Nowadays artemisinin is the most significant choice of anti-malarial drugs for treatment of falciparum malaria infection in the most malarious areas of the world. Reports about resistance to the drug is one of the greatest fears for international attempt to control and eliminate malaria parasites. Producing experimental P. falciparum resistant strain against antimalarial drugs in the laboratory provides an invaluable opportunity to investigate the mechanism of parasite resistance to drugs, moreover these strains facilitate investigations about those substances that bears potential antimalarial activities. Previous reports suggested that mutations in the Pf kelch13 gene can be resulted in P.falciparum resistance to artesunate, similarly mutation in this gene was reported as the responsible agent for slow parasite clearance in many cases. It is implied that this gene is the best candidate for the study of nucleotide mutations in the resistance of P. falciparum to artesunate [12–14]. On the other hand Mukherjee et al. in 2017 conducted a study at Harvard University, they reviewed the resistance of the parasite to artemisinin in Africa. Despite administration of the standard dosage, patients showed drug resistance of the parasite, but authors clarified that there was no mutation in the kelch13 gene in spite of resistance to artemisinin. They also claimed that it can be searched the cause of resistance in another gene locus except Pf kelch13[12].
It is recommended that there are other possible genes related to resistance in Plasmodium species. Scientists reported that mutations in pfmdr1, pftctp and PfATPase6 genes can be emerging with presence of higher concentrations of drug and resulted in resistance phenomenon of parasites [15–17]. Identifying the exact mechanism of resistance needs more investigation about those genes that involve in resistance phenomenon. It should be considered that parasites may show conflict with drugs via different ways. Reducing the sensitivity in one strain can be accompanied by over expression of a specific genes involved in the process of inducing resistance and producing a series of proteins and novel substances. As mentioned, resistance can be induced through the cultivation of P. falciparum parasite and its long exposure to artemisinin or its derivatives.
In the present study, a new strain of P. falciparum was created under the intermittent exposing the parasite to different concentrations of artesunate. The created strain cultured under the drug pressure from the least concentration to the maximum chosen dose for one month; then preserved in liquid nitrogen for further investigation. The line of Pfkelch13 Ar− 2 was the resistant line for the highest concentration of the drug which is exposed to it (10− 2 mol/l). Sequencing of the Pfkelch13 gene derived from produced strain indicated that there is no mutation in comparison with the original ones. This idea was confirmed when compared with the sequence of an isolated sample from an artemisinin treatment failed patient. It should be noted while some previous studies reported that nucleotide changes in this gene are responsible for phenotype resistance, others indicated that other interacting molecules may responsible for this mechanism, therefore, it can be suggested that sensitivity reduction to the drug likely occurred in the line used in this study, but in the Pfkelch13 piece has not happened any mutation.
During the past few years, scientists have tried to produce a resistant parasite line. Some of them used different procedures to produce it, Cui and colleagues in 2012 sought to establish a resistante line to dehydroartemisinin (DHA) in the 3D7 strain. They succeeded to produce of two parasite clones from Dd2 using in vitro method which showed 25-fold reduction in susceptibility to DHA. Moreover they declared that resistance of P.falciparum to dehydroartemisinin firstly occurred in ring stage, then propagate to schizont and trophozoite forms. Their results revealed that redundant proliferation of pfmdr1 gene locus of this new clone enhances production of antioxidant and expression of a chaperone in parasite. The outcome has important concept for development of ART resistance in Southeast Asia [18].
In general, resistance to artemisinin in P. falciparum can be promoted by different mechanisms that can rely on the foundation of the drug. Studies by Witkowski and colleagues in 2013 and 2014 led to the discovery of a mechanism for resistance to artemisinin using an experimental model. Results of the study yield a creation of a cell line called F32-ART, which was produced in the laboratory after 5 years exposure to increasing concentrations of artemisinin. The data from the F32-ART cell line, highly resistant to artemisinin, showed that the pressure of artemisinin induces a stop in extension of very young parasites population in the ring stage, which led to the displacement of this small population toward the silent phase, however, artemisinin kills all the parasites in other phases of the life cycle [6, 19]. On the other hand, further studies by Straimer et al. in 2015 revealed that mutations in the kelch13 gene was firstly associated with resistance to artemisinin. The authors demonstrated a crucial and fundamental function for K13-propeller mutations related to ART resistance in vitro and explained about molecular feature for slow parasite clearance rates in patients [20].
In a study by Rocamora et al. in 2018, two resistant clones (6A-R and 11C-R) against artemisinin were produced from P.falciparum 3D7 by sequential contacts of synchronized parasite cultures to artemisinin to detect and describe molecular factors that are responsible for resistance of P.falciparum to artemisinin. Both the above mentioned parasite lines showed decreases drug susceptibility and derived resistance phenotype(s) were predominant in the ring stage; although both of them carried the wild-type allele of the K13 gene [21]. Chavchich and his colleague investigated potential mechanisms related to increase of resistance, they declared that P. falciparum lines have the ability to improve resistance to artemisinin derivatives in vitro due to multiple mechanisms such as amplification and increased expression of Pfmdr1. The results of the study was difference with other studies which explain that mutations are responsible for resistant phenomen[6, 7, 22].
The method of the present study from this point of view that the parasite is intermittently exposed to drugs, was similar to the Rocamora’s study, the parasite was able to be exposed to a concentration of 10− 2mol/l, while sequencing was not showed any mutation in Pf kelch13. Rocamora sequenced the whole parasite genome and found 5 mutations in others than kelch13. The mutation changes that were found by him were related to genes that have an expressive role, such as AP2-like transcription factors, PHD finger protein, and RNA helicase. Of course, both clones produced had wild kelch13 genes confirmed by PCR and genotyping. As it mentioned above, molecular studies and alignment of the Pfkelch13 Ar− 2 produced in the present study showed a similarity of 100% with Kelch13 gene sequence in the susceptible 3D7 strain at the nucleotide level and no mutation was observed at this level [21].
Ten years after using combination therapy based on artemisinin against P. falciparum studies in Grande Comore Island released some polymorphisms of resistance markers of artemisinin in kelch13. The results of the study showed that only 3 samples collected from 2006 to 2007were SNP carriers in the kelch13 gene, one synonymous replacement sample (G538G) and two other non-synonymous replacements (D284E, S477Y) that these replacements led to creating two mutant haplotypes, also, in the obtained samples in the years 2013–2014, three mutations were found with synonymous replacement (G538G, Y500Y, R471R) and 7 mutations with a non-synonymous replacement, with a total of 9 mutated haplotypes, but none of them presented artemisinin-resistance in Southeast Asia. The study showed that gene changes in the kelch 13 region can occurred, but with any association to artemisinin-resistance [23].
Another report describes polymorphisms of the kelch13 gene in African parasite populations Authors declared that numerous K13-propeller coding polymorphisms were found to circulate in Africa, but it wasn’t observed any mutations associated with artemisinin resistance such as those reported in Southeast Asia. They resulted that these specific mutation are not prevalent in sub-Saharan Africa. Moreover all mutations occurring in K13 are not related to resistance to artemisinin, and secondary locus can be associated with a mutation that plays a role in resistance to artemisinin. Also researches in Southeast Asia stated that a group of genetic factors might be created artemisinin-resistant parasites [24–26]. In a study conducted in 2016, the polymorphisms of P. falciparum resistance to artemisinin were evaluated in K13 and PfATPase6 genes in Bioco Island, Equatorial Guinea. The results showed that 2.04% of cases possess A578S non-synonymous mutation in the K13 but resistance to artemisinin did not spread in the region, While this SNP, K13-propeller A578S, was reported as the essential mutation involved in artemisinin resistance and accompanied with a high percentage of the artemisinin treatment failure in Southeast Asia [15].The rapid detection of molecular networks from resistance to artemisinin or and identification of new therapies or development of new drugs can be an effective way to prevent emerging drug resistance in the parasite and entering the silence phase.
Finally, it is important to note that producing experimental resistance strain of P. falciparum parasite against antimalarial drugs in the laboratory, provides an invaluable opportunity for investigation the exact mechanism of parasite resistance to antimalarial drugs. Moreover, it offers an opportunity to investigate about new potential drugs with antimalarial activates. Furthermore, producing experimental resistant strains of malaria parasites facilitates considering different mechanisms of establishing resistance in the parasites, and even how such resistance can be broken down by novel antimalarial.