The current first-line antimalarials, artemisinin-based combination therapies (ACTs), are threatened by increasing drug failure of both the artemisinin and its partner drugs especially in Southeast Asia. Recently, an increase in the plasmepsin 2-3 (PfPM2) gene copy number was shown to be associated with decreased susceptibility of Plasmodium falciparum to piperaquine, a partner drug used in ACT. We have demonstrated before photo induced electron transfer real-time PCR (PET-PCR) is a field friendly method for endemic countries than conventional real-time PCR for malaria diagnosis. Here, we designed a PET-PCR based assay to quantitate the copy number of PfPM2 in this multiplex assay, the P. falciparum β –tubulin gene was used as a reference gene. The forward primers for PfPM2 and β –tubulin were modified with the PET-tag at the 5' end and labeled with FAM (PfPM2) and HEX (β –tubulin) fluorophores. Amplification efficiencies of the PfPM2 and the β-tubulin genes were 97% and 100%, respectively. The PET-PCR was optimized using samples with known numbers of PfPM2 gene. Using this assay five specimens from anonymized Cambodian and Ethiopian samples obtained from 2 patients who failed treatment with dihydroaretmisinin-piperaquine were analyzed. The 3 sequential samples (day 1, day 6 and day 23 post treatment) from the Cambodian patient were all found to have 3 copies of PfPM2 while the Ethiopian samples (day of treatment and day 13 post treatment) had a single copy gene. These results support the potential use of the PET-PCR method for the detection of piperaquine drug resistance.
Detection of piperaquine resistant marker plasmepsin-2 gene amplification using PET PCR real time assay / Samaly Santos Souza, ; L'Episcopia, Mariangela; Michela, Menengon; Russo, Gianluca; Annalisa, Saracino; Sergio Lo Caputo, ; Didier, Menard; Carlo, Severini; Venkatachalam, Udhayakumar; Naomi, Lucchi. - ELETTRONICO. - (2017). (Intervento presentato al convegno ASTMH (American Society Tropical Medicine & Hygiene) 66th annual meeting tenutosi a Baltimore, USA nel 5-9 November 2017).
Detection of piperaquine resistant marker plasmepsin-2 gene amplification using PET PCR real time assay
L'Episcopia, Mariangela;Gianluca Russo;
2017
Abstract
The current first-line antimalarials, artemisinin-based combination therapies (ACTs), are threatened by increasing drug failure of both the artemisinin and its partner drugs especially in Southeast Asia. Recently, an increase in the plasmepsin 2-3 (PfPM2) gene copy number was shown to be associated with decreased susceptibility of Plasmodium falciparum to piperaquine, a partner drug used in ACT. We have demonstrated before photo induced electron transfer real-time PCR (PET-PCR) is a field friendly method for endemic countries than conventional real-time PCR for malaria diagnosis. Here, we designed a PET-PCR based assay to quantitate the copy number of PfPM2 in this multiplex assay, the P. falciparum β –tubulin gene was used as a reference gene. The forward primers for PfPM2 and β –tubulin were modified with the PET-tag at the 5' end and labeled with FAM (PfPM2) and HEX (β –tubulin) fluorophores. Amplification efficiencies of the PfPM2 and the β-tubulin genes were 97% and 100%, respectively. The PET-PCR was optimized using samples with known numbers of PfPM2 gene. Using this assay five specimens from anonymized Cambodian and Ethiopian samples obtained from 2 patients who failed treatment with dihydroaretmisinin-piperaquine were analyzed. The 3 sequential samples (day 1, day 6 and day 23 post treatment) from the Cambodian patient were all found to have 3 copies of PfPM2 while the Ethiopian samples (day of treatment and day 13 post treatment) had a single copy gene. These results support the potential use of the PET-PCR method for the detection of piperaquine drug resistance.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
