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RDTs as a source of DNA to study Plasmodium falciparum drug resistance in isolates from Senegal and the Comoros Islands.

Papa Mze N, Ndiaye YD, Diedhiou CK, Rahamatou S, Dieye B, Daniels RF, Hamilton EJ, Diallo M, Bei AK, Wirth DF, Mboup S, Volkman SK, Ahouidi AD, Ndiaye D - Malar. J. (2015)

Bottom Line: A total of 201 samples were successfully genotyped at all codons by HRM; whereas, in 200 msp1 and msp2 genes were successfully amplified and genotyped by nested PCR.The utility of field-collected RDTs was validated as a source of DNA for genetic studies interrogating frequencies of drug resistance mutations, using two different molecular methods (PCR and High Resolution Melting).RDTs should not be discarded after use as they can be a valuable source of DNA for genetic and epidemiological studies in sites where filter paper or venous blood collected samples are nonexistent.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Bacteriology-Virology, Hospital Aristide Le Dantec, 7325, Dakar, Senegal. npapamze@gmail.com.

ABSTRACT

Background: The World Health Organization has recommended rapid diagnostic tests (RDTs) for use in the diagnosis of suspected malaria cases. In addition to providing quick and accurate detection of Plasmodium parasite proteins in the blood, these tests can be used as sources of DNA for further genetic studies. As sulfadoxine-pyrimethamine is used currently for intermittent presumptive treatment of pregnant women in both Senegal and in the Comoros Islands, resistance mutations in the dhfr and dhps genes were investigated using DNA extracted from RDTs.

Methods: The proximal portion of the nitrocellulose membrane of discarded RDTs was used for DNA extraction. This genomic DNA was amplified using HRM to genotype the molecular markers involved in resistance to sulfadoxine-pyrimethamine: dhfr (51, 59, 108, and 164) and dhps (436, 437, 540, 581, and 613). Additionally, the msp1 and msp2 genes were amplified to determine the average clonality between Grande-Comore (Comoros) and Thiès (Senegal).

Results: A total of 201 samples were successfully genotyped at all codons by HRM; whereas, in 200 msp1 and msp2 genes were successfully amplified and genotyped by nested PCR. A high prevalence of resistance mutations were observed in the dhfr gene at codons 51, 59, and 108 as well as in the dhps gene at codons 437 and 436. A novel mutant in dhps at codon positions 436Y/437A was observed. The dhfr I164L codon and dhps K540 and dhps A581G codons had 100 % wild type alleles in all samples.

Conclusion: The utility of field-collected RDTs was validated as a source of DNA for genetic studies interrogating frequencies of drug resistance mutations, using two different molecular methods (PCR and High Resolution Melting). RDTs should not be discarded after use as they can be a valuable source of DNA for genetic and epidemiological studies in sites where filter paper or venous blood collected samples are nonexistent.

No MeSH data available.


Related in: MedlinePlus

HRM peak profiles for wild-type and mutant dhps 436/437 alleles. a The 3D7 peak represents the profile for the wild-type at codon 436 and mutant profile for the 437 codon. The Dd2 peak presents the profile for the two mutant Codons 436/437. The HB3 peak presents the wild-type profile of the two codons. The 436A/437Apeaks, represented the mutations found by Daniels et al. in Senegal [18]. The blue peak from sample Th50 RDT represents a new mutant allele. b The peaks C03 and C05 represent mixed samples (mutant + wild). The Dd2 peak presents the mutant profile for both codons 436 and 437. The HB3 peak represents the wild-type profile for both codons 436 and 437. The 3D7 peak represents the profile for the wild-type at codon 436 and mutant profile for the 437 codon
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Fig1: HRM peak profiles for wild-type and mutant dhps 436/437 alleles. a The 3D7 peak represents the profile for the wild-type at codon 436 and mutant profile for the 437 codon. The Dd2 peak presents the profile for the two mutant Codons 436/437. The HB3 peak presents the wild-type profile of the two codons. The 436A/437Apeaks, represented the mutations found by Daniels et al. in Senegal [18]. The blue peak from sample Th50 RDT represents a new mutant allele. b The peaks C03 and C05 represent mixed samples (mutant + wild). The Dd2 peak presents the mutant profile for both codons 436 and 437. The HB3 peak represents the wild-type profile for both codons 436 and 437. The 3D7 peak represents the profile for the wild-type at codon 436 and mutant profile for the 437 codon

Mentions: In Thiès, for the dhfr gene, 90 % (54/60) of the samples had the mutant allele at codons dhfr N51C/C59R, and 95.8 % (68/71) had the mutant allele at codon dhfr S108 N. For the dhps gene 1.33 % (1/75) had the A613T mutant allele and 53.9 % (41/76) of the samples had the A437G mutant allele. At the S436 codon of dhps 12 % (9/77) of the samples had the mutant allele S436F, a recently characterized mutation described previously [18]. For all other codons (namely, dhfr I164, dhps K540, and dhps A581) 100 % of the samples had the wild-type allele. Previously unreported mutations were observed for dhps gene at codons 436/437 by HRM (Fig. 1a) and were confirmed to be S436Y/437A by Sanger sequencing (Fig. 2).Fig. 1


RDTs as a source of DNA to study Plasmodium falciparum drug resistance in isolates from Senegal and the Comoros Islands.

Papa Mze N, Ndiaye YD, Diedhiou CK, Rahamatou S, Dieye B, Daniels RF, Hamilton EJ, Diallo M, Bei AK, Wirth DF, Mboup S, Volkman SK, Ahouidi AD, Ndiaye D - Malar. J. (2015)

HRM peak profiles for wild-type and mutant dhps 436/437 alleles. a The 3D7 peak represents the profile for the wild-type at codon 436 and mutant profile for the 437 codon. The Dd2 peak presents the profile for the two mutant Codons 436/437. The HB3 peak presents the wild-type profile of the two codons. The 436A/437Apeaks, represented the mutations found by Daniels et al. in Senegal [18]. The blue peak from sample Th50 RDT represents a new mutant allele. b The peaks C03 and C05 represent mixed samples (mutant + wild). The Dd2 peak presents the mutant profile for both codons 436 and 437. The HB3 peak represents the wild-type profile for both codons 436 and 437. The 3D7 peak represents the profile for the wild-type at codon 436 and mutant profile for the 437 codon
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4587814&req=5

Fig1: HRM peak profiles for wild-type and mutant dhps 436/437 alleles. a The 3D7 peak represents the profile for the wild-type at codon 436 and mutant profile for the 437 codon. The Dd2 peak presents the profile for the two mutant Codons 436/437. The HB3 peak presents the wild-type profile of the two codons. The 436A/437Apeaks, represented the mutations found by Daniels et al. in Senegal [18]. The blue peak from sample Th50 RDT represents a new mutant allele. b The peaks C03 and C05 represent mixed samples (mutant + wild). The Dd2 peak presents the mutant profile for both codons 436 and 437. The HB3 peak represents the wild-type profile for both codons 436 and 437. The 3D7 peak represents the profile for the wild-type at codon 436 and mutant profile for the 437 codon
Mentions: In Thiès, for the dhfr gene, 90 % (54/60) of the samples had the mutant allele at codons dhfr N51C/C59R, and 95.8 % (68/71) had the mutant allele at codon dhfr S108 N. For the dhps gene 1.33 % (1/75) had the A613T mutant allele and 53.9 % (41/76) of the samples had the A437G mutant allele. At the S436 codon of dhps 12 % (9/77) of the samples had the mutant allele S436F, a recently characterized mutation described previously [18]. For all other codons (namely, dhfr I164, dhps K540, and dhps A581) 100 % of the samples had the wild-type allele. Previously unreported mutations were observed for dhps gene at codons 436/437 by HRM (Fig. 1a) and were confirmed to be S436Y/437A by Sanger sequencing (Fig. 2).Fig. 1

Bottom Line: A total of 201 samples were successfully genotyped at all codons by HRM; whereas, in 200 msp1 and msp2 genes were successfully amplified and genotyped by nested PCR.The utility of field-collected RDTs was validated as a source of DNA for genetic studies interrogating frequencies of drug resistance mutations, using two different molecular methods (PCR and High Resolution Melting).RDTs should not be discarded after use as they can be a valuable source of DNA for genetic and epidemiological studies in sites where filter paper or venous blood collected samples are nonexistent.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Bacteriology-Virology, Hospital Aristide Le Dantec, 7325, Dakar, Senegal. npapamze@gmail.com.

ABSTRACT

Background: The World Health Organization has recommended rapid diagnostic tests (RDTs) for use in the diagnosis of suspected malaria cases. In addition to providing quick and accurate detection of Plasmodium parasite proteins in the blood, these tests can be used as sources of DNA for further genetic studies. As sulfadoxine-pyrimethamine is used currently for intermittent presumptive treatment of pregnant women in both Senegal and in the Comoros Islands, resistance mutations in the dhfr and dhps genes were investigated using DNA extracted from RDTs.

Methods: The proximal portion of the nitrocellulose membrane of discarded RDTs was used for DNA extraction. This genomic DNA was amplified using HRM to genotype the molecular markers involved in resistance to sulfadoxine-pyrimethamine: dhfr (51, 59, 108, and 164) and dhps (436, 437, 540, 581, and 613). Additionally, the msp1 and msp2 genes were amplified to determine the average clonality between Grande-Comore (Comoros) and Thiès (Senegal).

Results: A total of 201 samples were successfully genotyped at all codons by HRM; whereas, in 200 msp1 and msp2 genes were successfully amplified and genotyped by nested PCR. A high prevalence of resistance mutations were observed in the dhfr gene at codons 51, 59, and 108 as well as in the dhps gene at codons 437 and 436. A novel mutant in dhps at codon positions 436Y/437A was observed. The dhfr I164L codon and dhps K540 and dhps A581G codons had 100 % wild type alleles in all samples.

Conclusion: The utility of field-collected RDTs was validated as a source of DNA for genetic studies interrogating frequencies of drug resistance mutations, using two different molecular methods (PCR and High Resolution Melting). RDTs should not be discarded after use as they can be a valuable source of DNA for genetic and epidemiological studies in sites where filter paper or venous blood collected samples are nonexistent.

No MeSH data available.


Related in: MedlinePlus