Pyrosequencing, a High-Throughput Method for Detecting Single Nucleotide Polymorphisms in the Dihydrofolate Reductase and Dihydropteroate Synthetase Genes of
            <i>Plasmodium falciparum</i>

Zhou, Zhansong; Poe, Amanda; Limor, Josef; Grady, Katharine K.; Goldman, Ira F.; McCollum, Andrea M.; Escalante, Ananías A.; Barnwell, John W.; Udhayakumar, Venkatachalam

doi:10.1128/jcm.01209-06

Cited by 57 publications

(56 citation statements)

References 30 publications

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“…We used the relatively novel approach of pyrosequencing to quantitatively determine the fraction of resistance alleles in individual samples at multiple SNPs in DHFR and DHPS (24). We found that resistance alleles at codons DHFR 51 and 59, and at DHPS 540, were close to saturation in parasite samples from Muheza.…”

Section: Discussionmentioning

confidence: 99%

“…Negative controls without DNA template were included for all PCR reactions. Cycling parameters for both primary and nested PCRs were as described by Zhou et al (24). Amplified bands were confirmed by running 5 l of the nested PCR on 1.5% agarose gel and visualized using UV to confirm successful amplification.…”

Section: Methodsmentioning

confidence: 99%

“…For all positive placental samples, DNA was extracted using a QiaAmp DNA Micro or Mini kit (Qiagen), either from filter paper spots or from blood pellets where filter paper extraction was not successful. Primary and nested PCR amplifications of DHFR and DHPS fragments were performed as described by Zhou et al (24), except that the forward primer for the nested reaction was modified at the 5Ј end to disrupt a template loop (primer sequence: 5Ј GGGTTCTAATGCATA-AAAGAGGAAATC 3Ј). Briefly, reaction mixtures in 50 l volumes were amplified using the Roche AmpliTaq PCR system.…”

Section: Methodsmentioning

confidence: 99%

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Competitive facilitation of drug-resistant Plasmodium falciparum malaria parasites in pregnant women who receive preventive treatment

Harrington

Mutabingwa

Muehlenbachs

et al. 2009

Proc. Natl. Acad. Sci. U.S.A.

188

246

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Intermittent preventive treatment in pregnancy (IPTp) is used to prevent Plasmodium falciparum malaria. However, parasites resistant to the IPTp drug sulfadoxine-pyrimethamine (SP) have emerged worldwide, and infections with mixed resistant and susceptible parasites are exacerbated by pyrimethamine in mice. In a prospective delivery cohort in Muheza, Tanzania, we examined the effects of SP IPTp on parasite resistance alleles, parasite diversity, level of parasitemia, and inflammation in the placenta. IPTp use was associated with an increased fraction of parasites carrying the resistance allele at DHPS codon 581, an increase in the level of parasitemia, and more intense placental inflammation. The lowest mean level of parasite diversity and highest mean level of parasitemia occurred in women after recent IPTp use. These findings support a model of parasite release and facilitation, whereby the most highly resistant parasites out-compete less fit parasite populations and overgrow under drug pressure. Use of partially effective anti-malarial agents for IPTp may exacerbate malaria infections in the setting of widespread drug resistance.evolution of drug resistance ͉ intermittent preventive treatment in pregnancy ͉ malaria drug resistance ͉ pregnancy malaria M alaria during pregnancy causes both maternal and fetal morbidity and mortality, including the deaths of an estimated 100,000 infants each year because of malaria-related low birth weight. The World Health Organization recommends that women living in sub-Saharan Africa receive at least two doses of intermittent preventive treatment (IPTp) with sulfadoxinepyrimethamine (SP) to prevent malaria and improve pregnancy outcomes (1-5) and this advice is widely followed. However, the rapid spread of SP-resistant parasites might undermine the efficacy of SP-IPTp, although it still confers benefits in areas with low to moderate levels of drug resistance (6). The effect of SP IPTp in areas where resistant parasites are more widespread has not been studied.Pyrimethamine and sulfadoxine target the parasite proteins DHFR and DHPS, respectively, and increasing resistance is conferred by ordered accumulating point mutations, of which DHPS codon 581 is one of the final to occur (7). The mutation at DHPS codon 581 has been associated with high-level in vitro resistance (8), as well as treatment failure in children (9, 10). Single-dose treatment of children with SP has been associated with an increase in parasite resistance alleles (11, 12), but little work has been done to examine the relationship between IPTp and selection for drug resistance alleles. One recent study observed an increased prevalence of placental infections harboring the DHFR triple mutant in women who had received pyrimethamine prophylaxis during pregnancy, as compared with women who had not (13). In contrast, IPTp use was not related to increased prevalence of resistance alleles at the same study site (14). Modeling studies have proposed that IPTp is less likely to contribute to accumulating drug resistance as...

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Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

See 1 more Smart Citation

Competitive facilitation of drug-resistant Plasmodium falciparum malaria parasites in pregnant women who receive preventive treatment

Harrington

Mutabingwa

Muehlenbachs

et al. 2009

Proc. Natl. Acad. Sci. U.S.A.

188

246

View full text Add to dashboard Cite

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“…Several papers have introduced interesting methods for evaluating the frequency of drug resistant genotypes within the context of heterogeneous pathogen populations [26,34,35]. Most techniques employ PCR-based amplification of SNP markers surrounding the resistance locus.…”

Section: Tracking Known Resistance Mutations Allele Identificationmentioning

confidence: 99%

Advances in understanding the genetic basis of antimalarial drug resistance

Ekland

Fidock

2007

Current Opinion in Microbiology

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SummaryThe acquisition of drug resistance by Plasmodium falciparum has severely curtailed global efforts to control malaria. Our ability to define resistance has been greatly enhanced by recent advances in Plasmodium genetics and genomics. Sequencing and microarray studies have identified thousands of polymorphisms in the P. falciparum genome, and linkage disequilibrium analyses have exploited these to rapidly identify known and novel loci that influence parasite susceptibility to antimalarials such as chloroquine, quinine, and sulfadoxine-pyrimethamine. Genetic approaches have also been designed to predict determinants of in vivo resistance to new antimalarials such as the artemisinins. Transfection methodologies have defined the role of determinants including pfcrt, pfmdr1 and dhfr. This knowledge can be leveraged to develop more efficient methods of surveillance and treatment.

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“…Existing molecular methods for analysis of these SNPs include allele-specific PCR, PCR-restriction fragment length polymorphism (RFLP) analysis, multiplex PCR (mPCR)-RFLP, DNA sequencing, dot blot hybridization techniques, the molecular beacon method, real-time PCR, PCR-enzymelinked immunosorbent assay, and pyrosequencing (1,2,6,8,10,28,31,37,38). Each of these techniques offers its own advantages and limitations.…”

mentioning

confidence: 99%

Multiplex PCR and Oligonucleotide Microarray for Detection of Single-Nucleotide Polymorphisms Associated with Plasmodium falciparum Drug Resistance

Zhang

Guan

Sheng³

et al. 2008

J Clin Microbiol

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Drug resistance in Plasmodium falciparum is a serious public health threat in the countries where this organism is endemic. Since resistance has been associated with specific single-nucleotide polymorphisms (SNPs) in parasite genes, molecular markers are becoming useful surrogates for monitoring the emergence and dispersion of drug resistance. In this study, a multiplex PCR (mPCR) and oligonucleotide microarray method was developed for the detection of these SNPs in genes encoding chloroquine resistance transporter (Pfcrt), multidrug resistance 1 (Pfmdr1), dihydrofolate reductase (Pfdhfr), dihydropteroate synthetase (Pfdhps), and ATPase 6 (PfATPase6) of P. falciparum. The results show that DNA microarray technology, combined with mPCR, is a promising and time-saving tool that supports conventional detection methods, allowing sensitive, accurate, simultaneous analysis of the SNPs associated with drug resistance in P. falciparum.

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Pyrosequencing, a High-Throughput Method for Detecting Single Nucleotide Polymorphisms in the Dihydrofolate Reductase and Dihydropteroate Synthetase Genes of Plasmodium falciparum

Cited by 57 publications

References 30 publications

Competitive facilitation of drug-resistant Plasmodium falciparum malaria parasites in pregnant women who receive preventive treatment

Competitive facilitation of drug-resistant Plasmodium falciparum malaria parasites in pregnant women who receive preventive treatment

Advances in understanding the genetic basis of antimalarial drug resistance

Multiplex PCR and Oligonucleotide Microarray for Detection of Single-Nucleotide Polymorphisms Associated with Plasmodium falciparum Drug Resistance

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