This study was conducted in ten different sites across DRC for monitoring molecular resistance of P. falciparum to CQ and to AQ. The study has shown that sixteen years after official CQ withdrawal from national treatment policy, CQ-resistance prevalence has decreased in DRC to 28.5% and is marked by a high variability from site to site with a significant difference (p<0.001). No molecular marker of AQ resistance has been found.
A decline in the prevalence of resistance has also been reported in several other African countries. An in vivo assay conducted in 2005  revealed the return of CQ efficacy to 99% in Malawi, the first country to change its national first-line malaria treatment policy from CQ to SP in 1993. More studies have shown the return of the efficacy of CQ several years after its withdrawal from the treatment policy. In Tanzania, CQ resistance decreased from 80% in 2001 to 5.7% in 2011 . In Kenya, it decreased from 76% to 6% between 2003 and 2015 . In Republic of Congo (Brazzaville), the decrease went from 100% in 2005, one year before the introduction and implementation of ACT in 2006, to 71% in 2015 . In Zambia, no CQ resistance marker was detected in clinical trial conducted from 2010 to 2013 .
The relationship between drug pressure and CQ sensitivity has been clearly reported by Feng et al and Frosch et al [15, 27]. When the pressure stops, the drug tends to recover its effectiveness against the parasite. Mutations are common in parasites. However, the fitness of the mutant, which is its ability to survive compared to the wild-type parasite, may be altered as shown by a deficit of reproductive potential in a number of mutants that are otherwise viable. Consequently, the majority of mutant parasites will gradually disappear from the natural population, allowing the emergence of wild-type population . This explanation could explain partly the observations of the very low prevalence of K76T mutations detected in Fungurume (1.5%), Lubumbashi (1.8%), Kalima (4.5%) and Kisangani (4.7%) in the present study. However, this prevalence was higher in other sites such as Bolenge (32%), Vanga (41.3%), Kinshasa (48.8%) and not the least at Katana (89.5%). Previously studies reported much higher K76T rates from Kinshasa in 2008 (83.8%)  and in 2010 (73.2%) , from Bolenge in 2014 (70.6%) .
The simultaneous presence of very low and high prevalence of CQ resistance would probably be related to different levels of CQ pressure depending from one site to another before and after the withdrawal of this molecule. Concerning the use of CQ in DRC, data from the DHS II in 2013—2014 revealed that in provinces where our sites are located (before territorial apportionment from 11 to 26 provinces) CQ was still in use despite its withdrawal from national policy of malaria management in 2001 . Unfortunately, these DHS data have not been yet updated in 26 current provinces and the low remaining use of CQ cannot alone explain the disparity of CQ resistance observed in different locations. Thus, further studies at the community level should be conducted to enrich the data.
The Katana site which had the highest rate of CQ resistance (89.5%) is located in the Eastern province of Sud-Kivu where several armed militias have been warring during the last two decades. This instability in the country security is an obstacle to good management of the use of antimalarial drugs as recommended by the national. Indeed, lack of control of the antimalarial supply chain could result in the use of non-recommended molecules such as CQ by the population. On the contrary, other sites such as Lubumbashi in the South-East of the country where the security situation has been calmer, have seen a significant decrease in prevalence of K76T mutation in the parasites. Another particularity of Katana is its epidemiological facies. The Katana site corresponds to the mountainous facies with high probability of mutations conservation; resistance appears and emerges from this kind of places as previously shown in South-East Asia, considered as the bastion of antimalarial resistance . In Africa, antimalarial drug resistance rises from the East and spreads to the rest of the continent . As a reminder, the first case of resistance to CQ was reported in Tanzania . The province of Kivu in the Eastern Congo (where Katana is located) was the first region to report CQ resistance in the DRC . In addition, a therapeutic efficacy study conducted in 2001 in DRC, found that the city of Bukavu in Kivu contained the highest percentage (80%) of patients who had a treatment failure to CQ (2). This finding has shown a high conservation of K76T mutation in a part of DRC sixteen years after discontinuance of CQ use as first-line therapy in of DRC.
The present study could be the base of prospects for more in depth research, which should accurately provide the explanation of persistent high CQ resistance rates in some sites several years after withdrawing the CQ from the country.
Concerning pfcrt haplotypes, we note that there are many possible combinations of polymorphisms in positions 72–76 that include the key mutation K76T in CQ-resistant P. falciparum with CVIET as the most common haplotype in Africa. The single mutation in codon 76 is rarely observed in nature suggesting that compensatory mutations in other codon positions than 76 could be required to restore the fitness of the CQ-resistant parasites bearing the K76T mutation .
The SVMNT haplotype associated with AQ resistance was not detected in the present study which is good news for ACT use. This haplotype has not been reported yet in DRC [19—21] whereas it was found in neighboring countries such as Tanzania and Angola [11—12]. AQ is one of the components of some ACTs, and regular monitoring of resistance to this molecule is required.
Out of 806 positive P. falciparum samples sequenced, 42 (5.2%) samples have not given interpretable sequences. The yield of sequencing depended on several factors including the concentration of template used in the reaction. The discordance between screening tests (RDT’s and thick blood test) results and those of PCR P. falciparum detection will be addressed in further publication.