Improved Protocol for Continuous Culture of Plasmodium falciparum Reference Strains

Abstract

Parasitic biobank of Plasmodium falciparum is almost germinal in Côte d’Ivoire. However, several high-level research topics on this parasite involve the taking into account of nature isolates but also chemo-sensitive or resistant reference strains for a better validation of results. In addition, acquisition of these reference strains is still arduous for laboratories in developing countries due to complexity of administrative procedures. For those reasons, this study aimed in to combine several procedures into a consolidated one in order to enhance the multiplication of P. falciparum reference strains. Continuous culture of plasmodial strains was based on the Trager and Jensen procedures. The CELL culture protocols used are those of the Swiss TPH described by Sergio Wittlin; the “Growing Plasmodium falciparum cultures at high parasitemia” and the “Stockholm sorbitol method” of Methods in Malaria Research-6th edition 2013; and the INV-01 and INV-02 procedures of the Worldwide Antimalarial Resistance Network (WWARN). Reference Plasmodium falciparum strains NF54 sensitive to chloroquine (CQs) and K1 resistant to chloroquine (CQr) were received from the Swiss Tropical Institute and Public Health (Swiss TPH). The CQs NF54 strain reacted more quickly to the protocol unlike the CQr K1 strain. Parasitic densities (DP) obtained with NF54 strain were ranged from 0.4% at day zero (D0) to 11.4% at day eight (D8). Strain K1 finally adapted successfully after one month of follow-up. Related DPs ranged from less than 0.1% to more than 20% in just three growth cycles after adaptation. A joint protocol (from this work) called “CRLP-SwissTPH-Pasteur_001” is available and allows to efficiently multiply reference strains NF54 and K1. It is planned to spread out the tests to other plasmodial strains as well as to wild isolates in order to standardize this procedure.