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      Deletions of pfhrp2 and pfhrp3 genes of Plasmodium falciparum from Honduras, Guatemala and Nicaragua

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          Abstract

          Background

          Malaria remains a public health problem in some countries of Central America. Rapid diagnostic tests (RDTs) are one of the most useful tools to assist in the diagnosis of malaria in remote areas. Since its introduction, a wide variety of RDTs have been developed for the detection of different parasite antigens. PfHRP2 is the most targeted antigen for the detection of Plasmodium falciparum infections. Genetic mutations and gene deletions are important factors influencing or affecting the performance of rapid diagnostic tests.

          Methods

          In order to demonstrate the presence or absence of the pfhrp2 and pfhrp3 genes and their flanking regions, a total of 128 blood samples from patients with P. falciparum infection from three Central American countries were analysed through nested or semi-nested PCR approaches.

          Results

          In total, 25.8 and 91.4% of the isolates lacked the region located between exon 1 and exon 2 of pfhrp2 and pfhrp3 genes, respectively. Parasites from the three countries showed deletions of one or both genes. The highest proportion of pfhrp2 deletions was found in Nicaragua while the isolates from Guatemala revealed the lowest number of pfhrp2 deletions. Parasites collected from Honduras showed the highest proportion of phfrp3 absence (96.2%). Twenty-one percent of isolates were double negative mutants for the exon 1–2 segment of both genes, and 6.3% of isolates lacked the full-length coding region of both genes.

          Conclusions

          This study provides molecular evidence of the existence of P. falciparum isolates lacking the pfhrp2 and pfhrp3 genes, and their flanking regions, in Honduras, Guatemala and Nicaragua. This finding could hinder progress in the control and elimination of malaria in Central America. Continuous evaluation of RDTs and molecular surveillance would be recommended.

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          Most cited references24

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          A genetic map and recombination parameters of the human malaria parasite Plasmodium falciparum.

          Genetic investigations of malaria require a genome-wide, high-resolution linkage map of Plasmodium falciparum. A genetic cross was used to construct such a map from 901 markers that fall into 14 inferred linkage groups corresponding to the 14 nuclear chromosomes. Meiotic crossover activity in the genome proved high (17 kilobases per centimorgan) and notably uniform over chromosome length. Gene conversion events and spontaneous microsatellite length changes were evident in the inheritance data. The markers, map, and recombination parameters are facilitating genome sequence assembly, localization of determinants for such traits as virulence and drug resistance, and genetic studies of parasite field populations.
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            Effect of sequence variation in Plasmodium falciparum histidine- rich protein 2 on binding of specific monoclonal antibodies: Implications for rapid diagnostic tests for malaria.

            The ability to accurately diagnose malaria infections, particularly in settings where laboratory facilities are not well developed, is of key importance in the control of this disease. Rapid diagnostic tests (RDTs) offer great potential to address this need. Reports of significant variation in the field performance of RDTs based on the detection of Plasmodium falciparum histidine-rich protein 2 (HRP2) (PfHRP2) and of significant sequence polymorphism in PfHRP2 led us to evaluate the binding of four HRP2-specific monoclonal antibodies (MABs) to parasite proteins from geographically distinct P. falciparum isolates, define the epitopes recognized by these MABs, and relate the copy number of the epitopes to MAB reactivity. We observed a significant difference in the reactivity of the same MAB to different isolates and between different MABs tested with single isolates. When the target epitopes of three of the MABs were determined and mapped onto the peptide sequences of the field isolates, significant variability in the frequency of these epitopes was observed. These findings support the role of sequence variation as an explanation for variations in the performance of HRP2-based RDTs and point toward possible approaches to improve their diagnostic sensitivities.
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              False-negative malaria rapid diagnostic tests in Rwanda: impact of Plasmodium falciparum isolates lacking hrp2 and declining malaria transmission

              Background Rapid diagnostic tests (RDTs) for histidine rich protein 2 (HRP2) are often used to determine whether persons with fever should be treated with anti-malarials. However, Plasmodium falciparum parasites with a deletion of the hrp2 gene yield false-negative RDTs and there are concerns the sensitivity of HRP2-based RDTs may fall when the intensity of transmission decreases. Methods This observational study enrolled 9226 patients at three health centres in Rwanda from April 2014 to April 2015. It then compared the sensitivity of RDTs based on HRP2 and the Plasmodium lactate dehydrogenase (pLDH) to microscopy (thick smears) for the diagnosis of malaria. PCR was used to determine whether deletions of the histidine-rich central repeat region of the hrp2 gene (exon 2) were associated with false-negative HRP2-based RDTs. Results In comparison to microscopy, the sensitivity and specificity of HRP2- and pLDH-based RDTs were 89.5 and 86.2% and 80.2 and 94.3%, respectively. When the results for both RDTs were combined, sensitivity rose to 91.8% and specificity was 85.7%. Additionally, when smear positivity fell from 46 to 3%, the sensitivity of the HRP2-based RDT fell from 88 to 67%. Of 370 samples with false-negative HRP2 RDT results for which PCR was performed, 140 (38%) were identified as P. falciparum by PCR. Of the isolates identified as P. falciparum by PCR, 32 (23%) were negative for the hrp2 gene based on PCR. Of the 32 P. falciparum isolates negative for hrp2 by PCR, 17 (53%) were positive based on the pLDH RDT. Conclusion This prospective study of RDT performance coincided with a decline in the intensity of malaria transmission in Kibirizi (fall in slide positivity from 46 to 3%). This decline was associated with a decrease in HRP2 RDT sensitivity (from 88 to 67%). While P. falciparum isolates without the hrp2 gene were an important cause of false-negative HRP2-based RDTs, most were identified by the pLDH-based RDT. Although WHO does not recommend the use of combined HRP2/pLDH testing in sub-Saharan Africa, these results suggest that combination HRP2/pLDH-based RDTs could reduce the impact of false-negative HRP2-based RDTs for detection of symptomatic P. falciparum malaria. Electronic supplementary material The online version of this article (doi:10.1186/s12936-017-1768-1) contains supplementary material, which is available to authorized users.
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                Author and article information

                Contributors
                gustavo.fontecha@unah.edu.hn
                Journal
                Malar J
                Malar. J
                Malaria Journal
                BioMed Central (London )
                1475-2875
                31 August 2018
                31 August 2018
                2018
                : 17
                : 320
                Affiliations
                [1 ]ISNI 0000 0001 2297 2829, GRID grid.10601.36, Microbiology Research Institute, , Universidad Nacional Autonoma de Honduras, ; Tegucigalpa, Honduras
                [2 ]Panamerican Health Organization, Tegucigalpa, Honduras
                [3 ]National Department of Surveillance, Ministry of Health, Tegucigalpa, Honduras
                [4 ]Panamerican Health Organization, Washington, USA
                Author information
                http://orcid.org/0000-0001-9756-4520
                Article
                2470
                10.1186/s12936-018-2470-7
                6119307
                30170596
                77b5de27-ee7d-491b-bfb3-2961228d2134
                © The Author(s) 2018

                Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License ( http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver ( http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

                History
                : 11 July 2018
                : 27 August 2018
                Funding
                Funded by: Scientific Research Office DICYP - UNAH
                Award ID: S/N
                Award Recipient :
                Funded by: FundRef http://dx.doi.org/10.13039/100011893, Pan American Health Organization;
                Award ID: S/N
                Award Recipient :
                Categories
                Research
                Custom metadata
                © The Author(s) 2018

                Infectious disease & Microbiology
                malaria,central america,plasmodium falciparum,rdt,pfhrp2,pfhrp3
                Infectious disease & Microbiology
                malaria, central america, plasmodium falciparum, rdt, pfhrp2, pfhrp3

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