Susceptibility of Plasmodium falciparum isolates to antimalarial drugs in a highly seasonal malaria endemic village in Mali

Background: In 2006, the National Malaria Control Program (NMCP) in Mali recommended artemisinin-based combination therapy (ACT) as the first-line treatment for uncomplicated malaria. Since the introduction of ACT, few reports are available on the level of resistance of Plasmodium falciparum (P. falciparum) to antimalarial drugs in Mali. Dihydroartermisinin is the active metabolite of artemisinin derivatives. Here, we conducted an ex-vivo drug sensitivity testing in a rural area of southern Mali, namely the Kéniéroba village from 2016 to 2017. Methods: Seventy-five (75) isolates of P. falciparum were successfully evaluated for ex-vivo sensitivity to key anti-malarial drugs, namely chloroquine (CQ), quinine (QN), amodiaquine (AQ), mefloquine (MQ), lumefantrine (LUM), dihydroartermisinin (DHA) , and piperaquine (PPQ). P. falciparum sensitivity to these drugs was assessed using the World Wide Antimalarial Resistance Network (WWARN) SYBR-GREEN method of inhibitory concentration of 50% (IC50) determination. Reduced sensitivity to antimalarial drugs was defined as IC50 less than the WWARN standard IC50. Results: The proportion of resistant P. falciparum isolates was 20.2% for CQ, 40.5% for QN, 6.8% for AQ, and 1.3% for MQ. All tested P. falciparum isolates were sensitive to LUM, DHA, and PPQ. A statistically significant correlation was found between QN and AQ IC50 values (r = 0.80; r2 = 0.64, P<0.0001). Conclusions: P. falciparum isolates were sensitive to all ACT derivates tested in Kenieroba in Mali. In contrast, P. falciparum isolates were resistant to, CQ, QN, and AQ as evidenced by high IC50 to these drugs.


4
Early treatment of a malaria episode with an efficient antimalarial drug is required to prevent life-threatening disease outcome. Thus, the resistance of P. falciparum to common antimalarial drugs is a serious hurdle for malaria control in endemic countries [1]. The development of P. falciparum resistance to low-cost and welltolerated antimalarial drugs such as CQ, AQ, antifolates, and MQ [2][3][4][5][6] has led the World Health Organisation (WHO) and the National Malaria Control Programs (NMCP) to recommend the artemisinin-based combination therapy (ACT) for malaria treatment [7]. The rationale for using ACT drugs is 2-folded. First, it relies on the high efficacy of artemisinin that early suppresses disease progression which could lead to life threatening manifestation. Second, there is a need to delay as long as possible the emergence and spread of artemisinin resistance worldwide. Nowadays, one of the major concerns is that we are witnessing the first signs of the emergence of parasites resistant to artemisinin derivatives in Southeast Asia [8,9]. However, no molecular marker of such resistance has yet been identified [10]. Some of the mutations in the gene kelch (K13) have been associated with resistance to artemisinin in vitro in Asia [11][12][13]. Up-to-date, resistance to artemisinin has not been reported in Africa and ACTs have remained very effective [11,14]. However, sub-Saharan Africa remains under threat because of the widespread use of ACTs which could lead to selective pressure on ACT, and also the increasing intercontinental human migrations. In addition, the circulation of sub-standard or counterfeit drugs coupled to the non-adherence of patients to treatment may contribute to rapid selection of resistant malaria parasites. In 2006, the NMCP revised the treatment policy of uncomplicated malaria. Chloroquine has been replaced by two ACTs: artemether-lumefantrine (AL) and amodiaquine-artesunate (AQ-AS). For severe malaria, artesunate, artemether and QN should be used using 5 intra-veinous route. The necessity for regular monitoring of these antimalarial resistance phenomenon is therefore essential. Ex-vivo drug susceptibility testing is one of the most efficient indirect approaches to assess the efficacy of antimalarial drugs. Few data on in-vitro and ex-vivo ACT efficacy are available from isolates circulating in Mali. Here, we report the results of an ex-vivo susceptibility of P. falciparum isolates to seven (7) antimalarial drugs used in Kéniéroba, Mali.

Ex-vivo drug sensitivity testing of antimalarial drugs
For ex-vivo drug sensitivity testing, spectrometry-based determination of parasite growth using SYBR-GREEN (SG) method was used as described previously [15]. Culture plates were incubated at 37°C and 5% CO 2 for 72 hours. At the end of the incubation period (which corresponds to the schizonts' stage), the plate was taken out from the incubator and frozen at -20°C to read by adding SG. After, the plate was thawed for 2 hours to lyse the cells, Prepare SG lysis buffer (per plate -10 mL lysis buffer, 2 µl SG (0.002% SG) and use immediately. Then, 100 µl SG lysis buffer was added to each well. The plate was later covered with aluminum foil, shake using a plate shaker, and incubated at room temperature in the dark for 30 minutes. The amount of SG incorporated into the nucleic acids of the parasite was determined by the Fluorometer plate Reader with excitation filter of 485 nm and emission filter 538 7 nm. The IC 50 , defined as a drug concentration at which the SG signal was 50% of that measured from drug-free control wells, was calculated from In-Vitro Analysis and Reporting Tool (IVART) software to fit the concentration-inhibition data. The threshold values for the reduced in ex-vivo susceptibility were as followed: 61 nM, 77 nM, 12 nM, 115 nM, 30 nM, 135 nM and 611 nM for AQ, CQ, dihydroartemisin, LUM, MQ, piperaquine and QN respectively [16,17].

Statistical analysis
Data were expressed as the geometric mean IC 50 and 95% confidence intervals (95 CIs) were calculated after logarithmic transformation. Cross-susceptibility was analyzed using the Pearson correlation using Graphpad Prism version8. Statistical analysis of IC 50 s was performed with Graphpad Prism. Two-tailed p-values were computed and any value less than 0.05 were considered significant.

Results
In total, 75 isolates of P. falciparum were successfully evaluated for ex-vivo sensitivity to CQ, QN, AQ, MQ, LUM, DHA, and PPQ. Figure 1 [19]. This difference with our results could be explained by the heterogeneity of the distribution of P. falciparum strains and the fluctuation of the phenomenon over time. Also, the proportion of QN-resistant P. falciparum isolates defined as having an IC 50 <611 nM reached up to 40.5% in our study. This proportion was much higher than 19.4% (6/31) [19] in Ghana in 2011 and 9.7% (3/31) [20] in Senegal in 2017. Inde found 8% reducted sensitivity to AQ [21]. Our study comfirmed the reduced sensitivity of P. falciparum to AQ in Kenieroba, which may quickly impact the clinical 10 efficacy of the artemisinin combinations.
One isolate had reduced sensitivity to MQ (1.3%). The geometric mean of IC 50 to MQ was 13.13 nM. The geometric mean of IC 50 to CQ was 46.07 nM. Our result was similar to 51nM by Phong, NC. et al. in 2019 in Vietnam [22] and much lower than A cross-resistance between CQ-AQ and CQ-QN and between QN-AQ has already been reported in previous studies [27][28][29]. A negative correlation between the IC 50 values of DHA-QN, LUM-QN reflects higher activity of DHA and LUM against the isolates of P. falciparum QN-resistant. A negative correlation was also observed between DHA and AQ implying that they have different mechanisms of action and that isolates resistant to AQ may be sensitive to DHA. This is reassuring considering the emergence of resistance to artemisinin derivatives [30][31][32][33].
In conclusion, we found that P. falciparum isolates from Kéniéroba had a reduced sensitivity to QN, AQ, and MQ. All clinical field isolates tested showed a very good sensitivity to DHA, LUM, and PPQ. Resistance to QN and AQ showed a positive correlation between IC 50 values, indicative of cross-resistance between these two important anti-malarial drugs.

Ethics approval and consent to participate
The study was approved by the ethics committee of the faculty of medicine and Pharmacy of the University of Sciences, Technics and Technologies of Bamako (USTTB), Mali. All study particiapnts signed a written consent or assent (for children) forms in order to participate to this study.

Consent for publications
All authors read and approved the final manuscript.    Figure 1 Median values of IC50 of different anti-malarial drugs