The present study has not detected molecular markers associated with resistance to first-lines ACT in use in the DRC in patients returning to health facilities for malaria retreatment.
Although none of the mutations associated with ART resistance in Southeast Asia has been found by the study, 7 coding substitutions that are of unknown phenotype were observed, among which 3 previously reported notably N498I in Kenya , N554K in Comoros and A557S in Togo and DRC  and 4 others not yet reported (F506L, E507V, D516E, G538S). Numerous pfk13-propeller mutations of unknown function are commonly reported in the Sub-Saharan African countries like in the DRC [17, 21, 22]. There are criteria for prioritizing further laboratory studies notably the frequent observation of a new allele with a non-synonymous mutation, the evidence of dissemination and preliminary association with clinical data whenever possible . Several independent single nucleotide polymorphisms (SNPs) could be responsible of the ART-R in Sub-Saharan Africa, the known mutations that confer drug resistance would differ from one location to another, depending on the parasite genetics. There is the possibility that pfk13 mutations do not cause ART-R in isolation but would act in combination with other genetic or non-genetic factors that are different in African and Southeast Asian parasite populations [23, 24]. Since African pfk13-propeller mutations were shown to be different from those found in Southeast Asia, further molecular and biochemical studies should investigate whether other factors such as additional mutations could be associated to alter the functions of PFK13 protein, resulting in altered ART sensibility. However, some of the validated and candidate mutations associated with ART resistance in Southeast Asia have been detected in some neighbouring countries such as in Rwanda [8, 9] and in Uganda , providing evidence of de novo emergence of ART resistance in Sub-Saharan Africa. Thus, surveillance must be strengthened to avoid the worst.
The global prevalence of the K76T mutation known to be associated with CQ resistance was 41.5%, but it remained variable from one site to another, ranging from 10.0% in Fungurume to 76.9% in Katana. In 2017, a study conducted in 10 sites including 6 sites of the present study reported a global prevalence of K76T mutation of 28.5% but with a high between regions variability ranging from 1.5% in Fungurume to 89.5% in Katana . In the present study, the prevalence of K76T in patients treated with ASAQ (48.9%) was higher than in those treated with AL (37.4%) with low statistical difference (p-value = 0.038). The possibility that AQ would continue to contribute to the selection for the K76T mutant even after discontinuance of CQ usage has been previously raised . The simultaneous presence of very low and high prevalence of CQ resistance could be related to between-regions difference of CQ pressure and also to effect of selection for CQ resistance depending on the genetic structure of parasite populations which have been shown to vary significantly across the country . Data concerning current CQ use in the country are not available, further studies at the community level and parasite genetic studies should be conducted to explain the persistence of high CQ resistance rate in some provinces despite the withdrawal of this molecule from the national policy of malaria treatment. The SVMNT haplotype associated with AQ resistance was not detected in the present study, which is encouraging for the DRC national policy for the continued use of AQ in ACT. This haplotype has not yet been reported in the DRC [16, 18, 27, 28] whereas it was found in neighbouring countries such as Tanzania and Angola [29, 30].
None of the P. falciparum isolates had multiple copies number of pfmdr1 gene in this study, consistent with the general absence [31, 32] or low frequencies [28, 33, 34] of pfmdr1 copy number variation in P. falciparum from sub-Saharan Africa. Multiple copy number of pfmdr1 gene has been postulated to confer resistance to LU , which is the associated drug in AL, one of the ACTs used in the DRC.
The ACT used to treat the initial episode was that available on each study site during investigation and best tolerated by the patient. In practice, AL tends to be primarily used in urban areas because patients have more options to obtain it from private pharmacies, while ASAQ is used in rural areas . The study assessed molecular markers of anti-malarial resistance in patients who returned to health structures for fever within 28 days of the ACT treatment, which is considered as treatment failure . Treatment failure is the inability to clear parasites from a patient’s blood or to prevent their recrudescence after the administration of an anti-malarial drug. When possible, treatment failure must be confirmed by microscopy, as histidin rich protein-2 (HRP-2)-based RDT may remain positive for weeks after successful treatment due to persistent antigenemia, even without recrudescence [35, 36]. In this study, P. falciparum real-time PCR assay was used afterwards to confirm P. falciparum malaria in patients returning to health structures for fever within 28 days of an initial malaria treatment.
The study contributes to the ongoing surveillance of the resistance to anti-malarial drugs in use in the DRC as recommended to track the emergence and spread of P. falciparum resistance to different molecules used in malaria management. However, although the study was carried out in six provinces of the DRC with varied geography and malaria endemicity, the results were not representative of either any one province or the entire country.