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.

Expanding the SAR of Nontoxic Antiplasmodial Indolyl‐3‐ethanone Ethers and Thioethers

“…It must be noted that these assays were conducted independently of one another in separate laboratories, where subtle inter‐lab variability may account for variations in the biological data. Furthermore, recent studies have demonstrated that phenotypic and genotypic characteristics of P. falciparum isolates are unstable in long‐term culture, which alter the susceptibility of different P. falciparum strains to different drugs . To illustrate, cultures of the 3D7 strain of P. falciparum have been found to express significantly higher levels of the adhesion protein encoding var genes than NF54, which is particularly active in the ring stage .…”

“…Furthermore, recent studies have demonstrated that phenotypic and genotypicc haracteristics of P. falciparum isolates are unstable in long-term culture, which alter the susceptibility of different P. falciparum strains to different drugs. [35,36] To illustrate, cultures of the 3D7 strain of P. falciparum have been found to express significantly higher levels of the adhesion protein encoding var genes than NF54, [37] which is particularly active in the ring stage. [38] While this specific alteration may not adequately answer our specific alterations in biological activity,i tdoes highlight the challenges in antimalarial drugd iscovery,a nd the need for robustm ultifaceted datasets to adequatelya ssess promising compounds.…”

Selective Expression of Variant Surface Antigens EnablesPlasmodium falciparumto Evade Immune Clearancein vivo

“…Since long-term continuous in vitro culture of P. falciparum leads to genetic heterogeneity (Yeda et al, 2016), we serially diluted the non-adapted 3D7 strain of P. falciparum to obtain a single clone, 3D7-B7, and repeated the adaptation experiment in RBC-NSG mice. Again there was a significant delay of 26 ± 5.1 days before 3D7-B7 was detected in the blood of inoculated mice ( Figure 1C).…”

Strains used in whole organism Plasmodium falciparum vaccine trials differ in genome structure, sequence, and immunogenic potential

“…Recently, longread sequencing technologies have been used to overcome some of these assembly challenges, as was shown with assemblies for 3D7, 7G8, and several other cultureadapted P. falciparum strains generated using Pacific Biosciences (PacBio) technology [31][32][33]. However, NF166.C8 and NF135.C10 still lack whole-genome assemblies; in addition, while an assembly for 7G8 is available [32], it is important to characterize the specific 7G8 clone used in heterologous CHMI, from Sanaria's working bank, as strains can undergo genetic changes over time in culture [34]. Here, reference assemblies for NF54, 7G8, NF166.C8, and NF135.C10 (hereafter referred to as PfSPZ strains) were generated using approaches to take advantage of the resolution power of long-read sequencing data and the low error rate of short-read sequencing platforms.…”