Impact of parasite genomic dynamics on the sensitivity of Plasmodium falciparum isolates to piperaquine and other antimalarial drugs

Study design, ethical statement, and subjects

This study was approved by the Kenya Medical Research Institute (KEMRI) Scientific and Ethics Review Unit and the Walter Reed Army Institute of Research (WRAIR) institutional review boards. Archived (2008–2017) and freshly collected (2018–2021) samples were collected under protocol numbers: KEMRI #1330 and #3628, WRAIR #1384, and #2454.

The samples were collected from Kenya Ministry of Health (MOH) sentinel hospital sites located in four of Kenya’s five different malaria ecological zones, namely; the lake endemic region (Kisumu East County and Kisumu West/Kombewa sub-county hospitals), Coastal endemic region (Malindi sub-county hospital), highland epidemic-prone areas (Kericho and Kisii county referral hospitals), and semi-arid/seasonal prone area (Marigat sub-county hospital) (Fig. 1) [2].

Fig. 1figure 1

Map of Kenya depicting the six hospital surveillance sites

Individuals aged ≥ 6 months, presenting at the health care facilities outpatient department with symptoms consistent with uncomplicated malaria and confirmed positive for Plasmodium falciparum by malaria rapid diagnostic test (mRDT) (Parascreen Pan/Pf; Zephyr Biomedicals, Verna, Goa, India) or microscopy were enrolled into the study upon obtaining a written informed consent. Written informed assent for individuals under 18 years was obtained in accordance with the laws of the government of Kenya. Individuals with diminished autonomy such as prisoners, individuals previously enrolled in the study during the same calendar year, infants weighing < 5 kg, and those individuals whom the opinion of the attending medical officer considered that they could be adversely affected by the drawing of 2.5 mL of blood were excluded. About 2–3 mL of venous blood was obtained from those enrolled before treatment. Subsequently, treatment for these individuals was based on the Kenya Ministry of Health guidelines on case management of uncomplicated malaria, as reported in our previous study [24].

Sample collection and preparation

The venous blood sample collected from each individual was distributed in different tubes as follows, 0.5 mL in acid citrate dextrose (ACD) tube (Becton-Dickinson, Franklin Lakes, NJ, USA) for immediate ex vivo growth inhibition assay and culture adaptation, 0.5 mL in sodium heparin tube (Becton-Dickinson, Franklin Lakes, NJ, USA) for in vitro studies, 1 mL in EDTA tube (Becton-Dickinson, Franklin Lakes, NJ, USA) for leukocyte depletion and genomic analysis, two drops on each of the two glass slides for microscopic parasite examination, and three blood spots of each 100 μL on FTA filter paper (Whatman Inc., Bound Brook, NJ) for DNA extraction and genomic analysis [23, 24]. Collected samples were transported to the central laboratory for processing, culture adaptation, genomic, growth inhibition assays, and storage, as described earlier [25].

Reference Plasmodium falciparum strains

The following reagents were obtained through BEI Resources, NIAID-NIH: Plasmodium falciparum, strain 3D7, and MRA-102 were contributed by Daniel J. Carucci; Strain D6, MRA-285, and Strain W2, MRA-157, contributed by Dennis E. Kyle.

Reference antimalarial drugs

The reference antimalarials drugs piperaquine (PPQ), dihydroartemisinin (DHA), lumefantrine (LM), artemether (ART), and chloroquine (CQ) were donated by the WorldWide Antimalarial Resistance Network (WWARN) External Quality Assurance Programme, Bangkok, Thailand [26].

In vitro parasite adaptation

Archived samples comprising previously collected field isolates and laboratory reference strains (3D7/D6 [sensitive to chloroquine and resistant to mefloquine] and W2 [chloroquine resistant and sensitive to mefloquine]) were pulled from the liquid nitrogen, thawed, and maintained in continuous culture in RPMI plus supplements consisting of 10.4 g RPMI 1640 medium [Gibco BRL, UK], 25 mM HEPES, 5.5 mM D-glucose, 10 mM hypoxanthine, 7.5% sodium bicarbonate, 15% (vol/vol) heat-inactivated pooled ABO human sera and 0.1 μl/mL; gentamycin per litre of distilled water, adjusted at 2% hematocrit with uninfected human red blood cells and maintained at 37°C, 5% CO2, 5% O2, 90% N2, and humidified environment as described [17, 25, 27, 28]. The cultures were monitored at 24 hourly intervals till attaining parasitemia of 3–8%. Cultures that could not attain this parasitemia were considered unsuccessful [17, 29].

Drug sensitivity testing by SYBR Green I-based assay

SYBR Green I-based in vitro and immediate ex vivo drug sensitivity assay was used to test each P. falciparum field isolate and control against a panel of five antimalarial drugs, namely, piperaquine (PPQ), dihydroartemisinin (DHA), lumefantrine (LM), artemether (ART), and chloroquine diphosphate (CQ) [25]. Drugs were prepared to the desired concentration as described earlier [25]. Freshly collected samples were subjected to immediate ex vivo assay [29, 30] while culture-adapted field isolates and reference clones that had attained 3–8% parasitemia in continuous culture were subjected to in vitro malaria SYBR Green I assay [25, 31]. Briefly, the sample parasitemia was adjusted to 1% at 2% hematocrit using complete media and uninfected O+ RBCs [25, 30] and mixed to homogeneity. 100 μl of this mixture was loaded to each well of the 96-well microtiter plates (catalog no: 167008 Nunc, Inc, Roskilde, Denmark) containing different concentrations of drug aliquots, incubated at 37°C under a 90% N2, 5% O2, and 5% CO2 humidified environment and terminated 72 h later [25, 31]. Lysis buffer containing SYBR Green I dye was added and then incubated for 24 h in the dark. Readings were then done using Tecan Genios Plus® (Tecan US, Inc., Durham, NC) which gave the relative fluorescence units (RFUs). Using these readouts, the strength of inhibition of each drug, and the 50% inhibition concentration (IC50) were calculated using Graphpad prism® 8.1 for Windows® software (Graphpad Software, San Diego, CA, USA) using non-linear regression analysis of the dose-response curve [25, 31].

Piperaquine survival assay (PSA)

Fresh samples collected from malaria patients presenting themselves at Kisumu West and Kisumu East hospital sites between 2019 and 2021 were analyzed using the immediate ex vivo PSA as described by Duru and coworkers [17]. Samples at equal or greater than 1% parasitemia, arriving at the central laboratory within 6 h after phlebotomy at ambient temperature, were reconstituted to 1% parasitemia, and 2% hematocrit, and then subjected to the assay. Similarly, the samples maintained in continuous culture were synchronized as described initially [30] to attain 0–3 h old rings prior to initiation of PSA [17]. Importantly, a blood smear was made from the reconstituted samples at 1% parasitemia for reference as initial parasitemia. Aliquot of 900 μl of the reconstitutes parasitized red blood cell sample were loaded to a 48-well culture plate (Nunc, Inc., Roskilde, Denmark) containing 100 μl of 2000 nM piperaquine tetraphosphate tetrahydrate giving a final concentration of 200 nM piperaquine (exposed culture) and 89.5% dimethyl sulfoxide (Sigma-Aldrich, ST. Louis, Germany) (non-exposed culture/control) in triplicates [17]. The assay plates were incubated for 48 h at 37°C in a mixture of 5% O2, 5% CO2, and 90% N2 in a humidified environment as earlier described [17]. After 48 h incubation, contents of the assay wells were separately washed three times in RPMI, then resuspended in a drug-free RPMI plus supplements medium for a further 24 h [17]. Thin blood smears were prepared, methanol-fixed, and stained by 10% Giemsa (Sigma-Aldrich, ST. Louis, Germany) for 20 min [17]. The percentage of viable P. falciparum which developed into second-generation rings or trophozoites in the exposed and non-exposed cultures was determined by assessing parasites with normal morphology in 10,000 red blood cells by two independent microscopists blinded to the clinical data [17]. Piperaquine (PPQ) sensitivity was expressed as median survival rate and computed as earlier described [17]. Clinical isolates with a piperaquine survival rate ≥10% were considered PPQ-resistant and vice versa. The assay was considered valid when the growth rate (parasite in the control at 72 h )/initial parasitemia before testing at 72 h) was ≥ 1 for ex vivo.

Analysis of Pfmdr1, Pfpm2, and Pfpm3 copy number variation

Parasite nucleic acids were extracted from whole blood or dry blood spots (DBS) using QIAamp® DNA mini kit (Qiagen, Inc., Germany) following the manufacturer's instructions. Extracted DNA was stored at -20°C. Plasmepsin-2 (Pfpm2), Plasmepsin-3 (Pfpm3), and P. falciparum multi-drug-resistant transporter 1 (Pfmdr1) primers for real-time PCR (Applied Biosystems Inc., Foster City, CA) method were designed using an online GeneScript tool as described earlier [23] and purchased from applied biosystems (Foster City, CA). Copy number variation in the genes was analyzed as described by Ansbro and coworkers with some modifications [23]. Briefly, each 20 μl PCR reaction composed of 10 μl of Taqman multiplex mastermix, 1 μl of forward and reverse primer for each gene and β-tubulin, 1 μl of each probe, 2 μl nuclease-free water, and 2 μl of template DNA. In each real-time PCR run, 3D7 and DD2 strains of P. falciparum were used as controls with one and multiple copies of Pfmdr1, respectively; nuclease-free water was also included as a negative control [23, 32]. A housekeeping gene, β-tubulin, and 3D7 were used as internal control and calibrator respectively for the assay [23, 26, 32]. Test samples were assayed in triplicates for real-time PCR [33]. The cycling conditions were; the initial holding step of 95°C for 5 min followed by 40 cycles of 95°C for 15 s and 60°C for 30 s [34]. PCR cycle threshold (CT) values were generated for all the DNA isolates. Only those with CT value ≤ 32 were considered for analysis by SDS software (version 2.0.6; Applied Biosystems Inc., Foster City, CA). The relative quantification method, 2-ΔΔCt was used to estimate copy number variation for each test gene [25, 30]. Field isolates with copy number ≥1.5 were interpreted as true multiple copies [35].

Genotyping of piperaquine resistance genes on the MassARRAY platform

A matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) coupled with a single-base extension PCR (iPLEX PCR) (Agena Biosciences, San Diego, CA) was used to analyze single nucleotide polymorphisms (SNPs) in Pfdhps, Pfdhfr, Pfcrt and Pfmdr1 genes of P. falciparum following the manufacturer’s protocol. Field isolates from all study sites collected between 2008–2013 and 2018–2021 study periods were analyzed. The primers for this assay were designed by the Agena Bioscience Assay Design Suite (ADS) version 2.0 (Agena Bioscience, San Diego, CA). The primary PCR reaction was run on a GeneAmp 9700 PCR system (Applied Biosystems, Foaster City, California, USA), using the Agena Bioscience PCR Reagent Set. Each reaction mixture comprised of 0.5 μL 10x PCR buffer, 1.8 μL HPLC-grade water, 0.40 μL MgCl2, 0.10 μL dNTPs mix, 1.00 μL primer mix, 0.20 μl Taq polymerase enzyme, and 1 μL of DNA. This PCR was set at the following conditions; initial denaturation at 95°C for 2 min, 44 cycles of 95°C for 30 s, 56°C for 30 S, and 72°C for 60 S, and a final extension at 72°C for 5 min. The unincorporated dNTPs from the primary PCR were dephosphorylated by the shrimp alkaline phosphatase enzyme (SAP). The iPLEX PCR was then performed on a GeneAmp 9700 PCR system with the iPLEX Gold Reaction Kit (Agena Bioscience), following the manufacturer's instructions. For PCR, 2 μL of the prepared extension primer cocktail was added to each well. Cycling conditions for this step were as follows; initial denaturation at 94°C for 30 S, 40 cycles of one step at 94°C for 5 S with five sub-cycles of 52°C for 5 S, and 80°C for 5 S, and final extension at 72°C for 3 min. Extended products from the later step were conditioned using a resin (Agena Bioscience) and HPLC-grade water. Approximately 10 nL of the extended products was dispensed into a 96-well SpecroCHIP (Agena Bioscience) using the MassARRAY Nano-dispenser RS1000 (Agena Bioscience) followed by automatic data acquisition on the mass spectrometer using SpectroAcquire software.

Data analysis

In vitro susceptibility and genotype alongside ex vivo PSA data were expressed as median IC50s with interquartile range (IQR) and proportions, respectively. The isolates were grouped into three periods, namely, 2008–2013, 2014–2017, and 2018–2021. Differences in IC50s between study periods and study sites were quantified using the Kruskal-Wallis (H-test). Post hoc analyses were done using Dunn's multiple comparison tests. Proportions were examined using the Chi-square test (χ2) and Fisher exact test. Associations between genotype and drug susceptibilities (IC50s) were calculated using the Kruskal-Wallis H-test and Mann-Whitney U test respectively. The drug in vitro activity correlations were calculated using the Spearman correlation coefficient. All statistical tests were carried out in GraphPad version 8.0 (GraphPad Software, Inc., San Diego, CA, USA). Two-sided P value < 0.05 was considered statistically significant.

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