Abstract
Malaria control and elimination are recognised to be significantly hampered by Plasmodium parasites that carry single nucleotide polymorphisms (SNPs) associated with antimalarial resistance. The lack of ASSURED (Accessible, Sensitive, Specific, User-friendly, Rapid and robust, Equipment-free, and Deliverable to end-users) point-of-care diagnostic tools is one
of the main reasons for the spread of drug-resistant parasites in endemic settings. We aim to develop an ASSURED biosensor based on isothermal Recombinase Polymerase Amplification (RPA) for the detection of SNPs associated with resistance of Plasmodium falciparum parasites to antifolates (sulfadoxine and pyrimethamine), which are widely used in chemopreventive
regimens for children and pregnant women in endemic areas. The method is centred on the optical detection of the isothermal extension of immobilised primers on activated maleimide plates. Target DNA hybridises to 5'-end thiolated primers carrying the desired SNP at the 3'-end nucleotide. Primer extension with biotin-labelled nucleotides occurs only if the target DNA has the
SNP of interest.
We have (i) selected the SNPs associated with pyrimethamine (N51I+C59R+S108N) and sulfadoxine (A437G+K540E) resistance located in the dihydrofolate reductase (pfdhfr) and dihydropteroate synthetase (pfdhps) P. falciparum genes, respectively, (ii) designed synthetic DNA templates and primers, (iii) prepared DNA templates from in vitro cultures of P. falciparum
clones containing the SNPs of interest, and (iv) optimised assays in liquid and solid phase, using synthetic DNA templates and appropriate DNA from parasite clones.
The methodology will be validated using DNA templates from field-collected P. falciparum isolates from Equatorial Guinea, previously characterized for the SNPs of interest. For the pyrimethamine-resistant associated SNPs (N51I, C59R, and S108N), we performed cross-reactivity assays using the RPA in the liquid phase. We found a common concentration of synthetic DNA, temperature, and reaction time for all three SNPs for specific reactions.
The results suggest that our approach is highly sensitive in detecting even low levels of parasitaemia, common in endemic areas. At the end of the project, we expect to have a proof-of-concept for all SNPs to adapt the isothermal amplification detection of sulfadoxinepyrimethamine-resistant P. falciparum to a portable device.
Keywords: malaria, diagnostic, drug resistance, isothermal-amplification, biosensors
Acknowledgments: Fundação para a Ciência e a Tecnologia for funds to GHTM - UID/04413/2020 (RESMALDETECT project) and LA-REAL – LA/P/0117/2020
of the main reasons for the spread of drug-resistant parasites in endemic settings. We aim to develop an ASSURED biosensor based on isothermal Recombinase Polymerase Amplification (RPA) for the detection of SNPs associated with resistance of Plasmodium falciparum parasites to antifolates (sulfadoxine and pyrimethamine), which are widely used in chemopreventive
regimens for children and pregnant women in endemic areas. The method is centred on the optical detection of the isothermal extension of immobilised primers on activated maleimide plates. Target DNA hybridises to 5'-end thiolated primers carrying the desired SNP at the 3'-end nucleotide. Primer extension with biotin-labelled nucleotides occurs only if the target DNA has the
SNP of interest.
We have (i) selected the SNPs associated with pyrimethamine (N51I+C59R+S108N) and sulfadoxine (A437G+K540E) resistance located in the dihydrofolate reductase (pfdhfr) and dihydropteroate synthetase (pfdhps) P. falciparum genes, respectively, (ii) designed synthetic DNA templates and primers, (iii) prepared DNA templates from in vitro cultures of P. falciparum
clones containing the SNPs of interest, and (iv) optimised assays in liquid and solid phase, using synthetic DNA templates and appropriate DNA from parasite clones.
The methodology will be validated using DNA templates from field-collected P. falciparum isolates from Equatorial Guinea, previously characterized for the SNPs of interest. For the pyrimethamine-resistant associated SNPs (N51I, C59R, and S108N), we performed cross-reactivity assays using the RPA in the liquid phase. We found a common concentration of synthetic DNA, temperature, and reaction time for all three SNPs for specific reactions.
The results suggest that our approach is highly sensitive in detecting even low levels of parasitaemia, common in endemic areas. At the end of the project, we expect to have a proof-of-concept for all SNPs to adapt the isothermal amplification detection of sulfadoxinepyrimethamine-resistant P. falciparum to a portable device.
Keywords: malaria, diagnostic, drug resistance, isothermal-amplification, biosensors
Acknowledgments: Fundação para a Ciência e a Tecnologia for funds to GHTM - UID/04413/2020 (RESMALDETECT project) and LA-REAL – LA/P/0117/2020
| Original language | English |
|---|---|
| Pages | P58 |
| Number of pages | 1 |
| Publication status | Published - 19 Sept 2024 |
| Event | Wellcome Connecting Science - Genomic Epidemiology of Malaria - Wellcome Genome Campus, Hinxton, United Kingdom Duration: 18 Sept 2024 → 20 Sept 2024 https://coursesandconferences.wellcomeconnectingscience.org/event/genomic-epidemiology-of-malaria-20240918/ |
Conference
| Conference | Wellcome Connecting Science - Genomic Epidemiology of Malaria |
|---|---|
| Country/Territory | United Kingdom |
| City | Hinxton |
| Period | 18/09/24 → 20/09/24 |
| Internet address |
Keywords
- malaria
- diagnostic
- drug resistance
- isothermal-amplification
- biosensors
