The National Malaria Control Programme in 2005 introduced ART-AMD as the first-line drug for the treatment of uncomplicated malaria after years of chloroquine therapy failures in Ghana . Since the introduction of this combination therapy for the treatment of uncomplicated malaria, the country has continued to monitor its efficacy in addition to other artemisinin-based combination therapies (ACTs) using in vitro methods. The strength of this study is the use of the newly developed DAPI DNA stain  to study the susceptibility of clinical isolates to the drugs currently in use in the country. This assay is robust and a very reproducible marker of parasite growth; it has been demonstrated to be very good in monitoring the development of resistance in parasites at field settings . The study therefore aimed at using this method to investigate the efficacy of commonly used antimalarial drugs in Ghana in addition to chloroquine (currently not in use in Ghana) against clinical parasite isolates causing uncomplicated malaria in different transmission zones of Ghana to aid in providing malaria-drug efficacy surveillance data. SYBR GREEN1 method have been used in Ghana in studying the susceptibility of clinical isolates to a panel of antimalarial drugs in Ghana  but to the best of our knowledge, this is the first time the DAPI assay is being used in Ghana and its advantage has been described elsewhere . One major limitation of DAPI assay is the fact that it does not work well at a very low DNA content 
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 , 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  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  to the 9% we saw over the period (2015–2017). Our data supports findings from Ghana and other parts of Africa which have demonstrated the return of Chloroquine-sensitive strains after the withdrawal of chloroquine as the first-line drug for the treatment of malaria [23, 34–37] with a continuous drop in the prevalence of CQ resistance markers since its withdrawal as the first-line drug for the treatment of uncomplicated malaria.
In Ghana, ART-AMD is one of the first-line drugs for the treatment of uncomplicated malaria and also used to treat pregnant women after the first trimester . This justifies the importance of monitoring the possible development of resistance among the parasites circulating in the country especially to the individual drugs that are used for the combination therapies. Setting the threshold for AMD at 60 nM, many of the parasite isolates were found to be resistant at Begoro between 2016 and 2017 with similar trend also occurring at Cape Coast. These observed in vitro resistant levels does not translate to drug failures as we did report on the therapeutic efficacy outcome of over 90% in the same communities . These in vitro observations could therefore be as a result of an increased tolerance of these parasites to AMD taking into account the slow acting nature of AMD .
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 . 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. In comparison, resistance levels among the Begoro isolates were lower 11.11% and 78% for 2016 and 2017 respectively. The sharp emergence of resistant isolates in Cape Coast as compared to Begoro is not surprising because Cape Coast is a Metropolitan with a lot of Tourist activities and therefore attracts a lot of non-immune travelers to the place as compared to Begoro with very little or no tourist activities .
It is interesting to note that the observed significantly higher geometric IC50 values of each of the drugs tested to be higher in Cape Coast as compared to Begoro for each of the years (2016 and 2017) was not surprising because the transmissions intensities differ in these two communities complicated with the differences in both environmental and socioeconomic conditions all of which have an impact on the development of drug resistance. Although data from this study seems to suggest an increased tolerance to most of the drugs tested, it does not suggest high treatment failures to the antimalarials currently in use in Ghana despite the fact that we do not have concrete data to support what has been found elsewhere . Again, in vitro studies alone cannot be used to explain treatment failures as it is very complex and involves other several factors including host genetic factors, poor adherence and exposure to subtherapeutic doses as well as management and administrative issues [44–45]
In conclusion, we have used the in vitro DAPI assay to assess the sensitivities of over 200 parasites isolates collected over 3 consecutive years from 2 sites of different transmission intensities in Ghana to five drugs (single individual drugs). Although we did see higher resistance levels to all the drugs used over the period (based on the cutoff poits from literature), this does not mean high drug treatment failures because these were tested for individual drugs most of which are used in combinations in Ghana except Chloroquine that is currently not in use in Ghana. This study has clearly demonstrated the continual return of chloroquine-sensitive parasites after its withdrawal as the first-line drug for the treatment of uncomplicated malaria over a decade ago, and higher tolerability of parasites to selected drugs used in artemisinin-based combination therapy. It is therefore prudent to use this method to continually monitor these individual drugs in Ghana.