Mutations Associated with Sulfadoxine-Pyrimethamine and Chlorproguanil Resistance in
            <i>Plasmodium falciparum</i>
            Isolates from Blantyre, Malawi

Alker, Alisa P.; Mwapasa, Victor; Purfield, Anne; Rogerson, Stephen J.; Molyneux, Malcolm E.; Kamwendo, Deborah; Tadesse, Eyob; Chaluluka, Ebbie; Meshnick, Steven R.

doi:10.1128/aac.49.9.3919-3921.2005

Cited by 58 publications

(57 citation statements)

References 17 publications

(13 reference statements)

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“…Of particular interest is the study by Alker et al (1), who used real-time PCR with fluorescent probes specific for the mutation and reported a 4.7% prevalence in parasites collected from human immunodeficiency virus (HIV)-positive pregnant women in Malawi between 2001 and 2003, a finding that they validated more recently using a heteroduplex tracking assay (9). However, on biological grounds, if the quadruple-mutant alleles were present in reasonable proportions, it is hard to imagine that they would not have been selected to high levels by this time.…”

mentioning

confidence: 99%

Plasmodium falciparum Strains Harboring Dihydrofolate Reductase with the I164L Mutation Are Absent in Malawi and Zambia Even under Antifolate Drug Pressure

Ochong

Bell

Johnson

et al. 2008

Antimicrob Agents Chemother

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The Plasmodium falciparum dihydrofolate reductase (PfDHFR) enzyme is the target of pyrimethamine, a component of the antimalarial pyrimethamine-sulfadoxine. Resistance to this drug is associated primarily with mutations in the Pfdhfr gene. The I164L mutant allele is of particular interest, because strains possessing this mutation are highly resistant to pyrimethamine and to chlorproguanil, a component of chlorproguanildapsone. A recent study from Malawi reported this mutation at a prevalence of 4.7% in parasites from human immunodeficiency virus-positive pregnant women by using a real-time PCR method. These observations have huge implications for the use of pyrimethamine-sulfadoxine, chlorproguanil-dapsone, and future antifolateartemisinin combinations in Africa. It was imperative that this finding be rigorously tested. We identified a number of critical limitations in the original genotyping strategy. Using a refined and validated real-time PCR strategy, we report here that this mutation was absent in 158 isolates from Malawi and 42 isolates from Zambia collected between 2003 and 2005.

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mentioning

confidence: 99%

Plasmodium falciparum Strains Harboring Dihydrofolate Reductase with the I164L Mutation Are Absent in Malawi and Zambia Even under Antifolate Drug Pressure

Ochong

Bell

Johnson

et al. 2008

Antimicrob Agents Chemother

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“…However, compared to those for the published TaqManbased real-time PCR assay by Alker et al (2,3), our assay demonstrated greater sensitivity and specificity. Although the reported sensitivity of the TaqMan assay is 10 copies of the target DNA (2), this was not observed in our experiments or by another laboratory that attempted to reproduce this method (1,2,13).…”

Section: Discussionmentioning

confidence: 68%

“…In previous publications, Alker et al reported a method using hydrolysis probes (TaqMan technology) to discriminate between wild-type and mutant genotypes in the Pfdhfr gene (2,3). We compared the sensitivity and specificity of genotyping by MCA with the TaqMan methodology as described previously.…”

Section: Vol 48 2010 High-throughput Genotyping For P Falciparum Dmentioning

confidence: 99%

“…The TaqMan-based real-time PCR method described by Alker et al (2,3) was performed on the ABI Prism 7000 sequence detection system using published primers (IDT) and probes (Applied Biosystems) (2,3).…”

Section: Plasmidsmentioning

confidence: 99%

“…Recently, the application of real-time PCR for genotyping malaria parasites has been described (19). Studies by Alker et al (2,3) used realtime PCR with sequence-specific probes to detect four of the Pfdhfr SNPs conferring resistance to pyrimethamine. Despite excellent sensitivity reported for this assay, cross-reactivity between probes has been observed (13).…”

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confidence: 99%

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High-Throughput Genotyping of Single Nucleotide Polymorphisms in the Plasmodium falciparum dhfr Gene by Asymmetric PCR and Melt-Curve Analysis

et al. 2010

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Mutations within the Plasmodium falciparum dihydrofolate reductase gene (Pfdhfr) contribute to resistance to antimalarials such as sulfadoxine-pyrimethamine (SP). Of particular importance are the single nucleotide polymorphisms (SNPs) within codons 51, 59, 108, and 164 in the Pfdhfr gene that are associated with SP treatment failure. Given that traditional genotyping methods are time-consuming and laborious, we developed an assay that provides the rapid, high-throughput analysis of parasite DNA isolated from clinical samples. This assay is based on asymmetric real-time PCR and melt-curve analysis (MCA) performed on the LightCycler platform. Unlabeled probes specific to each SNP are included in the reaction mixture and hybridize differentially to the mutant and wild-type sequences within the amplicon, generating distinct melting curves. Since the probe is present throughout PCR and MCA, the assay proceeds seamlessly with no further addition of reagents. This assay was validated for analytical sensitivity and specificity using plasmids, purified genomic DNA from reference strains, and parasite cultures. For all four SNPs, correct genotypes were identified with 100 copies of the template. The performance of the assay was evaluated with a blind panel of clinical isolates from travelers with low-level parasitemia. The concordance between our assay and DNA sequencing ranged from 84 to 100% depending on the SNP. We also directly compared our MCA assay to a published TaqMan real-time PCR assay and identified major issues with the specificity of the TaqMan probes. Our assay provides a number of technical improvements that facilitate the high-throughput screening of patient samples to identify SP-resistant malaria.

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Effect of Sulfadoxine-Pyrimethamine Resistance on the Efficacy of Intermittent Preventive Therapy for Malaria Control During Pregnancy

Kuile¹,

Eijk²,

Filler³

2007

JAMA

295

242

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ContextIn malaria-endemic regions, strategies to control malaria during pregnancy rely on case management of malaria illness and anemia, and preventive measures such as insecticide-treated nets and intermittent preventive therapy (IPT).Objective To determine the effect of increasing resistance to sulfadoxinepyrimethamine on the efficacy of IPT during pregnancy in Africa. Data Sources and Study SelectionThe 6 databases of MEDLINE, EMBASE, SCOPUS, LILACS, Cochrane CENTRAL, and the trial register and bibliographic database of the Malaria in Pregnancy Library were searched for relevant studies regardless of language, published between 1966 and December 2006. The reference lists of all trials identified were searched and researchers were contacted about relevant data. Nine trials of IPT with sulfadoxine-pyrimethamine during pregnancy in Africa were identified and matched by year and location with treatment studies of sulfadoxinepyrimethamine among symptomatic children. Data ExtractionData on the efficacy of IPT with sulfadoxine-pyrimethamine on placental and peripheral malaria, birth weight, and hemoglobin level/anemia were independently abstracted by 2 investigators. Sulfadoxine-pyrimethamine resistance was defined as the proportion of total treatment failures in symptomatic children by day 14.Data Synthesis Four trials compared 2-dose IPT with sulfadoxine-pyrimethamine to case management or placebo in women during their first or second pregnancy. The IPT reduced placental malaria (relative risk [RR], 0.48; 95% CI, 0.35-0.68), low birth weight (RR, 0.71; 95% CI, 0.55-0.92), and anemia (RR, 0.90; 95% CI, 0.81-0.99). The effect did not vary by sulfadoxine-pyrimethamine resistance levels (range, 19%-26%). Efficacy of IPT with sulfadoxine-pyrimethamine was lower among women using insecticide-treated nets. Three trials compared 2-dose with monthly IPT with sulfadoxinepyrimethamine during pregnancy. Among HIV-positive women in their first or second pregnancy, monthly IPT resulted in less placental malaria (RR, 0.34; 95% CI, 0.18-0.64) and higher birth weight (mean difference, 112 g; 95% CI, 19-205 g) over the range of sulfadoxine-pyrimethamine resistance tested (8%-39%). Among HIVnegative women, there was no conclusive additional effect of monthly dosing (2 trials; 24% and 39% resistance). ConclusionsIn areas in which 1 of 4 treatments with sulfadoxine-pyrimethamine fail in children by day 14, the 2-dose IPT with sulfadoxine-pyrimethamine regimen continues to provide substantial benefit to HIV-negative semi-immune pregnant women. However, more frequent dosing is required in HIV-positive women not using cotrimoxazole prophylaxis for opportunistic infections.

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Mutations Associated with Sulfadoxine-Pyrimethamine and Chlorproguanil Resistance in Plasmodium falciparum Isolates from Blantyre, Malawi

Cited by 58 publications

References 17 publications

Plasmodium falciparum Strains Harboring Dihydrofolate Reductase with the I164L Mutation Are Absent in Malawi and Zambia Even under Antifolate Drug Pressure

Plasmodium falciparum Strains Harboring Dihydrofolate Reductase with the I164L Mutation Are Absent in Malawi and Zambia Even under Antifolate Drug Pressure

High-Throughput Genotyping of Single Nucleotide Polymorphisms in the Plasmodium falciparum dhfr Gene by Asymmetric PCR and Melt-Curve Analysis

Effect of Sulfadoxine-Pyrimethamine Resistance on the Efficacy of Intermittent Preventive Therapy for Malaria Control During Pregnancy

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