Real-time quantitative PCR with SYBR Green I detection for estimating copy numbers of nine drug resistance candidate genes in Plasmodium falciparum

BACKGROUND: Evaluating copy numbers of given genes in Plasmodium falciparum parasites is of major importance for laboratory-based studies or epidemiological surveys. For instance, pfmdr1 gene amplification has been associated with resistance to quinine derivatives and several genes involved in anti-oxidant defence may play an important role in resistance to antimalarial drugs, although their potential involvement has been overlooked. METHODS: The (ΔΔ)Ct method of relative quantification using real-time quantitative PCR with SYBR Green I detection was adapted and optimized to estimate copy numbers of three genes previously indicated as putative candidates of resistance to quinolines and artemisinin derivatives: pfmdr1, pfatp6 (SERCA) and pftctp, and in six further genes involved in oxidative stress responses. RESULTS: Using carefully designed specific RT-qPCR oligonucleotides, the methods were optimized for each gene and validated by the accurate measure of previously known number of copies of the pfmdr1 gene in the laboratory reference strains P. falciparum 3D7 and Dd2. Subsequently, Standard Operating Procedures (SOPs) were developed to the remaining genes under study and successfully applied to DNA obtained from dried filter blood spots of field isolates of P. falciparum collected in São Tomé & Principe, West Africa. CONCLUSION: The SOPs reported here may be used as a high throughput tool to investigate the role of these drug resistance gene candidates in laboratory studies or large scale epidemiological surveys

Analysis of genetic mutations associated with anti-malarial drug resistance in Plasmodium falciparum from the Democratic Republic of East Timor

<p>Abstract</p> <p>Background</p> <p>In response to chloroquine (CQ) resistance, the policy for the first-line treatment of uncomplicated malaria in the Democratic Republic of East Timor (DRET) was changed in early 2000. The combination of sulphadoxine-pyrimethamine (SP) was then introduced for the treatment of uncomplicated falciparum malaria.</p> <p>Methods</p> <p>Blood samples were collected in two different periods (2003–2004 and 2004–2005) from individuals attending hospitals or clinics in six districts of the DRET and checked for <it>Plasmodium falciparum </it>infection. 112 PCR-positive samples were inspected for genetic polymorphisms in the <it>pfcrt</it>, <it>pfmdr1</it>, <it>pfdhfr </it>and <it>pfdhps </it>genes. Different alleles were interrogated for potential associations that could be indicative of non-random linkage.</p> <p>Results</p> <p>Overall prevalence of mutations associated with resistance to CQ and SP was extremely high. The mutant form of <it>Pfcrt </it>(76T) was found to be fixed even after five years of alleged CQ removal. There was a significant increase in the prevalence of the <it>pfdhps </it>437G mutation (X²= 31.1; p = 0.001) from the first to second survey periods. A non-random association was observed between <it>pfdhfr</it>51/<it>pfdhps</it>437 (p = 0.001) and <it>pfdhfr </it>59/<it>pfdhps </it>437 (p = 0.013) alleles.</p> <p>Conclusion</p> <p>Persistence of CQ-resistant mutants even after supposed drug withdrawal suggests one or all of the following: local <it>P. falciparum </it>may still be inadvertently exposed to the drug, that mutant parasites are being "imported" into the country, and/or reduced genetic diversity and low parasite transmission help maintain mutant haplotypes. The association between <it>pfdhfr</it>51/<it>pfdhps</it>437 and <it>pfdhfr </it>59/<it>pfdhps </it>437 alleles indicates that these are undergoing concomitant positive selection in the DRET.</p

Evaluation of prevalence's of pfdhfr and pfdhps mutations in Angola

<p>Abstract</p> <p>Background</p> <p>Malaria is the major cause of morbidity and mortality in Angola. The most vulnerable groups to <it>Plasmodium falciparum </it>infection are pregnant women and children under five years of age. The use of an intermittent preventive treatment (IPT) with sulphadoxine/pyrimethamine (SP) in pregnant women was introduced in Angola in 2006 by the National Malaria Control Programme, and currently this strategy has been considered to be used for children malaria control. Considering the previous wide use of SP combination in Angola, together to the reported cases of SP treatment failure it is crucial the evaluation of the prevalence of five mutations in <it>pfdhfr </it>and <it>pfdhps </it>genes associated to <it>P</it>. <it>falciparum </it>resistance to SP before the introduction of S/P IPT in children.</p> <p>Methods</p> <p>The study was conducted in five provinces, with different transmission intensities: Huambo, Cabinda, Uíge, Kwanza Norte, and Malanje. The detection of the mutations in <it>pfdhfr </it>and <it>pfdhps </it>genes was carried out in 452 <it>P</it>. <it>falciparum </it>blood samples by PCR RFLP.</p> <p>Results</p> <p>For <it>pfdhfr </it>gene, 90,3% of the samples carried the mutation 51<b>I</b>, with 7.5% of mixed infections; 51% carried wild type allele 59<b>C</b>, with 29.2% mixed infections and; 99.1% of isolates harboured the mutant allele 108<b>N</b>. Concerning, <it>pfdhps </it>gene, 83,1% were mutant type 437<b>G </b>with 11% mixed infections , while 87% of the studied isolates were wild type for codon 540.</p> <p>Discussion</p> <p>This is the first representative epidemiological study of the whole Angola country on the prevalence of the genotypes associated with SP chemoresistance. A high frequency of individual mutations in both genes (51<b>I </b>and 108<b>N </b>in <it>pfdhfr</it>, and 437<b>G </b>in <it>pfdhps</it>) was found, besides a low prevalence of the quintuple mutation.</p> <p>Conclusion</p> <p>The data showed that the implementation IPT using SP in children needs to be reviewed.</p

Epidemiological characterization of Plasmodium falciparum in the Republic of Cabo Verde: implications for potential large-scale re-emergence of malaria

BACKGROUND: Malaria has come near eradication at archipelago of Cabo Verde in 1970. Infections are now only observed in Santiago, where outbreaks occur. In these islands, malaria is considered by the international community as being of limited risk and, therefore, no prophylaxis is recommended. Since the understanding of factors that determine malaria outbreaks are crucial for controlling the disease, the present study aimed to investigate if the malaria infections observed in Santiago Island are maintained in isolated foci and in asymptomatic individuals. METHODS: The occurrence of asymptomatic carriers in villages with history of malaria as well as the level of exposure of these populations were investigated using PCR and serological analyses. RESULTS: Results indicate that malaria is maintained as asymptomatic and sub-patent infections and that the majority of the circulating parasite populations harbour chloroquine-resistant mutations. CONCLUSION: These observations highlight the alarming prospect of malaria to become a serious public health problem and underscore the need for a tighter surveillance

Molecular evolution of the three short PGRPs of the malaria vectors <em>Anopheles gambiae</em> and <em>Anopheles arabiensis</em> in East Africa

BACKGROUND: Immune responses to parasites, which start with pathogen recognition, play a decisive role in the control of the infection in mosquitoes. Peptidoglycan recognition proteins (PGRPs) are an important family of pattern recognition receptors that are involved in the activation of these immune reactions. Pathogen pressure can exert adaptive changes in host genes that are crucial components of the vector's defence. The aim of this study was to determine the molecular evolution of the three short PGRPs (PGRP-S1, PGRP-S2 and PGRP-S3) in the two main African malaria vectors - Anopheles gambiae and Anopheles arabiensis. RESULTS: Genetic diversity of An. gambiae and An. arabiensis PGRP-S1, PGRP-S2 and PGRP-S3 was investigated in samples collected from Mozambique and Tanzania. PGRP-S1 diversity was lower than for PGRP-S2 and PGRP-S3. PGRP-S1 was the only gene differentiated between the two species. All the comparisons made for PGRP-S1 showed significant P-values for Fst estimates and AMOVA confirming a clear separation between species. For PGRP-S2 and PGRP-S3 genes it was not possible to group populations either by species or by geographic region. Phylogenetic networks reinforced the results obtained by the AMOVA and Fst values. The ratio of nonsynonymous substitutions (Ka)/synonymous substitutions (Ks) for the duplicate pair PGRP-S2 and PGRP-S3 was very similar and lower than 1. The 3D model of the different proteins coded by these genes showed that amino acid substitutions were concentrated at the periphery of the protein rather than at the peptidoglycan recognition site. CONCLUSIONS: PGRP-S1 is less diverse and showed higher divergence between An. gambiae and An. arabiensis regardless of geographic location. This probably relates to its location in the chromosome-X, while PGRP-S2 and PGRP-S3, located in chromosome-2L, showed signs of autosomal introgression. The two short PGRP genes located in the chromosome-2L were under purifying selection, which suggests functional constraints. Different types of selection acting on PGRP-S1 and PGRP-S2 and S3 might be related to their different function and catalytic activity

Evaluation of Antiplasmodial activity of extracts and constituents from Ampelozizyphus amazonicus

PMID: 26664012 WOS: 000362879000003BACKGROUND: Ampelozizyphus amazonicus Ducke, a plant that is widely used by the population of the Amazonian region to prevent and treat malaria, was investigated in this work, which describes, for the first time, the antiplasmodial activity of its extracts and associates this activity with its isolated constituents. METHODS: Different extracts with solvents of increasing polarity (hexane, chloroform, ethanol, and water) were obtained of the root bark. This procedure resulted in extracts that were characterized for their constituents. The cytotoxicity and activity of the extracts against Plasmodium berghei (schizontocidal activity, liver stage) and Plasmodium falciparum (3D7 and Dd2 strains, erythrocyte stage) were assessed in vitro. RESULTS: Of the four extracts assayed against P. berghei, the chloroform extract showed the greatest activity, with an inhibitory concentration 50% (IC50) value of 30.1 µg/mL, followed by the aqueous extract (IC50 = 39.9 µg/mL). The chloroform extract exhibited the highest antiplasmodial activity in the erythrocyte stage of P. falciparum, with an IC50 value lower than 15 µg/mL. Fractionation of this more active extract led to the isolation and elucidation of pentacyclic triterpenes, lupeol, betulin and betulinic acid, which showed antiplasmodial activities with IC50 values ranging from 5.6 to 80.30 µM. The most active of these, betulinic acid, was further quantified in the extracts by high-performance liquid chromatography-photodiode array detector analyzes. The higher amount was found in the chloroform extract, which was the most active one against P. falciparum. CONCLUSION: The results obtained in this work may partly explain the popular intake of A. amazonicusas an antimalarial remedy in the Amazon region.publishersversionpublishe

Genetic diversity and signatures of selection of drug resistance in Plasmodium populations from both human and mosquito hosts in continental Equatorial Guinea

BACKGROUND: In Plasmodium, the high level of genetic diversity and the interactions established by co-infecting parasite populations within the same host may be a source of selection on pathogen virulence and drug resistance. As different patterns have already been described in humans and mosquitoes, parasite diversity and population structure should be studied in both hosts to properly assess their effects on infection and transmission dynamics. This study aimed to characterize the circulating populations of Plasmodium spp and Plasmodium falciparum from a combined set of human blood and mosquito samples gathered in mainland Equatorial Guinea. Further, the origin and evolution of anti-malarial resistance in this area, where malaria remains a major public health problem were traced. METHODS: Plasmodium species infecting humans and mosquitoes were identified by nested-PCR of chelex-extracted DNA from dried blood spot samples and mosquitoes. Analysis of Pfmsp2 gene, anti-malarial-resistance associated genes, Pfdhps, Pfdhfr, Pfcrt and Pfmdr1, neutral microsatellites (STR) loci and Pfdhfr and Pfdhps flanking STR was undertaken to evaluate P. falciparum diversity. RESULTS: Prevalence of infection remains high in mainland Equatorial Guinea. No differences in parasite formula or significant genetic differentiation were seen in the parasite populations in both human and mosquito samples. Point mutations in all genes associated with anti-malarial resistance were highly prevalent. A high prevalence was observed for the Pfdhfr triple mutant in particular, associated with pyrimethamine resistance.Analysis of Pfdhps and Pfdhfr flanking STR revealed a decrease in the genetic diversity. This finding along with multiple independent introductions of Pfdhps mutant haplotypes suggest a soft selective sweep and an increased differentiation at Pfdhfr flanking microsatellites hints a model of positive directional selection for this gene. CONCLUSIONS: Chloroquine is no longer recommended for malaria treatment in Equatorial Guinea but sulphadoxine-pyrimethamine (SP) remains in use in combination with artesunate and is the only drug recommended in preventive chemotherapy in pregnancy. The high prevalence of point mutations in Pfdhfr and Pfdhps points to the danger of an eventual reduction in the efficacy of SP combined therapy in P. falciparum populations in Equatorial Guinea and to the essential continuous monitoring of these two genes.This study was supported by PEst-OE/SAU/LA0018/2011 - Proj. Estratégico LA0018 2011/2012 (http://cmdt.ihmt.unl.pt/index.php/pt/) and PTDC/SAU-EPI/113326/2009, “Fundacão para a Ciência e Tecnologia/Ministério da Educação e Ciência”, FCT/MEC (http://alfa.fct.mctes.pt/index.phtml.pt), Portugal and by “Instituto de Salud Carlos III, Ministerio de Ciencia e Innovación”, Madrid, Spain. C. Mendes and P. Salgueiro hold FCT grants (SRFH/BD/41473/2007 and SFRH/BPD/72532/2010, respectively).S

Molecular markers of antifolate resistance in Plasmodium falciparum isolates from Luanda, Angola

Abstract Background Plasmodium falciparum malaria remains a leading health problem in Africa and its control is seriously challenged by drug resistance. Although resistance to the sulphadoxine-pyrimethamine (SP) is widespread, this combination remains an important component of malaria control programmes as intermittent preventive therapy (IPT) for pregnant women and children. In Angola, resistance patterns have been poorly characterized, and IPT has been employed for pregnant women since 2006. The aim of this study was to assess the prevalence of key antifolate resistance mediating polymorphisms in the pfdhfr and pfdhps genes in P. falciparum samples from Angola. Methods Plasmodium falciparum samples collected in Luanda, in 2007, were genotyped by amplification and DNA forward and reverse sequencing of the pfdhfr and pfdhps genes. Results The most prevalent polymorphisms identified were pfdhfr 108N (100%), 51I (93%), 59R (57%) and pfdhps 437G (93%). Resistance-mediating polymorphisms in pfdhps less commonly observed in West Africa were also identified (540E in 10%, 581G in 7% of samples). Conclusion This study documents an important prevalence of 4 P. falciparum polymorphisms that predicts an antifolate resistance in Luanda. Further, some samples presented additional mutations associated to high-level resistance. These results suggest that the use of SP for IPT may no longer be warranted in Angola

Genetic and phenotypic variation of the malaria vector Anopheles atroparvus in southern Europe

<p>Abstract</p> <p>Background</p> <p>There is a growing concern that global climate change will affect the potential for pathogen transmission by insect species that are vectors of human diseases. One of these species is the former European malaria vector, <it>Anopheles atroparvus</it>. Levels of population differentiation of <it>An. atroparvus </it>from southern Europe were characterized as a first attempt to elucidate patterns of population structure of this former malaria vector. Results are discussed in light of a hypothetical situation of re-establishment of malaria transmission.</p> <p>Methods</p> <p>Genetic and phenotypic variation was analysed in nine mosquito samples collected from five European countries, using eight microsatellite loci and geometric morphometrics on 21 wing landmarks.</p> <p>Results</p> <p>Levels of genetic diversity were comparable to those reported for tropical malaria vectors. Low levels of genetic (0.004 <<it>F</it>_{<it>ST </it>}<0.086) and phenotypic differentiation were detected among <it>An. atroparvus </it>populations spanning over 3,000 km distance. Genetic differentiation (0.202 <<it>F</it>_{<it>ST </it>}<0.299) was higher between the sibling species <it>An. atroparvus </it>and <it>Anopheles maculipennis </it>s.s. Differentiation between sibling species was not so evident at the phenotype level.</p> <p>Conclusions</p> <p>Levels of population differentiation within <it>An. atroparvus </it>were low and not correlated with geographic distance or with putative physical barriers to gene flow (Alps and Pyrenées). While these results may suggest considerable levels of gene flow, other explanations such as the effect of historical population perturbations can also be hypothesized.</p