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Abstract
BackgroundThe ability to culture Plasmodium falciparum continuously in vitro has enabled stable access to asexual and sexual parasites for malaria research. The portfolio of isolates has remained limited and research is still largely based on NF54 and its derived clone 3D7. Since 1978, isolates were collected and cryopreserved at Radboudumc from patients presenting at the hospital. Here, procedures are described for culture adaptation of asexual parasites, cloning and production of sexual stage parasites responsible for transmission (gametocytes) and production of oocysts in Anopheles mosquitoes. This study aimed to identify new culture-adapted transmissible P. falciparum isolates, originating from distinct geographical locations.MethodsOut of a collection of 121 P. falciparum isolates stored in liquid nitrogen, 21 from different geographical origin were selected for initial testing. Isolates were evaluated for their ability to be asexually cultured in vitro, their gametocyte production capacity, and consistent generation of oocysts.ResultsOut of 21 isolates tested, twelve were excluded from further analysis due to lack of mature gametocyte production (n = 1) or generation of satisfactory numbers of oocysts in mosquitoes (n = 11). Nine isolates fulfilled selection criteria and were cloned by limiting dilution and retested. After cloning, one isolate was excluded for not showing transmission. The remaining eight isolates transmitted to Anopheles stephensi or Anopheles coluzzii mosquitoes and were categorized into two groups with a reproducible mean oocyst infection intensity above (n = 5) or below five (n = 3).ConclusionsThese new P. falciparum culture-adapted isolates with reproducible transmission to Anopheles mosquitoes are a valuable addition to the malaria research tool box. They can aid in the development of malaria interventions and will be particularly useful for those studying malaria transmission.
Highlights
The ability to culture Plasmodium falciparum continuously in vitro has enabled stable access to asexual and sexual parasites for malaria research
This was a case of airport malaria in a patient residing near Schiphol international airport in the Netherlands in 1979 [5]. This isolate, thought to have originated from West-Africa [6], and its clone 3D7 [7] are arguably the most widely used laboratory strains of P. falciparum and have become a standard resource for malaria research. They have formed the basis of whole sporozoite vaccination approaches [8], and the basis of many functional assays for evaluation of malaria interventions, such as the standard membrane feeding assay (SMFA) [9]
Collection and selection of patient isolates Since 1978, P. falciparum isolates derived from various geographical locations were collected from patients prior to anti-malarial drug treatment at the Radboudumc and other hospitals in the Netherlands
Summary
The ability to culture Plasmodium falciparum continuously in vitro has enabled stable access to asexual and sexual parasites for malaria research. In search of an isolate for stable continuous in vitro culture and transmission experiments, the Nijmegen falciparum strain 54 (NF54) was selected This was a case of airport malaria in a patient residing near Schiphol international airport in the Netherlands in 1979 [5]. This isolate, thought to have originated from West-Africa [6], and its clone 3D7 [7] are arguably the most widely used laboratory strains of P. falciparum and have become a standard resource for malaria research. This study describes selection, clonal adaptation, and evaluation of P. falciparum isolates for in vitro culture and sporogonic stage development
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