P. falciparum clinical isolates were collected from 25 symptomatic children, aged 2 to 14 years old, visiting the LEKMA Hospital, in Accra between February 2017 and January 2018. LEKMA is a sub-urban area of Accra, the capital city of Ghana, a low malaria transmission setting, with an estimated entomological inoculation rate of <50 infective mosquito bites per person/year. The study was approved by the Institutional Review Board of the Noguchi Memorial Institute for Medical Research, University of Ghana (IRB00001276) and the Ghana Health Service Ethical Review Committee (GHC-ERC: 005/12/2017). All guidelines and principles contained in the approved protocol were duly followed in the execution of the project. A written informed consent was obtained from a parent and/or legal guardian and assent obtained for older children. Venous blood samples were collected in ACD vacutainers (BD Biosciences) and transported to the laboratory for processing within two hours after collection. At the laboratory, the infected erythrocytes were separated from the leucocytes through centrifugation at 2000 rpm and washed twice with RPMI1640 medium (Sigma). About 200µL of packed erythrocytes were put straight in culture, while the remaining sample was resuspended in glycerolyte in ~500 µL vials following the standard protocol (EVIMalaR, 2013) and stored in liquid nitrogen. Frozen vials were thawed at different time intervals using two distinct sodium chloride-based protocols. Vials were thawed using either a two-step protocol (12% NaCl and 1.6% NaCl) or a three-step protocol (12% NaCl, 1.8% NaCl and 0.9% NaCl supplemented with 0.2% Glucose) as per standard procedure (EVIMalaR, 2013). To minimize the effect of possible confounders, all reagents used in this study were prepared from single batches and stored as single-use aliquots.

P. falciparum clinical isolates were cultured as per standard protocols (EVIMalaR, 2013). In brief, isolates were maintained at 37°C in RPMI1640 medium (Sigma), supplemented with 5% Albumax (Gibco), 2 mg/ml sodium bicarbonate, 50 µg/ml gentamycin (Sigma) and 2% AB⁺ heat-inactivated normal human serum (PAN Biotech, UK). All cultures were adjusted to 4% hematocrit using O⁺ erythrocytes from a single donor and incubated in an atmosphere of 2% O_2, 5% CO₂ and balanced with Nitrogen. For isolates cultured upon arrival to the lab, the parasites multiplication rate (PMR), defined as the ratio of the parasitemia before and after re-invasion, was monitored for the first two in vitro cycles and fresh erythrocytes were only added after 96 hours in culture, while fresh erythrocytes were immediately added upon thawing of cryopreserved isolates. Following the addition of fresh erythrocytes, growth tests were performed to assess the PMR every 48 hours for the first three in vitro replicative cycles for the cryopreserved isolates and for up to 12 cycles for freshly culture adapted isolates. After each cycle, cultures were diluted to 0.5% parasitemia for the next cycle. Sample aliquots, taken following each replicative cycle were stained with 1 µM of Hoechst 33342 dye (Sigma Aldrich, UK) and the resulting parasitemia was assessed using flow cytometry. The growth test was considered successful only when the resulting PMR, measured after 48 hours, was greater than one (>1) ( Figure 1 ). The median PMR of successful growth tests after twelve successive replicative cycles following the addition of fresh erythrocytes were considered as PMR of culture-adapted isolates.

P. falciparum genomic DNA (gDNA) was extracted from filter paper spots using the QIAamp DNA Blood Mini Kit (Qiagen, Hilden, Germany) and eluted in 30 µL elution buffer following the manufacturer’s instructions. The concentration and purity of the eluted gDNA were estimated using a NanoDrop One (Thermo Fisher Scientific, Madison, WI, USA). For each isolate, the presence of single or multiple parasite clones was assessed using a nested PCR approach as described earlier. The assays were performed using primers targeting the highly polymorphic regions of msp1 (block 2) and msp2 (block 3). All isolates that grew successfully during culture adaptation were genotyped after 7, 15, 21 and 28 days in culture and the number of clones was compared to that of the fresh isogenic isolate. The expected heterozygosity (H_E) defined as the probability of being infected by at least two distinct alleles at a given locus was calculated as follows: H_E = [n/(n-1)] [(1-Σpi)], with n being the number of isolates and pi the allele frequency at a given locus (Diouf et al., 2019).

Invasion phenotyping assays were performed using enzyme-treated erythrocytes (targets) from a single donor. Target erythrocytes were treated with either 250 mU/mL of neuraminidase, 1 mg/mL of trypsin or 1 mg/mL of chymotrypsin for 1 hour at 37° C with gentle shaking and washed thrice with RPMI1640 medium. The efficiency of enzyme treatment was assessed as previously described (Thiam et al., 2021). Treated erythrocytes were then labelled with 20 µM of carboxyfluorescein diacetate, succinimidyl ester (CFDA-SE) (Thermo Fisher Scientific) for two hours at 37° C with gentle shaking and protected from light exposure. For each isolate, schizont-infected erythrocytes were adjusted to 2% parasitemia and co-incubated with an equal volume of target erythrocytes in 96 well plates. All assays were performed in triplicates in a total volume of 100 µL at 2% haematocrit and incubated at 37° C for 24 hours. Parasitemia were adjusted to 1% for all isolates and invasion assays were considered successful only when invasion efficiency into control erythrocytes (untreated) was at least two-fold greater than the starting parasitemia. Plates were removed from the incubator and spun at 2,000 rpm for 3 minutes, after which the supernatant was discarded, replaced with a solution of 1 mM Hoechst 33342 to label the parasites’ DNA and incubated for an hour at 37° C. Plates were subsequently washed with 1X PBS and flow cytometry analyses were performed on a BD LSR Fortessa X-20 cytometer (BD Biosciences, Belgium). Invasion into target erythrocytes was determined by analysis of the proportion of Hoechst positive erythrocytes in 50,000 counted CFDA-SE positive cells. Percent invasion into enzyme-treated erythrocytes was expressed as a percentage of the invasion efficiency into labelled untreated erythrocytes.

Samples used in this study were collected from Ghanaian children with uncomplicated malaria visiting the LEKMA hospital in Accra. To monitor the in vitro growth patterns of freshly collected P. falciparum clinical isolates, parasites were initially allowed to grow in the patient-derived erythrocytes for a minimum period of 96 hours after which freshly washed O⁺ erythrocytes (from a single donor) (Thiam et al., 2021) were added and growth tests were performed for isolates that successfully grew. Of the 25 isolates used in this study, 19 (76%) yielded detectable parasitemia 48 hours following in vitro adaptation. As measured by flow cytometry, P. falciparum clinical isolates showed different growth patterns during the first 96 hours of in vitro adaptation ( Figure 2A ). Monitoring of the PMR during these first two in vitro cycles revealed that most of the isolates had less than two-fold increase in parasitemia from one cycle to another. Isolates with successful growth patterns were further diluted with fresh erythrocytes and the parasitemia was used to assess the parasite multiplication rate through successive growth tests for a maximum period of twelve successive replication cycles. Overall, all isolates showed a minimum of 8 successful growth tests (66.67% success rate) with a median PMR of 1.77 (range 1.13 – 2.43) ( Figure 2B ).

Unlike laboratory strains, P. falciparum clinical isolates frequently harbour multiple parasite clones, which could modulate the parasite’s in vitro adaptability. Here, we assessed the presence of multiple parasite clones in each of the tested isolates using the highly polymorphic regions of msp1 and msp2 genes. All 25 isolates were shown to be polyclonal and the number of alleles detected were 74 and 44 for msp1 and msp2, respectively ( Table 1 ). For msp1, the allele frequencies were 36.47% (27/74), 32.43% (24/74) and 31.10% (23/74) for K1, MAD20 and RO33, respectively, while those for the msp2 allelic families were 54.54% (24/44) and 45.46% (20/44) for 3D7 and FC27, respectively. The prevalence of multiple infections was respectively 16 and 14 for msp1 and msp2, while the multiplicity of infections was 2.6 and 1.76, respectively for the two genes ( Table 1 ). Besides, the H_E was 0.35 and 0.52 for msp1 and msp2, respectively.

To measure the effect of short-term culture adaptation on the parasites’ genotypes, ten of the culture-adapted isolates were genotyped at days 7, 15, 21 and 28 post-adaptation using the msp1 and 2 allelic families. Overall, there was little variation in the proportion of allelic families across the different time points ( Figure 3 ). However, our analysis showed a reduction in the number of clones per isolate at day 28 as compared to day 0 ( Table 2 ). The maximum number of clones per isolate was reduced from 4 to 1 for the msp1 gene, while that of msp2 was reduced from 2 to 1 ( Table 2 ). Of all msp1 allelic families, K1 was the most predominant at both day 0 and day 28, with a percentage of 41.94% and 58.33%, respectively ( Table 2 ). MAD20 and RO33, which were present at the same proportion at day 0 (29.03%, each), represented respectively 16.67% and 25.00% of the total number of alleles ( Table 2 ). For msp2, there were little changes in the proportions of the respective allelic families, with 3D7 being the most predominant allele representing 63.16% and 61.55% at day 0 and 28, respectively ( Table 2 ). Out of the eight isolates that harboured the MAD20 allelic family at day 0, only two were detected with a copy of the allele at day 28, while the RO33 allele which was initially present in nine isolates at day 0, was detected in only three isolates at day 28. K1, initially detected in all ten isolates, was still present in seven of them at day 28. Of all three msp1 allelic families, only K1 and RO33 were simultaneously detected in the same isolates at day 28, while MAD20 was only detected as single infections ( Table 2 ).

For the msp2 gene, out of the seven isolates harbouring the FC27 allelic family at day 0, only four persisted at day 28, while the 3D7 allelic family was initially present in all ten isolates at day 0 but was detected in eight of them at day 28. As for msp1, both allelic families of msp2 were present in three isolates as co-infections, while two and four isolates presented single infections of FC27 and 3D7, respectively at day 28. However, there was no specific dominant combination of msp1 and msp2 detected in our isolates at day 28. Moreover, aside those detected at day 0, there were no newly detected alleles in our isolates at day 28, therefore suggesting the absence of detectable cross-contamination during culture adaptation.

Culture-adaptation of P. falciparum clinical isolates has been reported as more labor-intensive than immediate ex vivo processing. Moreover, there are earlier reports of P. falciparum clonal selection during in vitro adaptation (Chen et al., 2014). To ascertain the effect of cryopreservation on P. falciparum early in vitro adaptation, we compared the PMR of short-term cryopreserved clinical isolates to that of their freshly cultured isogenic counterparts. Two vials of the same isolate were simultaneously revived using two distinct NaCl-based thawing protocols. Parasitemia were adjusted to 0.5-1% using erythrocytes from a single donor and the PMR was monitored during the first three asexual replicative cycles. Successful monitoring of the PMR of isolates prior to and following cryopreservation (one-year interval) revealed differences in PMR between cryopreserved isolates and the matched freshly culture-adapted counterparts. The median PMR was 1.62 for fresh isolates while that of cryopreserved isolates was 1.12 and 1.27 following two-step and three-step thawing, respectively ( Figure 4A ). However, the difference in PMR was only significant when comparing fresh isolates and cryopreserved parasites thawed using a two-step protocol (P = 0.03; Figure 4A ), while no significant difference was observed between isogenic isolates thawed using distinct protocols ( Figure 4A, B ). Flow cytometric analysis of the DNA content of revived cryopreserved parasites revealed different fluorescence peaks following Hoechst 33342 staining suggesting the presence of different parasite stages after 48-, 96- and 144-hours post-incubation. However, no significant difference was observed in the proportions of the different parasite stages when comparing the parasites’ growth after thawing using different protocols ( Figure 4C ).

P. falciparum invasion phenotyping has mostly been conducted following short-term culture-adaptation (Okoyeh et al., 1999; Lobo et al., 2004; Nery et al., 2006; Lopez-Perez et al., 2012). Invasion phenotypes are classified as either resistant (r) or susceptible (s) to the different enzyme treatment of the erythrocytes with invasion efficiency set at 50%. To this, we assessed the effect of short-term culture adaptation on P. falciparum invasion phenotype in the different enzyme treated erythrocytes. To allow for easy inference, we compared the ex vivo invasion phenotype of four isolates when the samples were collected to that obtained after 28 days in culture to see if there are changes. Of the four isolates tested, only one (ACC015) had a significant change in the sensitivity to enzyme treatment following culture adaptation ( Figure 5 ), while significant changes were observed in ACC003 and ACC014 following treatment with neuraminidase and trypsin, respectively.

Moreover, changes in invasion profile, defined as the combination of sensitivity to the three enzymes, was observed in two isolates (ACC014: NrTsCs → NsTsCs and ACC015: NrTsCr → NrTsCs), where N: neuraminidase, T: trypsin, C: chymotrypsin, s: sensitive and r: resistant; while → depicts the changes in profile). Interestingly, although all three msp1 alleles were initially present in these two isolates, only K1 persisted at Day 28-post adaptation, while both the 3D7 and FC27 alleles of msp2 persisted throughout the 28-day period post adaptation.

Cryopreserved isolates were thawed at different time intervals (from 3 to 12 months after cryopreservation) and assayed for their ability to invade enzyme-treated erythrocytes as compared to their fresh uncultured counterparts. Given the challenges associated with the parasites’ in vitro adaptability during the first rounds of asexual replication, and to avoid clonal selection following long-term culture adaptation, all assays were set during the first two replicative cycles. Of the 25 isolates collected in this study, seven had enough numbers of cryopreserved vials (six vials) to be thawed at all the time-points and successfully phenotyped over the course of one-year post cryopreservation. All isolates showed a sialic acid independent phenotype with the invasion of neuraminidase treated erythrocytes greater than 50% relative to that of untreated control erythrocytes ( Figure 6 ). Overall, there was no significant change in sialic acid dependency in the parasites after cryopreservation relative to fresh uncultured isolates ( Figure 6 ). The most common invasion profile was neuraminidase resistant, trypsin sensitive and chymotrypsin sensitive (NrTsCs). However, three out of the seven isolates showed changes in invasion profiles in trypsin and chymotrypsin treated erythrocytes following cryopreservation with the apparition of three novel phenotypes (NrTrCs, NrTsCr and NrTrCr) after six months post cryopreservation ( Figure 6 ). Nevertheless, none of the novel phenotypes persisted after twelve months post cryopreservation, suggesting a technical or random effect associated with these changes.