Geographic Differences in the Sensitivity of a Polymerase Chain Reaction for the Detection of Plasmodium falciparum Infection

Jelı́nek, Tomáš; Pröll, Simon; Hess, F; Kabagambe, G.; F, von Sonnenburg; Löscher, Thomas; Ah, Kilian

doi:10.4269/ajtmh.1996.55.647

Cited by 34 publications

(26 citation statements)

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“…Isolation of DNA from filter paper was performed as previously described in detail. 14,15 Following a modified procedure used for DNA extraction from material derived from Egyptian mummies, 16 blood slides taken at day 7 were incubated in 10 mM Tris-HCl, 2 mM EDTA, 0.1% sodium dodecyl sulfate, dithiothreitol (10 mg/ml), and proteinase K (0.5 mg/ml) for 16 hr at 37ЊC. Subsequently, the dissolved material was extracted with phenol-chloroform.…”

Section: Patients Materials and Methodsmentioning

confidence: 99%

Plasmodium falciparum resistance to sulfadoxine/pyrimethamine in Uganda: correlation with polymorphisms in the dihydrofolate reductase and dihydropteroate synthetase genes.

Jelı́nek

Kilian²,

Kabagambe³

et al. 1999

The American Journal of Tropical Medicine and Hygiene

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Abstract. The efficacy of sulfadoxine/pyrimethamine (S/P) in treatment of uncomplicated falciparum malaria in Africa is increasingly compromised by development of resistance. The occurrence of active site mutations in the Plasmodium falciparum gene sequences coding for dihydrofolate reductase (DHFR) and dihydropteroate synthetase (DHPS) is known to confer resistance to pyrimethamine and sulfadoxine. This study investigated the occurrence of these mutations in infected blood samples taken from Ugandan children before treatment with S/P and their relationship to parasite breakthrough by day 7. The results confirm the occurrence of mutations in DHFR and DHPS that were significantly selected under S/P pressure at day 7: a combination of alleles 51-isoleucine and 108-asparagine in DHFR, and 436-serine, 437-alanine, 540-lysine and 581-alanine in DHPS, appears to play a major role in the development of in vivo resistance in P. falciparum strains against S/P. Therefore, earlier results derived from isolates from hyperendemic areas in Tanzania were confirmed by this investigation.The antifolate combination of sulfadoxine/pyrimethamine (S/P) is experiencing an increasingly widespread use in Africa, making it to one of the major first-and second-line drugs. After replacement of chloroquine, clinical resistance to S/P has arisen rapidly in other areas of the world. Monitoring and, if possible, delaying the spread of S/P-resistance is therefore a major public health objective in affected African countries. All efforts to control resistance will require methods that make it possible to map S/P-resistant malaria quickly and accurately in epidemiologic studies. Currently used in vitro and in vivo methods for the measurement of drug resistance in Plasmodium falciparum are not well-suited for fast epidemiologic surveillance. Techniques that facilitate screening of a large number of samples without unduly straining scarce financial resources are needed.Drug resistance of P. falciparum to pyrimethamine has been associated with point mutations in the gene coding for dihydrofolate reductase (DHFR), in particular with the occurrence of asparagine in position 108. 1 Polymorphisms at 5 highly conserved positions within dihydropteroate synthetase (DHPS) have been reported in sulfadoxine-resistant isolates of P. falciparum: codons 436, 437, 581, 613 2-5 and also at 540. 6 Recently, alteration of 437-alanine to glycine has been identified as key residue on DHPS for the development of sulfadoxine resistance. 7

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Section: Patients Materials and Methodsmentioning

confidence: 99%

Plasmodium falciparum resistance to sulfadoxine/pyrimethamine in Uganda: correlation with polymorphisms in the dihydrofolate reductase and dihydropteroate synthetase genes.

Jelı́nek

Kilian²,

Kabagambe³

et al. 1999

The American Journal of Tropical Medicine and Hygiene

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“…DNA was prepared from the dried blood spots as previously described. 17 Half of the bloodspot (corresponding to approximately 5 l of blood) was cut from the filter, transferred to a tube containing 180 l of 5% Chelex-100 (Bio-Rad Laboratories, Munich, Germany) and mixed intensively. After incubation in boiling water for 5 minutes, the tube was vortexed for 30 seconds and incubated in boiling water for 10 minutes more.…”

Section: Patients Materials and Methodsmentioning

confidence: 99%

Diagnostic value of molecular markers in chloroquine-resistant falciparum malaria in Southern Mauritania.

Jelı́nek¹,

Aida²,

Peyerl‐Hoffmann³

et al. 2002

The American Journal of Tropical Medicine and Hygiene

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Despite its diminishing efficacy because of increased resistance, chloroquine remains the primary antimalarial agent in many endemic areas. Evidence is mounting that point mutations on the Pfcrt and possibly the Pfmdr1 genes are conferring plasmodial resistance to chloroquine. In 1998, atypically strong rainfalls led to an increased activity of falciparum malaria in Mauritania that affected non-endemic regions bordering the Saharan desert. An in vivo study on chloroqine resistance was combined with studies for molecular markers of drug resistance. Detection of Pfmdr1-76-tyrosine showed an increased odds ratio (2.91) for resistance (P ‫ס‬ 0.0195). However, by use of this codon alone, sensitivity for detection of resistance was 60.6%, and specificity was 65.3%. In comparison, detection of the K76T mutation at Pfcrt showed a very high sensitivity (100%) while specificity remained relatively low (65.4%). For the combination of mutations on both genes, the odds ratio for detection of resistance increased to 5.31 (P ‫ס‬ 0.0005). Here, sensitivity was again decreased to 60.6% while specificity increased to 76.9%. The results of this study suggest that detection of Pfcrt T76 can be applied for predicting chloroquine resistance in epidemiologic settings with sufficiently high sensitivity to make it an attractive alternative to time-and labor-consuming in vivo trials. Additional testing for Pfmdr Y76 provides increased specificity to this approach.

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“…Preparation of DNA from filter paper was performed as previously described in detail. 22,23 Following a modified procedure used for DNA extraction from material derived from Egyptian mummies, 24 blood slides taken at days 3 or 7 after treatment were incubated in 10 mM Tris-HCl, 2 mM EDTA, 0.1% sodium dodecyl sulfate, 10 mg/ml of dithiothreitol, and 0.5 mg/ml of proteinase K for 16 hr at 37ЊC. Subsequently, the dissolved material was extracted with phenol-chloroform.…”

Section: Patients Materials and Methodsmentioning

confidence: 99%

Plasmodium falciparum: selection of serine 108 of dihydrofolate reductase during treatment of uncomplicated malaria with co-trimoxazole in Ugandan children.

Jelı́nek¹,

Kilian²,

Curtis³

et al. 1999

Am J Trop Med Hyg

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Abstract. In vivo testing for resistance of Plasmodium falciparum to co-trimoxazole (trimethoprim/sulfamethoxazole) was performed in Uganda in 41 children with uncomplicated malaria, and blood samples were screened before and after treatment for polymorphisms in the antifolate target genes for dihydrofolate reductase (DHFR) and dihydropteroate synthetase (DHPS). Selection towards a specific genotype at some codons of the DHFR and DHPS genes was observed in samples collected after exposure to co-trimoxazole drug pressure. The alleles 51-isoleucine, 59-arginine, and 108-serine of DHFR were significantly associated with clinical resistance, as was allele 581-alanine of DHPS. Resistance against antifolate combinations probably requires resistance-related polymorphisms in both the DHFR and the DHPS genes. In addition, it appears that the trimethoprim-resistant DHFR genotype differs from that for pyrimethamine at residue 108.Falciparum malaria and acute respiratory tract infection, two of the most common causes of childhood morbidity and mortality in sub-Saharan Africa, show an overlap of symptoms in a high proportion of cases. 1 The World Health Organization (WHO) recommends co-trimoxazole (trimethoprim and sulfamethoxazole) as treatment for children in malaria-endemic areas presenting with fever and respiratory symptoms. 2 This drug has been shown in the past to be effective against malaria as well as pneumonia. 3,4 The components of co-trimoxazole target selectively dihydrofolate reductase (DHFR) and dihydropteroate synthetase (DHPS), the same folate pathway enzymes as pyrimethamine/sulfadoxine, which is used in many African countries for treatment of chloroquine-resistant infections with Plasmodium falciparum. In vitro drug resistance of P. falciparum to pyrimethamine has been associated with point mutations in the gene coding for DHFR, in particular with the presence of asparagine in position 108.5 Likewise, polymorphisms at 5 highly conserved positions within DHPS have been reported in sulfadoxine-resistant isolates of P. falciparum: codons 436, 437, 581, 613, 6,7 and 540. [8][9][10] Unlike DHFR, no single polymorphism has been associated with all resistant strains and only limited data are available on the global distribution of these polymorphisms.11 In vivo testing for resistance to co-trimoxazole was performed in rural health centers in the 2 districts of Kabarole and Bundibugyo in western Uganda. Results of the clinical and parasitologic outcomes have been reported elsewhere.12 Fortyone children with symptomatic malaria from the Kabarole and Bundibugyo Districts of western Uganda were recruited for the study. Data were obtained about polymorphisms in the DHFR and DHPS genes that have been associated with antifolate resistance [5][6][7][13][14][15][16][17][18][19] by extraction and amplification of parasite DNA from filter paper and thick blood films. We report on a comparison of the prevalence of polymorphisms of the antifolate target genes at days 0 and 3/7 and their association with in vivo results in one...

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Geographic Differences in the Sensitivity of a Polymerase Chain Reaction for the Detection of Plasmodium falciparum Infection

Cited by 34 publications

References 0 publications

Plasmodium falciparum resistance to sulfadoxine/pyrimethamine in Uganda: correlation with polymorphisms in the dihydrofolate reductase and dihydropteroate synthetase genes.

Plasmodium falciparum resistance to sulfadoxine/pyrimethamine in Uganda: correlation with polymorphisms in the dihydrofolate reductase and dihydropteroate synthetase genes.

Diagnostic value of molecular markers in chloroquine-resistant falciparum malaria in Southern Mauritania.

Plasmodium falciparum: selection of serine 108 of dihydrofolate reductase during treatment of uncomplicated malaria with co-trimoxazole in Ugandan children.

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