title: The Genotypic and Phenotypic Stability of Plasmodium falciparum Field Isolates in Continuous In Vitro Culture.
abstract: The Plasmodium falciparum in vitro culture system is critical for genotypic and phenotypic analyses of the parasites. For genotypic analysis, the genomic DNA can be obtained directly from the patient blood sample or from culture adapted parasites whereas for phenotypic analysis, immediate ex vivo or in vitro culture adapted parasites are used. However, parasite biology studies have not investigated whether culture adaptation process affects genotypic and/or phenotypic characteristics of the parasites in short- or long-term cultures. Here, we set out to study the dynamics and stability of parasite genetic and phenotypic profiles as field isolate parasites were adapted in continuous cultures. Parasites collected from three different patients presenting with uncomplicated malaria were adapted and maintained in drug-free continuous cultures. Aliquots from the continuous cultures were collected every 24-48 hours for analyses. Each aliquot was treated as a separate parasite sample. For genetic analysis, microsatellite (MS) typing and single nucleotide polymorphism (SNP) analyses of 23 drug resistance markers were done. The 50% inhibitory concentrations (IC50) for some of the samples were also established for four antimalarial drugs. Samples from each patient (parasite-line) were compared as they were passed through the continuous culture. Data revealed genotypic and phenotypic profiles for the three parasite-lines fluctuated from one generation to the next with no specific pattern or periodicity. With few exceptions, multilocus analysis revealed samples from each parasite-line had high genetic diversity with unique haplotypes. Interestingly, changes in MS and SNP profiles occurred simultaneously. The difference in the IC50s of samples in each parasite-line reached statistical significance. However, phenotypic changes did not correspond or correlate to genotypic changes. Our study revealed parasite genetic and phenotypic characteristics fluctuates in short- and long-term cultures, which indicates parasite genetic information obtained even in short cultures is likely to be different from the natural infection parasites.
authors.affiliation: Department of Emerging Infectious Diseases-Global Emerging Infections Surveillance and Response System (DEID-GEIS) Program, United States Army Medical Research Directorate-Kenya (USAMRD-K), Kenya Medical Research Institute (KEMRI)-Walter Reed Project, Kisumu, Kenya.
authors.forename: Redemptah
authors.initials: R
authors.lastname: Yeda
authors.affiliation: Department of Emerging Infectious Diseases-Global Emerging Infections Surveillance and Response System (DEID-GEIS) Program, United States Army Medical Research Directorate-Kenya (USAMRD-K), Kenya Medical Research Institute (KEMRI)-Walter Reed Project, Kisumu, Kenya.
authors.forename: Luicer A
authors.initials: LA
authors.lastname: Ingasia
authors.affiliation: Department of Emerging Infectious Diseases-Global Emerging Infections Surveillance and Response System (DEID-GEIS) Program, United States Army Medical Research Directorate-Kenya (USAMRD-K), Kenya Medical Research Institute (KEMRI)-Walter Reed Project, Kisumu, Kenya.
authors.forename: Agnes C
authors.initials: AC
authors.lastname: Cheruiyot
authors.affiliation: Department of Emerging Infectious Diseases-Global Emerging Infections Surveillance and Response System (DEID-GEIS) Program, United States Army Medical Research Directorate-Kenya (USAMRD-K), Kenya Medical Research Institute (KEMRI)-Walter Reed Project, Kisumu, Kenya.
authors.forename: Charles
authors.initials: C
authors.lastname: Okudo
authors.affiliation: Department of Emerging Infectious Diseases-Global Emerging Infections Surveillance and Response System (DEID-GEIS) Program, United States Army Medical Research Directorate-Kenya (USAMRD-K), Kenya Medical Research Institute (KEMRI)-Walter Reed Project, Kisumu, Kenya.|Institute of Tropical Medicine and Infectious Diseases, College of Health Sciences, Jomo Kenyatta University of Agriculture and Technology, Nairobi, Kenya.
authors.forename: Lorna J
authors.initials: LJ
authors.lastname: Chebon
authors.affiliation: Department of Emerging Infectious Diseases-Global Emerging Infections Surveillance and Response System (DEID-GEIS) Program, United States Army Medical Research Directorate-Kenya (USAMRD-K), Kenya Medical Research Institute (KEMRI)-Walter Reed Project, Kisumu, Kenya.
authors.forename: Jelagat
authors.initials: J
authors.lastname: Cheruiyot
authors.affiliation: Department of Emerging Infectious Diseases-Global Emerging Infections Surveillance and Response System (DEID-GEIS) Program, United States Army Medical Research Directorate-Kenya (USAMRD-K), Kenya Medical Research Institute (KEMRI)-Walter Reed Project, Kisumu, Kenya.
authors.forename: Hoseah M
authors.initials: HM
authors.lastname: Akala
authors.affiliation: Department of Emerging Infectious Diseases-Global Emerging Infections Surveillance and Response System (DEID-GEIS) Program, United States Army Medical Research Directorate-Kenya (USAMRD-K), Kenya Medical Research Institute (KEMRI)-Walter Reed Project, Kisumu, Kenya.
authors.forename: Edwin
authors.initials: E
authors.lastname: Kamau
mesh_terms: D000483:Alleles; D000962:Antimalarials / Q000494:pharmacology; D016054:DNA, Protozoan / Q000235:genetics*; D004351:Drug Resistance; D004912:Erythrocytes / Q000187:drug effects / Q000469:parasitology; D005787:Gene Frequency; D018503:Genome, Protozoan; D042822:Genomic Instability; D005838:Genotype; D006801:Humans; D020128:Inhibitory Concentration 50; D007630:Kenya; D018895:Microsatellite Repeats; D058885:Multilocus Sequence Typing; D010641:Phenotype; D010802:Phylogeny; D010963:Plasmodium falciparum / Q000145:classification / Q000187:drug effects / Q000235:genetics*; D020641:Polymorphism, Single Nucleotide; D061251:Primary Cell Culture
chemical_list: D000962:Antimalarials; D016054:DNA, Protozoan