Drug sensitivity of Plasmodium falciparum field isolates to selected antimalarial drugs in Ghana using the in-vitro DAPI assay

Background: Malaria continues to be a major health issue globally with nine out of ten cases reported in Africa. Although the current artemisinin derived combination therapies in Ghana are still efficacious against the Plasmodium falciparum parasite, compounding evidence of artemisinin and amodiaquine resistance in the African region establish the need for a full, up-to-date understanding and monitoring of antimalarial resistance to provide evidence for planning control strategies. Methods: The study was cross-sectional and was conducted during the peak transmission seasons of 2015, 2016, and 2017 in two study sites located in different ecological zones of Ghana involving children aged 0.5-14 years presenting with symptomatic uncomplicated Plasmodium falciparum (Pf) malaria with parasitaemia greater than 1000 parasites/µl of blood. Using in vitro 4-,6-diamidino-2-phenylindole (DAPI) drug sensitivity assays, 328 Pf parasites collected were used to investigate susceptibility to five selected antimalarial drugs: chloroquine, amodiaquine, dihydroartemisinin, artesunate and mefloquine. Results: The geometric mean B (GMIC 50 ) of five drugs against the parasites collected from Cape Coast were 9.6, 23.6, 9.1, 3.5 and 8.1nM for chloroquine, amodiaquine, artemisinin, artesunate, and mefloquine respectively in 2015. There was a 2 fold increase in the GMIC 50 levels of all the drugs against the isolates collected in 2016 as compared to the 2015 data from Cape Coast .The a of the five drugs against the parasites collected from Cape Coast were significantly higher than those isolates collected from Begoro in 2016 and 2017 (P<0.001) . The chloroquine resistance ranged between 1.9% and 9.1% among isolates collected from Cape Coast but remained 0% in Begoro over the period. High amodiaquine resistance levels were recorded at both sites whilst that of artesunate resistance ranged between 4 and 10% over the study period. Conclusions: The study has assessed the antimalarial drug sensitivities of Ghanaian Pf isolates collected over 3 consecutive years. The parasites showed variable resistance levels to all the drugs used over the period. The study has demonstrated the continual return of chloroquine-sensitive parasites. The in vitro DAPI assay is a useful method for monitoring individual drugs used in combinations in Ghana for the generation of data on their sensitivities over time.

Coast Metropolis within the coastal savannah zone of Ghana where transmission is low to moderate and also perennial with an annual rainfall of 750 -1000 mm [27]. The malaria transmission pattern is similar to what pertains in other parts of Ghana with two seasonal peaks -major and minor. The major season occurs in April -July with the minor occurring in September-November [28]. The study involved children aged 6 months -14 years presenting with a history of fever, mono-infection with Plasmodium falciparum parasite density of 1,000 -250,000/µL blood; and absence of severe malaria.
After meeting the inclusion criteria and informed consent obtained from parents, 4ml of blood was aseptically obtained from each study participant of which 400 µl was used for the DAPI drug assays that was performed within one hour after sample collection. In vitro drug sensitivity assay. Parasitized blood samples collected from patients enrolled were washed twice with 10ml incomplete medium (without normal human serum). The erythrocytes were resuspended at 2% hematocrit in complete medium containing RPMI 1640, supplemented with 2% normal human serum, 10% Albumax and 10mg/ml Gentamycin. Blood samples with parasitemia higher than 1% were adjusted to 1% with uninfected O + erythrocytes. For each sample, aliquots of 180µl of parasite culture were added to the pre-dosed drugs with a negative control and incubated at 37°C for 72 hours in an air tight chamber containing a gas mixture of 5.5% CO 2 , 2% O 2 and 92.5% N 2 .
The remaining parasite cultures were maintained under the same conditions in T25 culture flasks for daily monitoring which included the change of media and preparation of smears from the culture for observing Pf growth using a light microscope. After 72 hours, the processed samples in the plates were harvested by preparing smears from a control well in each plate after which the plates were wrapped in aluminum foil and stored in a freezer at -20 o C until ready to be read. DAPI P. falciparum growth assay. This procedure was conducted using a protocol adapted by Ndiaye and colleagues [29] for frozen plate assay. The frozen plates were allowed to thaw at room temperature and spun for 30minutes at 4000rpm. The contents of these plates were discarded and padded on tissue to absorb all well content except the Pf DNA which settled as pellets in the wells.
Aliquots of 100µl of fluorochrome mixture containing 20 mM Tris-HCl (pH 7.5) (MP Biomedicals, LLC), 5 mM EDTA (Sigma-Aldrich), 0.004% Saponin (Sigma Life Science), 0.01% Trition X-100 (Sigma Life Science), and a 1:75,000 final dilution of 5 mg/ml DAPI (Sigma Life Science) were added to each well containing Pf DNA using a multi-channel pipette. This was carried out in the dark since the stain is light sensitive. The DAPI-Buffer-Pf DNA mixture was re-suspended in each well using a multi-channel pipette after which the plates were wrapped in aluminium foil to protect them from light and left to incubate at room temperature in the dark for 30 minutes. The incubation was followed with spinning for another 30minutes at 4000rpm and well contents were discarded. Aliquots of 200µl of 1×PBS were added per well using a multi-channel pipette with re-suspension. These plates were kept covered with aluminium foil until reading. The fluorescence from the stained parasite DNA in each plate well was measured using Tecan infinite M200PRO (Ex/Em: 358/461nm, bound to DNA).
Statistical Analysis. The drug concentration inhibiting 50% of parasite activity (IC 50 ) for each drug was estimated from the fluorescence data generated using the online tool, ICE estimator 1.2 generated by Le Nagard and colleagues [30]. The IC 50 values with their 95% confidence intervals (CI) were calculated by using an Emax model available at https://www.antimalarial-icestimator.net, as RE = 100 -[(100*Cγ )/(Cγ + ICγ )], where γ is a sigmoidicity factor which expresses the steepness of the curve, RE is the relative effect of the drug (in percent, Y-axis), and C is the drug concentration (Xaxis). The estimated drug IC 50 s were compared over a 3-year period between two study sites. The population means were compared by the Kruskal -Wallis test and the mean difference over time were compared by analysis of variance (ANOVA). The proportions of Pf isolates with IC 50 greater than the resistance threshold from literature [23,[42][43] between the two study sites and between different time points were compared for significant differences using the Chi-square test and Fishers exact test. All statistical analyses and figures were conducted using the software R and graphpad prism respectively. Statistical test were assumed significant at P < 0.05.

Baseline characteristics
A DAPI-based ex vivo assay was used to monitor the drug sensitivity of Pf parasite population circulating in two ecological zones in Ghana with different transmission intensities and over a period of 3 years. The validity of this assay was assessed using the Pf laboratory strain 3D7 against the selected drugs under field conditions. The IC 50 values obtained were comparable to those from other studies. The assay successfully tested for over 71% of the parasite isolates collected in 2015, 73% in 2016 and 54% in 2017. By site, the assay success rate ranged between 73.7and 88.5% for Cape Coast with a similar trend observed in Begoro for all parasites collected in 2016 whereas for those parasites collected in 2017 the success rates ranged between 54 and 88% for both sites ( Table 1).
The results of the in vitro susceptibility testing are shown in Table 2   Begoro, for all drugs except CQ (Table 2).

Discussion
The In this study, CQ was included as one of the test drugs although it is currently not in use in Ghana and interestingly but not surprising, we found a sharp drop of resistance to CQ ranging between 2 and 9% over the three years monitoring at Cape Coast in comparison with a study done in Ghana in 2012 by Quashie and colleagues [23], where they reported 13% of resistance to CQ. We did not record any resistance to CQ from Begoro which has a higher malaria transmission but that was not surprising because it has been shown that in areas of low transmission such as Cape Coast, the development of resistance is slower but at the same time, it takes a longer time for the resistance to disappear as compared to the high transmission areas [32] such as Begoro and therefore seeing remnants of resistance at Cape Coast is not surprising. Again, it is a drastic drop from the national average of resistance of 56% in 2004 [33] to the 9% we saw over the period ( Again, we observed a resistance ranging between 3.7% and 10% among all the parasites collected in 2016 and 2017 in Begoro when the threshold for the other partner drug, artesunate was set at 12 nM.
However, a very interesting observation was made with respect to parasite isolates collected from Cape Coast where we observed a steady increase over the study period In this study we found a very high DHA resistance in all the parasite isolates studied over the period in both study sites ( Table 2). The high DHA and ART in-vitro resistance did not significantly reflect in in-vivo therapeutic efficacy outcomes as earlier described [20]. It has been well documented that artemisinin resistance alone does not necessarily lead to treatment failures [38][39].
Mefloquine is not a recommended drug for the treatment of malaria in Ghana but then it is one of the major prophylactic drugs used by visitors to the country, so it was necessary that it was included in the assessment. In Cape Coast, we did not observe resistance among the isolates tested in 2015 but found 59% and 89% in 2016 and 2017 respectively when the cut off for resistance was set at 45 nM. Abbreviations and staff of the Begoro Government hospital and Ewim Polyclinic for their support. We are also grateful to all the study participants and especially their parents/Guardians at both sites.