A GENTES DE C ONTROL E STRUCTURAL

Once the staining procedure had been validated, using flask cultures of P. falciparum, it was necessary to optimise growth and drug treatment in a 96 well plate format to achieve medium-throughput efficiency for the SG-MicroPlate assay. In order to overcome issues with poor parasite growth in the 96 well plates, gas exchange was improved through the use of gas permeable films and replacement of old gassing chambers. Different haematocrits were also selected to determine the optimal plate growth conditions. The Giemsa results indicated that parasite growth was sufficient at both the 5 % and 2.5 %

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haematocrits and clear dose response to both antimalarials used in the study were observed. At the lower haematocrit, however, plate growth was either equal to or greater than flask growth at the standard 5 % haematocrit. Furthermore, there appeared to be no benefit bestowed by a 5 % haematocrit condition in the plate, even though the starting number of parasites would have been double. Taken together the results suggested that 2.5 % haematocrit may be optimal in the plate system. This is consistent with the literature, lower haematocrits are commonly used for plate growth experiments to permit a better surface area to blood volume ratio. This allows more efficient gas exchange and nutrient acquisition between parasites and the above culture media (Karl et al., 2009; Vossen et al., 2010). A further issue was that there was a discrepancy between SG-MicroPlate and Giemsa results as a comparable dose response pattern was not observed with the SG- MicroPlate readings. Despite previous in-house optimisation of the SYBR Green MicroPlate assay, other experiments have shown inconsistencies between the SG- MicroPlate and both the Giemsa and Flow cytometer assays (data not shown). The SG- Microplate data only showed absolute fluorescence differences between the variable haematocrits (Plate 2.5 % and Plate 5 %) and varying amounts of complete media (Plate 2.5 % and Flask 2.5 %). The earlier experimental method (Figure 3.1) that showed a parasite density dilution effect, represented by the CM/CM condition, also displayed a high level of background fluorescence for both infected and non-infected blood in the latter study, that overcome any SG staining that represented bound parasite DNA. One possibility was that the albumin from bovine serum (Sigma) used in the latter experiments contributed to higher background fluorescence than the AlbuMAX (lipid rich albumin from bovine serum, Gibco) used in the initial optimisation work. The trend observed in the CM/RPMI condition most likely represented a serial dilution of complete media with RPMI. However, the clear difference in the level of absolute fluorescence between the infected and non-infected blood indicated notable detection of parasite DNA. The RPMI/RPMI condition showed that when all of the complete media had been removed, the dilution of non-infected blood had no effect on fluorescence intensity. Therefore, the modest density dilution effect observed in infected blood series (RPMI/RMPI P) accurately represented parasite DNA. It was also possible that in the original SG- MicroPlate protocol the DNA from dead or dying parasites, outside the erythrocytes, may be recorded as live. Washing would also help reduce this confounding factor, improving the accuracy of the SG-MicroPlate assay and concordance with Giemsa staining and SG- FCM. Taken together, the data highlighted the importance of the inclusion of washing

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steps in the SG-MicroPlate protocol in order to achieve true parasite density readings. Indeed, variable volumes of complete media could drastically alter the results this is particularly important for growth inhibition studies where parasitaemia may be low.

3.4.4 SG-FCM for detailed in vitro investigations of parasite killing

Unlike any other currently available objective method, the unique advantage of the SG- FCM technique is that it offers the opportunity to gain more information about intra- erythrocytic parasite growth and development and toaccurately monitor stage-specific effects in response to drug treatment (Izumiyama et al., 2009; Grimberg, 2011.) Indeed, the corroboration of the SG-FCM data and Giemsa images during time-course analyses provided further confirmation that the method can be used to accurately monitor mononuclear and multinuclear parasite transitions. The results of the current time-course and stage-specific investigations were in agreement, showing delayed progression to schizont formation under DHA drug pressure. The stage-specific data clearly demonstrated that this disruption in schizont formation is dose dependent. This is in line with previous findings that suggest artemisinins arrest parasite development at the ring stage (Klonis et al., 2011). However, although differences in stage-specific detection can be reported here, stage-specific inhibition cannot be inferred, as the drug was present throughout the erythrocytic cycle and transition through parasite stages. To obtain true stage specific-data using the SG-FCM method any future work should direct drug exposure against a single parasite stage. This could be achieved by altering the treatment regime to a short 6 hr window of drug exposure, followed by washing, removal of the drug and subsequent re- incubation to permit completion of the 48 hr erythrocytic cycle. Wein et al (2010) suggest that a 72 hour treatment period is needed to obtain reliable data using the SG-based methods. Conversely, the data from both the time-course analysis and IC50 determination of four existing antimalarials: namely, atovaquone, dihydroartemisin, chloroquine and proguanil, suggests that so long as the 48 hr treatment period spans the transition into the subsequent erythrocytic cycle, accurate drug inhibition and IC50 data can be generated. Data presented here, irrespective of whether parasites were grown under flask or plate conditions (all analysed using SG-FCM) shows that 48 hr exposure initiated at trophozoite stage yields the expected dose response for the four existing antimalarials selected. The IC50 values obtained were all within the range of the values reported elsewhere in the literature. The advantages of this treatment protocol are that it allows all stages of the parasite to be exposed to the drug, permits entry into a subsequent erythrocytic cycle and

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ensures that early ring stages are not overlooked as all parasites should have entered the trophozoite stage at the time of the analysis. The data presented, therefore, indicates that parasite maturation rather than treatment period is important for analyzing the efficacy of antimalarials with different modes of action. Indeed focusing on multiplication only may overlook drugs with novel modes of action, for example targeting other organelles such as those involved in invasion. It is evident that a number of findings presented here highlight the importance of timing in the basic drug susceptibility assays. Firstly, the comparison study of the Giemsa microscopic test, the SG-MicroPlate and the SG-FCM assays revealed a slight shift in IC50 values between the 48 h and 72 h time points. Secondly, during the extended time-course analysis there was an increase in parasitaemia from the 48 hr to the 72 h reading. This suggests that some parasites may have become unsynchronized and had not entered the subsequent erthrocytic cycle at 48 hours, but had successfully invaded new erythrocytes by the 72 h time point. Thirdly, without alterations in the treatment and analysis protocol, the dose-dependent effects of DHA on schizont formation would have been missed.