Identification and analysis of dihydrofolate reductase alleles from Plasmodium falciparum present at low frequency in polyclonal patient samples.

Mookherjee, Somnath; Howard, Vincent; Nzila-Mouanda, Alexis; Watkins, William A.; Sibley, Carol Hopkins

doi:10.4269/ajtmh.1999.61.131

Cited by 17 publications

(12 citation statements)

References 58 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The presence of minority-variant drug-resistant mutations in P. falciparum has been previously demonstrated by subcloning dihydrofolate reductase genes into yeast vectors and growing them under drug pressure (17). However, this technique cannot determine the frequency of minority variants either in a single host or in a population.…”

Section: Discussionmentioning

confidence: 99%

Minority-Variant pfcrt K76T Mutations and Chloroquine Resistance, Malawi

Juliano

Kwiek

Cappell

et al. 2007

Emerg. Infect. Dis.

View full text Add to dashboard Cite

Genotyping of the chloroquine-resistance biomarker pfcrt (Plasmodium falciparum chloroquine resistance transporter gene) suggests that, in the absence of chloroquine pressure, Plasmodium falciparum parasites in Malawi have reverted to chloroquine sensitivity. However, malaria infections in Africa are commonly polyclonal, and standard PCRs cannot detect minority genotypes if present in <20% of the parasites in an individual host. We have developed a multiple site-specifi c heteroduplex tracking assay (MSS-HTA) that can detect pfcrt 76T mutant parasites consisting of as little as 1% of the parasite population. In clinical samples, no pfcrt 76T was detected in 87 pregnant Malawian women by standard PCR. However, 22 (25%) contained minority-variant resistant genotypes detected by the MSS-HTA. These results were confi rmed by subcloning and sequencing. This fi nding suggests that the chloroquine-resistant genotype remains common in Malawians and that PCR-undetectable drug-resistant genotypes may be present in disease-endemic populations. Surveillance for minority-variant drugresistant mutations may be useful in making antimalarial drug policy. D rug-resistant Plasmodium falciparum malaria continues to be a growing health problem throughout most of the world (1). To combat this threat, governments and aid agencies need accurate drug resistance surveillance data. The World Health Organization has stressed the need for methods of detecting molecular markers of drug resistance that will be useful in predicting responses to both clinical and public health interventions (2). This has been diffi cult in highly malaria-endemic areas, where infections are almost always polyclonal (3,4). In patients with polyclonal infections, small drug-resistant parasite populations (minority variants) may be masked by larger drug-sensitive populations because standard PCRs are relatively insensitive to minority variants (2). Therefore, new methods capable of detecting these subpopulations may lead to better drug resistance surveillance and provide a better tool to predict outcome.We describe a new multiple site-specifi c heteroduplex tracking assay (MSS-HTA) for detecting the pfcrt (Plasmodium falciparum chloroquine resistance transporter gene) K76T mutation. This mutation in a putative transporter gene is well-associated with chloroquine resistance in P. falciparum (5). The MSS-HTA was compared with a standard allele-restricted PCR (ARPCR) in clinical samples from Malawi, a country where standard PCR analyses and a recent clinical trial have suggested that chloroquine-resistant malaria has disappeared (6-9). Materials and Methods Study SamplesInformed consent, as approved by the ethics committees of the University of North Carolina and the Malawi College of Medicine/Ministry of Health, was obtained from all participants in this research study. The Malaria and HIV in Pregnancy Study (MHP) patient samples originated from a study of pregnant women attending Queen Elizabeth Central Hospital, an urban hospital in Blantyre. The complete characte...

show abstract

Section: Discussionmentioning

confidence: 99%

Minority-Variant pfcrt K76T Mutations and Chloroquine Resistance, Malawi

Juliano

Kwiek

Cappell

et al. 2007

Emerg. Infect. Dis.

View full text Add to dashboard Cite

show abstract

“…This might reflect greater biological variability in IEV P. falciparum isolates, including better preserved sub-populations of drug-resistant and sensitive parasites. 11,25,26 It may be useful to determine multiplicity of infection of each P. falciparum isolate, processed IEV, and culture adapted, along with comparative IEV and culture-adapted SYBR Green I assays.…”

Section: Discussionmentioning

confidence: 99%

Antimalarial Drug Sensitivity Profile of Western Kenya Plasmodium falciparum Field Isolates Determined by a SYBR Green I in vitro Assay and Molecular Analysis

Akala¹,

Eyase²,

Cheruiyot³

et al. 2011

The American Society of Tropical Medicine and Hygiene

View full text Add to dashboard Cite

In vitro drug sensitivity and molecular analyses of Plasmodium falciparum track drug resistance. DNA-binding fluorescent dyes like SYBR Green I may allow field laboratories, proximal to P. falciparum collection sites, to conduct drug assays. In 2007–2008, we assayed 121 P. falciparum field isolates from western Kenya for 50% inhibitory concentrations (IC50) against 6 antimalarial drugs using a SYBR Green I in vitro assay: 91 immediate ex vivo (IEV) and 30 culture-adapted, along with P. falciparum reference clones D6 (chloroquine [CQ] sensitive) and W2 (CQ resistant). We also assessed P. falciparum mdr1 (Pfmdr1) copy number and single nucleotide polymorphisms (SNPs) at four codons. The IC50s for IEV and culture-adapted P. falciparum isolates were similar, and approximated historical IC50s. For Pfmdr1, mean copy number was 1, with SNPs common at codons 86 and 184. The SYBR Green I assay adapted well to our field-based laboratory, for both IEV and culture-adapted P. falciparum, warranting continued use.

show abstract

“…This mutation has previously been reported only in two samples from Africa. 29 The spread of the dhfr 164 mutation is believed to be a major threat to the use of antifolate drugs for treatment of malaria in Africa.…”

Section: Discussionmentioning

confidence: 99%

Polyclonal Plasmodium falciparum malaria in travelers and selection of antifolate mutations after proguanil prophylaxis.

Färnert¹,

Tengstam²,

Palme³

et al. 2002

The American Journal of Tropical Medicine and Hygiene

View full text Add to dashboard Cite

Abstract. The polymorphism of malaria parasites will greatly influence the efficiency of antimalarial drugs and vaccines. This study determined the genetic diversity of Plasmodium falciparum infections in 107 travelers and estimated the importance of mutations in the parasite dihydrofolate reductase (dhfr) gene for clinical breakthrough during proguanil prophylaxis. Genotyping with regards to the three highly polymorphic antigen-coding regions (merozoite surface protein-1 [msp-1], msp-2, and the glutamate-rich protein [glurp]) revealed multiple genotypes (up to five) in 64% of the patients. Single genotype infections were mainly associated with prior intake of antimalarial drugs, but also with a shorter stay in a malaria-endemic area and low parasite density. Malaria breakthrough despite proguanil prophylaxis was always associated with mutations in the dhfr gene; always the Asn-108 mutation and often the Ile-51 and Arg-59 mutations. The Leu-164 mutation was found in four travelers from Africa. Travelers with limited time in an endemic area were often infected with polyclonal P. falciparum infections, which suggests that single mosquito inoculations are often composed of several genetically diverse parasites. Chemoprophylaxis reduces the number of infecting clones and selects for resistant parasites as shown for proguanil through mutations in the dhfr gene.

show abstract

Identification and analysis of dihydrofolate reductase alleles from Plasmodium falciparum present at low frequency in polyclonal patient samples.

Cited by 17 publications

References 58 publications

Minority-Variant pfcrt K76T Mutations and Chloroquine Resistance, Malawi

Minority-Variant pfcrt K76T Mutations and Chloroquine Resistance, Malawi

Antimalarial Drug Sensitivity Profile of Western Kenya Plasmodium falciparum Field Isolates Determined by a SYBR Green I in vitro Assay and Molecular Analysis

Polyclonal Plasmodium falciparum malaria in travelers and selection of antifolate mutations after proguanil prophylaxis.

Contact Info

Product

Resources

About