statistical models are needed to assign individual infections to locations where no reference DNA sequences are available. Conservation biologists apply spatial smoothing methods to infer allele frequencies across the entire area of distribution of African elephants, using genotypes of reference samples from a limited number of geo-referenced sites [2]. Validating analogous geo-statistical approaches for tracing malaria parasites worldwide would be crucial for further advancing the field. Genetic analyses can help to elucidate the origin and routes of dissemination of parasite strains and to define the target area for public health interventions during malaria outbreaks (Box 1). However, the 23-SNP barcode is unlikely to reveal subtle differences between local and imported P. falciparum strains. In other words, it may lack genetic resolution to distinguish between ongoing autochthonous transmission and malaria infections imported from one or
466
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more nearby locations. Highly polymorphic microsatellites (tandem repeats of motifs of one to six nucleotides) potentially offer a greater genetic resolution for outbreak investigation. These are fast-evolving markers, with 10 3 to 10 4 mutations per locus per generation mostly caused by strandslippage events during DNA replication. Microsatellites are useful to characterize community-, country- or region-level genetic diversity over relatively short periods of time, as often required in outbreak investigations, but they do not necessarily match everyone’s needs. More stable markers, such as nuclear and organellar SNPs, are better suited for tracing the origin and dispersal of parasite strains across and between continents. It is now clear that no single barcoding strategy will provide all answers. Public health specialists must be familiar with the wide range of parasite typing methods and their potential applications in different settings. The 23-SNP barcode developed by Preston and colleagues [3] represents a welcome complement to our arsenal of genotyping tools for malaria parasites. Acknowledgments The authors received scholarships from the Conselho Nacional de Desenvolvimento Cientı´fico e Tecnolo´gico (CNPq), Brazil.
References 1 Nkhoma, S.C. et al. (2013) Population genetic correlates of declining transmission in a human pathogen. Mol. Ecol. 22, 273–285 2 Wasser, S.K. et al. (2007) Using DNA to track the origin of the largest ivory seizure since the 1989 trade ban. Proc. Natl. Acad. Sci. U.S.A. 104, 4228–4233 3 Preston, M.D. et al. (2014) A barcode of organellar genome polymorphisms identifies the geographic origin of Plasmodium falciparum strains. Nat. Commun. 5, 4052 4 Yalcindag, E. et al. (2012) Multiple independent introductions of Plasmodium falciparum in South America. Proc. Natl. Acad. Sci. U.S.A. 109, 511–516 5 Rodrigues, P.T. et al. (2014) Using mitochondrial genome sequences to track the origin of imported Plasmodium vivax infections diagnosed in the United States. Am. J. Trop. Med. Hyg. 90, 1102–1108 6 Pe´rez-Losada, M. et al. (2013) Pathogen typing in the genomics era: MLST and the future of molecular epidemiology. Infect. Genet. Evol. 16, 38–53 7 Batista, C.L. et al. (2014) Genetic diversity of Plasmodium vivax over time and space: a community-based study in rural Amazonia. Parasitology http://dx.doi.org/10.1017/S0031182014001176 8 Spanakos, G. et al. (2013) Genotyping Plasmodium vivax isolates from the 2011 outbreak in Greece. Malar. J. 12, 463 9 Gunawardena, S. et al. (2014) The Sri Lankan paradox: high genetic diversity in Plasmodium vivax populations despite decreasing levels of malaria transmission. Parasitology 141, 880–890 10 Orjuela-Sa´nchez, P. et al. (2010) Single-nucleotide polymorphism, linkage disequilibrium and geographic structure in the malaria parasite Plasmodium vivax: prospects for genome-wide association studies. BMC Genet. 11, 65
