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

Failure to detect Plasmodium vivax in West and Central Africa by PCR species typing

<p>Abstract</p> <p>Background</p> <p><it>Plasmodium vivax </it>is estimated to affect 75 million people annually. It is reportedly absent, however, from west and central Africa due to the high prevalence of the Duffy negative phenotype in the indigenous populations. Despite this, non-African travellers consistently return to their own countries with <it>P. vivax </it>malaria after visiting this region. An attempt was made, therefore, to detect the presence of <it>P. vivax </it>parasites in blood samples collected from the indigenous populations of west and central Africa.</p> <p>Methods</p> <p>Parasite species typing (for all four human malaria parasites) was carried out by PCR on 2,588 blood samples collected from individuals from nine African malaria-endemic countries.</p> <p>Results</p> <p>Most infections (98.5%) were <it>Plasmodium falciparum</it>, <it>Plasmodium malariae </it>was identified in 8.5% of all infections, and <it>Plasmodium ovale </it>in 3.9%. The prevalence of both parasites varied greatly by country. Only one case of <it>P. vivax </it>was detected from Sao Tome, an island off the west coast of Africa, confirming the scarcity of this parasite in Africa.</p> <p>Conclusion</p> <p>The prevalence of <it>P. vivax </it>in local populations in sub-Saharan Africa is very low, despite the frequent identification of this parasite in non-African travellers.</p

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

BackgroundEvaluating 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.MethodsThe DeltaDeltaCt 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.ResultsUsing 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 &amp; Principe, West Africa.ConclusionThe 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

An AFLP-based genetic linkage map of <it>Plasmodium chabaudi chabaudi</it>

<p>Abstract</p> <p>Background</p> <p><it>Plasmodium chabaudi chabaudi </it>can be considered as a rodent model of human malaria parasites in the genetic analysis of important characters such as drug resistance and immunity. Despite the availability of some genome sequence data, an extensive genetic linkage map is needed for mapping the genes involved in certain traits.</p> <p>Methods</p> <p>The inheritance of 672 Amplified Fragment Length Polymorphism (AFLP) markers from two parental clones (AS and AJ) of <it>P. c. chabaudi </it>was determined in 28 independent recombinant progeny clones. These, AFLP markers and 42 previously mapped Restriction Fragment Length Polymorphism (RFLP) markers (used as chromosomal anchors) were organized into linkage groups using Map Manager software.</p> <p>Results</p> <p>614 AFLP markers formed linkage groups assigned to 10 of 14 chromosomes, and 12 other linkage groups not assigned to known chromosomes. The genetic length of the genome was estimated to be about 1676 centiMorgans (cM). The mean map unit size was estimated to be 13.7 kb/cM. This was slightly less then previous estimates for the human malaria parasite, <it>Plasmodium falciparum</it></p> <p>Conclusion</p> <p>The <it>P. c. chabaudi </it>genetic linkage map presented here is the most extensive and highly resolved so far available for this species. It can be used in conjunction with the genome databases of <it>P. c chabaudi</it>, <it>P. falciparum </it>and <it>Plasmodium yoelii </it>to identify genes underlying important phenotypes such as drug resistance and strain-specific immunity.</p