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. 2016 Sep 16;11(9):e0163137.
doi: 10.1371/journal.pone.0163137. eCollection 2016.

Genetic Characterisation of Plasmodium falciparum Isolates with Deletion of the pfhrp2 and/or pfhrp3 Genes in Colombia: The Amazon Region, a Challenge for Malaria Diagnosis and Control

Erika Jimena Dorado  1 , Sheila Akinyi Okoth  2   3 , Lidia Madeline Montenegro  1 , Gustavo Diaz  1 , John W Barnwell  2 , Venkatachalam Udhayakumar  2 , Claribel Murillo Solano  1
Affiliations

Genetic Characterisation of Plasmodium falciparum Isolates with Deletion of the pfhrp2 and/or pfhrp3 Genes in Colombia: The Amazon Region, a Challenge for Malaria Diagnosis and Control

Erika Jimena Dorado et al. PLoS One. .

Abstract

Most Plasmodium falciparum-detecting rapid diagnostic tests (RDTs) target histidine-rich protein 2 (PfHRP2). However, P. falciparum isolates with deletion of the pfhrp2 gene and its homolog gene, pfhrp3, have been detected. We carried out an extensive investigation on 365 P. falciparum dried blood samples collected from seven P. falciparum endemic sites in Colombia between 2003 and 2012 to genetically characterise and geographically map pfhrp2- and/or pfhrp3-negative P. falciparum parasites in the country. We found a high proportion of pfhrp2-negative parasites only in Amazonas (15/39; 38.5%), and these parasites were also pfhrp3-negative. These parasites were collected between 2008 and 2009 in Amazonas, while pfhrp3-negative parasites (157/365, 43%) were found in all the sites and from each of the sample collection years evaluated (2003 to 2012). We also found that all pfhrp2- and/or pfhrp3-negative parasites were also negative for one or both flanking genes. Six sub-population clusters were established with 93.3% (14/15) of the pfhrp2-negative parasites grouped in the same cluster and sharing the same haplotype. This haplotype corresponded with the genetic lineage BV1, a multidrug resistant strain that caused two outbreaks reported in Peru between 2010 and 2013. We found this BV1 lineage in the Colombian Amazon as early as 2006. Two new clonal lineages were identified in these parasites from Colombia: the genetic lineages EV1 and F. PfHRP2 sequence analysis revealed high genetic diversity at the amino acid level, with 17 unique sequences identified among 53 PfHRP2 sequences analysed. The use of PfHRP2-based RDTs is not recommended in Amazonas because of the high proportion of parasites with pfhrp2 deletion (38.5%), and implementation of new strategies for malaria diagnosis and control in Amazonas must be prioritised. Moreover, studies to monitor and genetically characterise pfhrp2-negative P. falciparum parasites in the Americas are warranted, given the extensive human migration occurring in the region.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. Sample collection sites in Colombia.
On the map the sample collection sites are highlighted. The names in black indicate the seven Colombian Departments from which the isolates studied were collected: Antioquia (N = 42), Choco (N = 74), Valle (N = 27), Cauca (N = 44), Nariño (N = 122), Guaviare (N = 26) and Amazonas (N = 39). The study site highlighted in red, Amazonas, indicates the Department where pfhrp2/pfhrp3 double negative isolates were found. Map source: DIVA-GIS, available in http://www.diva-gis.org/.
Fig 2
Fig 2. Clusters (C1—C6) defined by STRUCTURE v2.3.3. for the population of pfhrp2- and/or pfhrp3-negative parasites in Colombia (N = 132).
Each isolate (x-axis) is represented by a single vertical bar, and this bar is broken down into various coloured segments whose length varies according to the membership fraction (y- axis) of each of the six inferred clusters (K = 6). Cluster 1 (red) contained 22.7% of the total isolates evaluated (30 isolates). These were mainly samples from the Pacific coast and northern Colombia. Cluster 2 (green) contained 14.4% of the samples evaluated (19 isolates). Ninety-five percent of the samples in Cluster 2 were from northern Colombia. Cluster 3 (blue) contained 25.8% of the samples evaluated (34 isolates), all of which were collected in in the Colombian Pacific coast. Cluster 4 (yellow) was the smallest cluster with 6.8% of the samples (9 isolates). These originated from Guaviare, Nariño and Choco. Cluster 5 (pink) contained 18.2% of the samples (24 isolates) and they were collected in southern and south-eastern Colombia. This cluster contained the pfhrp2/pfhrp3 double negative samples. Cluster 6 (aqua) included 12.1% of the samples evaluated (16 isolates) and they originated from all the sites studied except Nariño.
Fig 3
Fig 3. PfHRP2 sequence types identified in Colombia.
Fifty three PfHRP2 sequences were evaluated and 17 unique sequences were identified. The heatmap shows the frequencies of the repeats for each type of PfHRP2 sequence identified. Study sites where the respective PfHRP2 sequence type (from 1 to 17) was found: AMA = Amazonas, GUA = Guaviare, ANT = Antioquia, CHO = Choco, NAR = Nariño, VAL = Valle. N = number of isolates that showed the PfHRP2 sequence type and (%) = percentage of the PfHRP2 sequence type in the population studied.
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