Independent Emergence of the Plasmodium falciparum Kelch Propeller Domain Mutant Allele C580Y in Guyana

International audienceSuspected artemisinin resistance in Plasmodium falciparum can be explored by examining polymorphisms in the Kelch (PfK13) propeller domain. Sequencing of PfK13 and other gene resistance markers was performed on 98 samples from Guyana. Five of these samples carried the C580Y allele in the PfK13 propeller domain, with flanking microsatellite profiles different from those observed in Southeast Asia. These molecular data demonstrate independent emergence of the C580Y K13 mutant allele in Guyana, where resistance alleles to previously used drugs are fixed. Therefore, in Guyana and neighboring countries , continued molecular surveillance and periodic assessment of the therapeutic efficacy of artemisinin-based combination therapy are warranted

Suspected locations where <i>P</i>. <i>falciparum</i> infections were acquired based on patient travel histories in regions with malaria transmission two weeks prior to malaria diagnosis in Guyana (Top; total N = 100), and Suriname (Bottom; total N = 78).

<p>The number of patients who reported travelling to a particular region (Guyana) or district (Suriname) is indicated in parentheses. The travel history of seven Suriname patients is unknown. Country maps reprinted from d-maps.com under a CC BY license, with permission from Daniel Dalet, original copyright 2007(<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0126805#pone.0126805.s001" target="_blank">S1 Supporting Information</a>).</p

Variation in <i>Plasmodium falciparum</i> Histidine-Rich Protein 2 (<i>Pfhrp2</i>) and <i>Plasmodium falciparum</i> Histidine-Rich Protein 3 (<i>Pfhrp3</i>) Gene Deletions in Guyana and Suriname

<div><p>Guyana and Suriname have made important progress in reducing the burden of malaria. While both countries use microscopy as the primary tool for clinical diagnosis, malaria rapid diagnostic tests (RDTs) are useful in remote areas of the interior where laboratory support may be limited or unavailable. Recent reports indicate that histidine-rich protein 2 (PfHRP2)-based diagnostic tests specific for detection of <i>P</i>. <i>falciparum</i> may provide false negative results in some parts of South America due to the emergence of <i>P</i>. <i>falciparum</i> parasites that lack the <i>pfhrp2</i> gene, and thus produce no PfHRP2 antigen. <i>Pfhrp2</i> and <i>pfhrp3</i> genes were amplified in parasite isolates collected from Guyana and Suriname to determine if there were circulating isolates with deletions in these genes. <i>Pfhrp3</i> deletions were monitored because some monoclonal antibodies utilized in PfHRP2-based RDTs cross-react with the PfHRP3 protein. We found that all 97 isolates from Guyana that met the inclusion criteria were both <i>pfhrp2-</i> and <i>pfhrp3</i>-positive. In Suriname (N = 78), 14% of the samples tested were <i>pfhrp2</i>-negative while 4% were <i>pfhrp3</i>-negative. Furthermore, analysis of the genomic region proximal to <i>pfhrp2</i> and <i>pfhrp3</i> revealed that genomic deletions extended to the flanking genes. We also investigated the population substructure of the isolates collected to determine if the parasites that had deletions of <i>pfhrp2</i> and <i>pfhrp3</i> belonged to any genetic subtypes. Cluster analysis revealed that there was no predominant <i>P</i>. <i>falciparum</i> population substructure among the isolates from either country, an indication of genetic admixture among the parasite populations. Furthermore, the <i>pfhrp2</i>-deleted parasites from Suriname did not appear to share a single, unique genetic background.</p></div

Proportion of deletions in (A) <i>pfhrp2</i>, (B) <i>pfhrp3</i> and their respective neighboring genes in <i>P</i>. <i>falciparum</i> isolates collected in Guyana (N = 97) and Suriname (N = 78).

<p>The three pie charts to the left of each figure illustrate the proportion of parasite isolates with gene deletions in Suriname samples, while the three pie charts to the right of each Fig show the proportion of isolates with gene deletions in Guyana samples. The percentages shown represent proportions of samples out of the total samples that were <i>18S RNA</i>- and <i>msp-2</i> positive.</p

Median joining network analysis of <i>P</i>. <i>falciparum</i> isolates collected in (A) Guyana (N = 97) and Suriname (N = 57) and (B) Suriname alone (N = 57).

<p>The genetic relationships among parasites were constructed using seven neutral microsatellite loci, which have been used previously to genetically characterize <i>P</i>. <i>falciparum</i> parasite populations in South America. <i>P</i>. <i>falciparum</i> parasite isolates from Guyana are shown in green while those collected in Suriname are rendered in red. Dotted circles indicate <i>pfhrp2</i>-negative isolates.</p

Implementation of basic quality control tests for malaria medicines in Amazon Basin countries: results for the 2005–2010 period

<p>Abstract</p> <p>Background</p> <p>Ensuring the quality of malaria medicines is crucial in working toward malaria control and eventual elimination. Unlike other validated tests that can assess all critical quality attributes, which is the standard for determining the quality of medicines, basic tests are significantly less expensive, faster, and require less skilled labour; yet, these tests provide reproducible data and information on several critical quality attributes, such as identity, purity, content, and disintegration. Visual and physical inspection also provides valuable information about the manufacturing and the labelling of medicines, and in many cases this inspection is sufficient to detect counterfeit medicines. The Promoting the Quality of Medicines (PQM) programme has provided technical assistance to Amazon Malaria Initiative (AMI) countries to implement the use of basic tests as a key screening mechanism to assess the quality of malaria medicines available to patients in decentralized regions.</p> <p>Methods</p> <p>Trained personnel from the National Malaria Control Programmes (NMCPs), often in collaboration with country’s Official Medicine Control Laboratory (OMCL), developed country- specific protocols that encompassed sampling methods, sample analysis, and data reporting. Sampling sites were selected based on malaria burden, accessibility, and geographical location. Convenience sampling was performed and countries were recommended to store the sampled medicines under conditions that did not compromise their quality. Basic analytical tests, such as disintegration and thin layer chromatography (TLC), were performed utilizing a portable mini-laboratory.</p> <p>Results</p> <p>Results were originally presented at regional meetings in a non-standardized format that lacked relevant medicines information. However, since 2008 information has been submitted utilizing a template specifically developed by PQM for that purpose. From 2005 to 2010, the quality of 1,663 malaria medicines from seven AMI countries was evaluated, mostly collected from the public sector, 1,445/1,663 (86.9%). Results indicate that 193/1,663 (11.6%) were found not to meet quality specifications. Most failures were reported during visual and physical inspection, 142/1663 (8.5%), and most of these were due to expired medicines, 118/142 (83.1%). Samples failing TLC accounted for 27/1,663 (1.6%) and those failing disintegration accounted for 24/1,663 (1.4%). Medicines quality failures decreased significantly during the last two years.</p> <p>Conclusions</p> <p>Basic tests revealed that the quality of medicines in the public sector improved over the years, since the implementation of this type of quality monitoring programme in 2005. However, the lack of consistent confirmatory tests in the quality control (QC) laboratory, utilizing methods that can also evaluate additional quality attributes, could still mask quality issues. In the future, AMI countries should improve coordination with their health authorities and their QC lab consistently, to provide a more complete picture of malaria medicines quality and support the implementation of corrective actions. Facilities in the private and informal sectors also should be included when these sectors constitute an important source of medicines used by malaria patients.</p