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Background: Malaria cases were estimated to 207 million in 2013. One of the problems of malaria control is the emergence and spread of Plasmodium falciparum strains that become resistant to almost all drugs available. Monitoring drug resistance is essential for early detection and subsequent prevention of the spread of drug resistance by timely changes of treatment policy. This review was performed to gather all data available on P. falciparum molecular resistance in DR Congo, as baseline for future assessments. Methods: The search for this review was undertaken using the electronic databases PubMed and Google Scholar using the terms "malaria", "Congo", "resistance", "molecular", "antimalarial", "efficacy". Articles were classified based on year of collecting, year of publication, sample size and characteristics, molecular markers analysed and polymorphisms detected. Results: Thirteen articles were included and five genes have been analysed in these studies: pfcrt, pfdhps, pfdhfr, pfmdr1 and K13-propeller. The majority of studies included were not representative of the whole country. Conclusion: This systematic review demonstrates the lack of molecular resistance studies in DRC. Only 13 studies were identified in almost 15 years. The MOH must implement a national surveillance system for monitoring malaria drug resistance and this surveillance should be conducted frequently and country-representative. ; Peer reviewed

Artemisinin-based combination therapies (ACTs) have been recommended by the World Health Organization (WHO) as first-line treatment of uncomplicated Plasmodium falciparum (P. falciparum) malaria since 2005 in Democratic Republic of Congo (DRC) and a regular surveillance of the ACT efficacy is required to ensure the treatment effectiveness. Mutations in the propeller domain of the pfk13 gene were identified as molecular markers of artemisinin resistance (ART-R). This study investigated the pfk13-propeller gene polymorphism in clinical isolates of P. falciparum collected in the DRC. In 2017, ten geographical sites across DRC were selected for a cross-sectional study that was conducted first in Kinshasa from January to March, then in the nine other sites from September to December. Dried blood samples were collected from patients attending health centers for fever where diagnosis of Malaria was first made by rapid diagnostic test (RDT) available on site (SD Bioline malaria Ag Pf or CareStart Malaria Pf) or by thick blood smear and then confirmed by a P. falciparum real-time PCR assay. A pfk13-propeller segment containing a fragment that codes for amino acids at positions 427-595 was amplified by conventional PCR before sequencing. In total, 1070 patients were enrolled in the study. Real-time PCR performed confirmed the initial diagnosis of P. falciparum infection in 806 samples (75.3%; 95% CI: 72.6%- 77.9%). Of the 717 successfully sequenced P. falciparum isolates, 710 (99.0%; 95% CI: 97.9% - 99.6) were wild-type genotypes and 7 (1.0%; 95% CI: 0.4% - 2.1%) carried non-synonymous (NS) mutations in pfk13-propeller including 2 mutations (A578S and V534A) previously detected and 2 other (M472I and A569T) not yet detected in the DRC. Mutations associated with ART-R in Southeast Asia were not observed in DRC. However, the presence of other mutations in pfk13-propeller gene calls for further investigations to assess their implication in drug resistance. ; Peer reviewed

Plasmodiums are protozoa that may infect various hosts. Only five species are now recognized as naturally parasitizing humans: Plasmodium falciparum, Plasmodium vivax, Plasmodium malariae, Plasmodium ovale and Plasmodium knowlesi. This fifth species, P. knowlesi, previously identified as naturally parasitizing the monkey Macaca fascicularis, has been microscopically confused for a long time with P. malariae or P. falciparum and it was not possible to correctly differentiate them until the advent of molecular biology. To date, natural human infections with P. knowlesi only occur in Southeast Asia and a similar phenomenon of natural transmission of simian plasmodium to humans has not been reported elsewhere. This study was conducted to investigate a possible transmission of African small monkey's plasmodium to humans in populations living near the rainforest of the Democratic Republic of Congo (DRC) where several species of non-human primates are living. Two successive real-time PCRs were identified in the literature and used in combination for purpose. Only P. falciparum was found in this study. However, studies with larger samples and with more advanced techniques should be conducted. ; Peer reviewed

Malaria remains a major public health problem in the Democratic Republic of Congo (DRC) with 14 million cases reported by the WHOMalaria Report in 2014. Asymptomaticmalaria cases are known to be prevalent in endemic areas and are generally untreated,resulting in a significant source of gametocytes that may serve as reservoir of disease transmission. Considering that microscopycertainly underestimates the prevalence of Plasmodium infections within symptomatic carriers and that PCR assays are currentlyrecognized as the most sensitive methods for Plasmodium identification, this study was conducted to weigh the asymptomatic carriage in DRC by a molecular method. Six provinces were randomly selected for blood collection in which 80 to 100 individuals were included in the study. Five hundred and eighty blood sampleswere collected andmolecular diagnosiswas performed. Globally, almost half of the samples collected from asymptomatic individuals (280/580; 48.2%) had Plasmodium infections and the most species identified was P. falciparum alone in combination with P. malariae. The high prevalence reported here should interpellate the bodies involved in malaria control in DR Congo to take into account asymptomatic carriers in actions taken and consider asymptomatic malaria as a major hurdle for malaria elimination. ; Peer reviewed

Background: The loss of chloroquine (CQ) efectiveness has led to its withdrawal from national policies as a frst-line treatment for uncomplicated malaria in several endemic countries, such as the Democratic Republic of Congo (DRC). The K76T mutation on the pfcrt gene has been identifed as a marker of CQ resistance and the SVMNT haplotype in codons 72–76 on the same gene has been associated with resistance to amodiaquine (AQ). In the DRC, the prevalence of K76T has decreased from 100% in 2000 to 63.9% in 2014. The purpose of this study was to determine the prevalence of K76T mutations in circulating strains of Plasmodium falciparum, 16 years after CQ withdrawal in the DRC and to investigate the presence of the SVMNT haplotype. Methods: In 2017, ten geographical sites across the DRC were selected. Dried blood samples were collected from patients attending health centres. Malaria was frst detected by a rapid diagnostic test (RDT) available on site (SD Bioline Malaria Ag Pf or CareStart Malaria Pf) or thick blood smear and then confrmed by a P. falciparum species-specifc real-time PCR assay. A pfcrt gene segment containing a fragment that encodes amino acids at positions 72–76 was amplifed by conventional PCR before sequencing. Results: A total of 1070 patients were enrolled. Of the 806 PCR-confrmed P. falciparum positive samples, 764 were successfully sequenced. The K76T mutation was detected in 218 samples (28.5%; 95% CI 25.4%–31.9%), mainly (96%) with the CVIET haplotype. Prevalence of CQ resistance marker was unequally distributed across the country, ranging from 1.5% in Fungurume to 89.5% in Katana. The SVMNT haplotype, related to AQ resistance, was not detected. Conclusion: Overall, the frequency of the P. falciparum CQ resistance marker has decreased signifcantly and no resistance marker to AQ was detected in the DRC in 2017. However, the between regions variability of CQ resistance remains high in the country. Further studies are needed for continuous monitoring of the CQ resistance level for its prospective re-use in malaria management. The absence of the AQ resistance marker is in line with the use of this drug in the current DRC malaria treatment policy. ; Peer reviewed

Objectives: In the Democratic Republic of Congo (DRC), the neuromeningeal cryptococcosis (NMC) hospital prevalence varies between 8.8 and 11% with a death rate of about 50%. However, the Cryptococcus isolates circulating are poorly and formerly described. We describe the molecular characterization of Cryptococcus spp. isolated from people living with HIV (PLWHIV), and the association between NMC severity factors and the Cryptococcus multilocus sequence typing (MLST) profiles. Materials & Methods: Cerebral spinal fluid (CSF) was collected from PLWHIV with neuromeningeal syndrome hospitalized in three Kinshasa public hospitals from 1 February 2019 to 29 February 2020, and cultured on Sabouraud dextrose agar + chloramphenicol medium afterwards. Serotyping PCR, internal transcribed spacer (ITS) sequencing, and MLST from next-generation sequencing (NGS) data were performed to genotype the Cryptococcus isolates. Raw NGS data were processed and the loci alleles were manually extracted using Geneious software. NMC severity factors such as hypoglycorrhachia (30 cm water), and the patient pejorative outcome were compared to the Cryptococcus sequence type (ST)-MLST profile which was identified, using Pearson chi-square test or Fisher exact test. Results: Out of 29 isolates included in the present study, 23 (79.3%; 95% IC: 65.5–93.1) have been identified as serotype A using serotyping PCR, while 6 (20.7%; 95% IC: 6.9–34.5) had unidentifiable profile. Moreover, all the 29 isolates have been characterized by ITS sequencing as follow: Cryptococcus neoformans var. grubii (23 of 29 isolates, 79.3%), Cryptococcus curvatus (5 of 29, 17.2%), and Cryptococcus laurentii (1 of 29, 3.5%). Apart from this highlighted species diversity, C. neoformans var. grubii isolates were all identified as molecular type VNI using the MLST ISHAM scheme, with seven different STs including ST93 (14 out of 23, 60.9%), ST53 (1 out of 23, 4.3%), ST31 (1 out of 23, 4.3%), ST5 (1 out of 23, 4.3%), ST4 (1 out of 23, 4.3%), ST69 (1 out 23, 4.3%), and one novel ST not yet reported worldwide (2 out of 23, 8.6%). Furthermore, two isolates (8.6%) showed a mixed molecular profile (under investigation). Phylogenetic analysis carried out by the unweighted pair group method with arithmetic mean algorithm (UPGMA) of concatenated gene sequences from the seven MLST loci indicated that the main ST isolated in this study is very close to the only isolate previously reported from the DRC. The others are distant from it, even more so for ST69. All the STs identified in the present study have been isolated in the neighbouring countries of the DRC except for ST53, which has only been reported in Southeast Asia. Among NMC severity factors, only the patient pejorative outcome was associated with the minority STs infections (87.5%, p = 0.02) (ST53, ST31, ST5, ST4, STA, ST69). Conclusions: Molecular analysis of Cryptococcus spp. isolates showed wide species diversity, and ST-MLST heterogenicity within the only one molecular type identified. The existence of the STs isolated in this study in the DRC neighbouring countries suggests probable close-to-home spread. Furthermore, infections due to minority STs were associated with a pejorative outcome than those due to ST93. ; Peer reviewed

Introduction: The emergence of Plasmodium falciparum (Pf) resistance to artemisinin (PfART-R) in Africa is a worrisome situation that would annihilate the progress made in reducing the global burden of malaria. The discovery of mutations occurring in portions of the Pf gene sequence encoding kelch 13 (PfK13) – propeller domain and inducing PfART-R, has provided unprecedented opportunities for monitoring such resistance at large scale. Aim: This study aimed to review the PfK13 allelic polymorphism and its spatial distribution in Africa for drawing a baseline for subsequent epidemiological surveillance and containment efforts of PfART-R. Methods: A systematic review was performed according to PRISMA guidelines through six electronic databases consulted up to December 2018. Studies assessing the PfK13 gene in any of the 54 African countries were explored and data related to individual single nucleotide polymorphisms from each report and sampling location were geo-referenced and locus-referenced to be uploaded on maps displaying spatial and molecular patterns. Results: From 8,678 screened records, 50 reports were identified as eligible providing 22,739 Pf isolates successfully sequenced for the PfK13 and originating from 109 sites surveyed in 41 African countries. Overall 619 nonsynonymous (NS) mutants (2.7% of sequenced isolates) were reported at varied relative frequencies (0.5 to 50%) most often being K189T and A578S.Intermediate proportions (30 to 50%) of NS mutants were found in Western and Eastern Africa, moderate proportions (10 to 20%) in Middle Africa and low to very low proportions (<5%), elsewhere. NS mutations were not detected in 11 of 41 sampling countries. A total of 8 PfK13 NS mutations (F446I, C469Y, R515K, S522C, P553L, V568G, P574L, and A675V) out of 24 known as "associated molecular markers" for PfART-R were noticed at relative frequencies from 0.08 to 10.2%. One NS mutation (M476I) out of 6 established as "validated molecular markers" for PfART-R was reported at a relative frequency of 0.42%. Possible foci of NS mutations were noticed in Eastern, Western, and Middle Africa. Conclusion: Africa has noticed rare but alarming signals of possible emergence of Pf-ART-R. Proactive surveillance strategies are needed to be established in different African regions to refrain from massive development of resistance. ; Peer reviewed

Artemisinin-based combination therapies (ACTs) have been recommended by the World Health Organization (WHO) as first-line treatment of uncomplicated Plasmodium falciparum (P. falciparum) malaria since 2005 in Democratic Republic of Congo (DRC) and a regular surveillance of the ACT efficacy is required to ensure the treatment effectiveness. Mutations in the propeller domain of the pfk13 gene were identified as molecular markers of artemisinin resistance (ART-R). This study investigated the pfk13-propeller gene polymorphism in clinical isolates of P. falciparum collected in the DRC. In 2017, ten geographical sites across DRC were selected for a cross-sectional study that was conducted first in Kinshasa from January to March, then in the nine other sites from September to December. Dried blood samples were collected from patients attending health centers for fever where diagnosis of Malaria was first made by rapid diagnostic test (RDT) available on site (SD Bioline malaria Ag Pf or CareStart Malaria Pf) or by thick blood smear and then confirmed by a P. falciparum real-time PCR assay. A pfk13-propeller segment containing a fragment that codes for amino acids at positions 427–595 was amplified by conventional PCR before sequencing. In total, 1070 patients were enrolled in the study. Real-time PCR performed confirmed the initial diagnosis of P. falciparum infection in 806 samples (75.3%; 95% CI: 72.6%– 77.9%). Of the 717 successfully sequenced P. falciparum isolates, 710 (99.0%; 95% CI: 97.9% - 99.6) were wildtype genotypes and 7 (1.0%; 95% CI: 0.4% - 2.1%) carried non-synonymous (NS) mutations in pfk13-propeller including 2 mutations (A578S and V534A) previously detected and 2 other (M472I and A569T) not yet detected in the DRC. Mutations associated with ART-R in Southeast Asia were not observed in DRC. However, the presence of other mutations in pfk13-propeller gene calls for further investigations to assess their implication in drug resistance.

Artemisinin-based combination therapies (ACTs) have been recommended by the World Health Organization (WHO) as first-line treatment of uncomplicated Plasmodium falciparum (P. falciparum) malaria since 2005 in Democratic Republic of Congo (DRC) and a regular surveillance of the ACT efficacy is required to ensure the treatment effectiveness. Mutations in the propeller domain of the pfk13 gene were identified as molecular markers of artemisinin resistance (ART-R). This study investigated the pfk13-propeller gene polymorphism in clinical isolates of P. falciparum collected in the DRC. In 2017, ten geographical sites across DRC were selected for a cross-sectional study that was conducted first in Kinshasa from January to March, then in the nine other sites from September to December. Dried blood samples were collected from patients attending health centers for fever where diagnosis of Malaria was first made by rapid diagnostic test (RDT) available on site (SD Bioline malaria Ag Pf or CareStart Malaria Pf) or by thick blood smear and then confirmed by a P. falciparum real-time PCR assay. A pfk13-propeller segment containing a fragment that codes for amino acids at positions 427–595 was amplified by conventional PCR before sequencing. In total, 1070 patients were enrolled in the study. Real-time PCR performed confirmed the initial diagnosis of P. falciparum infection in 806 samples (75.3%; 95% CI: 72.6%– 77.9%). Of the 717 successfully sequenced P. falciparum isolates, 710 (99.0%; 95% CI: 97.9% - 99.6) were wildtype genotypes and 7 (1.0%; 95% CI: 0.4% - 2.1%) carried non-synonymous (NS) mutations in pfk13-propeller including 2 mutations (A578S and V534A) previously detected and 2 other (M472I and A569T) not yet detected in the DRC. Mutations associated with ART-R in Southeast Asia were not observed in DRC. However, the presence of other mutations in pfk13-propeller gene calls for further investigations to assess their implication in drug resistance.

Plasmodiums are protozoa that may infect various hosts. Only five species are now recognized as naturally parasitizing humans: Plasmodium falciparum, Plasmodium vivax, Plasmodium malariae, Plasmodium ovale and Plasmodium knowlesi. This fifth species, P. knowlesi, previously identified as naturally parasitizing the monkey Macaca fascicularis, has been microscopically confused for a long time with P. malariae or P. falciparum and it was not possible to correctly differentiate them until the advent of molecular biology. To date, natural human infections with P. knowlesi only occur in Southeast Asia and a similar phenomenon of natural transmission of simian plasmodium to humans has not been reported elsewhere. This study was conducted to investigate a possible transmission of African small monkey's plasmodium to humans in populations living near the rainforest of the Democratic Republic of Congo (DRC) where several species of non-human primates are living. Two successive real-time PCRs were identified in the literature and used in combination for purpose. Only P. falciparum was found in this study. However, studies with larger samples and with more advanced techniques should be conducted.

BACKGROUND: Because of the loss of chloroquine (CQ) effectiveness, the Democratic Republic of Congo (DRC)'s malaria treatment policy replaced CQ by sulfadoxine–pyrimethamine (SP) as first-line treatment of uncomplicated malaria in 2003, which in turn was replaced by artemisinin-based combination therapies (ACT) in 2005. The World Health Organization (WHO) recommends monitoring of anti-malarial drug resistance every 2 years. The study aimed to provide baseline data for biennial molecular surveillance of anti-malarial drug resistance by comparing data from a study conducted in 2019 to previously published data from a similar study conducted in 2017 in the DRC. METHODS: From July to November 2019, a cross-sectional study was conducted in ten sites which were previously selected for a similar study conducted in 2017 across the DRC. P. falciparum malaria was diagnosed by a rapid diagnostic test (RDT) or by microscopy and dried blood samples (DBS) were taken from patients who had a positive test. Segments of interest in pfcrt and pfk13 genes were amplified by conventional PCR before sequencing. RESULTS: Out of 1087 enrolled patients, 906 (83.3%) were PCR-confirmed for P. falciparum. Like in the 2017-study, none of the mutations known to be associated with Artemisinine (ART) resistance in Southeast Asia was detected. However, non-synonymous (NS) mutations with unknown functions were observed among which, A578S was detected in both 2017 and 2019-studies. The overall prevalence of pfcrt-K76T mutation that confers CQ-resistance was 22.7% in 2019-study compared to 28.5% in 2017-study (p-value = 0.069), but there was high variability between sites in the two studies. Like in 2017-study, the pfcrt 72–76 SVMNT haplotype associated with resistance to amodiaquine was not detected. CONCLUSION: The study reported, within 2 years, the non-presence of molecular markers currently known to be associated with resistance to ART and to AQ in P. falciparum isolated in the DRC. However, the presence of polymorphisms with as-yet unknown ...

Background: Because of the loss of chloroquine (CQ) effectiveness, the Democratic Republic of Congo (DRC)'s malaria treatment policy replaced CQ by sulfadoxine–pyrimethamine (SP) as first‑line treatment of uncomplicated malaria in 2003, which in turn was replaced by artemisinin‑based combination therapies (ACT) in 2005. The World Health Organization (WHO) recommends monitoring of anti‑malarial drug resistance every 2 years. The study aimed to provide baseline data for biennial molecular surveillance of anti‑malarial drug resistance by comparing data from a study conducted in 2019 to previously published data from a similar study conducted in 2017 in the DRC. Methods: From July to November 2019, a cross‑sectional study was conducted in ten sites which were previously selected for a similar study conducted in 2017 across the DRC. P. falciparum malaria was diagnosed by a rapid diagnostic test (RDT) or by microscopy and dried blood samples (DBS) were taken from patients who had a positive test. Segments of interest in pfcrt and pfk13 genes were amplified by conventional PCR before sequencing. Results: Out of 1087 enrolled patients, 906 (83.3%) were PCR‑confirmed for P. falciparum. Like in the 2017‑study, none of the mutations known to be associated with Artemisinine (ART) resistance in Southeast Asia was detected. How‑ ever, non‑synonymous (NS) mutations with unknown functions were observed among which, A578S was detected in both 2017 and 2019‑studies. The overall prevalence of pfcrt‑K76T mutation that confers CQ‑resistance was 22.7% in 2019‑study compared to 28.5% in 2017‑study (p‑value = 0.069), but there was high variability between sites in the two studies. Like in 2017‑study, the pfcrt 72–76 SVMNT haplotype associated with resistance to amodiaquine was not detected. Conclusion: The study reported, within 2 years, the non‑presence of molecular markers currently known to be associated with resistance to ART and to AQ in P. falciparum isolated in the DRC. However, the presence of polymorphisms with as‑yet unknown functions was observed, requiring further characterization. Moreover, an overall decrease in the prevalence of CQ‑resistance marker was observed in the DRC, but this prevalence remained highly variable from region to region

Background: Because of the loss of chloroquine (CQ) effectiveness, the Democratic Republic of Congo (DRC)'s malaria treatment policy replaced CQ by sulfadoxine–pyrimethamine (SP) as first‑line treatment of uncomplicated malaria in 2003, which in turn was replaced by artemisinin‑based combination therapies (ACT) in 2005. The World Health Organization (WHO) recommends monitoring of anti‑malarial drug resistance every 2 years. The study aimed to provide baseline data for biennial molecular surveillance of anti‑malarial drug resistance by comparing data from a study conducted in 2019 to previously published data from a similar study conducted in 2017 in the DRC. Methods: From July to November 2019, a cross‑sectional study was conducted in ten sites which were previously selected for a similar study conducted in 2017 across the DRC. P. falciparum malaria was diagnosed by a rapid diagnostic test (RDT) or by microscopy and dried blood samples (DBS) were taken from patients who had a positive test. Segments of interest in pfcrt and pfk13 genes were amplified by conventional PCR before sequencing. Results: Out of 1087 enrolled patients, 906 (83.3%) were PCR‑confirmed for P. falciparum. Like in the 2017‑study, none of the mutations known to be associated with Artemisinine (ART) resistance in Southeast Asia was detected. How‑ ever, non‑synonymous (NS) mutations with unknown functions were observed among which, A578S was detected in both 2017 and 2019‑studies. The overall prevalence of pfcrt‑K76T mutation that confers CQ‑resistance was 22.7% in 2019‑study compared to 28.5% in 2017‑study (p‑value = 0.069), but there was high variability between sites in the two studies. Like in 2017‑study, the pfcrt 72–76 SVMNT haplotype associated with resistance to amodiaquine was not detected. Conclusion: The study reported, within 2 years, the non‑presence of molecular markers currently known to be associated with resistance to ART and to AQ in P. falciparum isolated in the DRC. However, the presence of polymorphisms with as‑yet unknown functions was observed, requiring further characterization. Moreover, an overall decrease in the prevalence of CQ‑resistance marker was observed in the DRC, but this prevalence remained highly variable from region to region

Background: The national policy for malaria treatment of the Democratic Republic of Congo recommends two first-line artemisinin-based combinations for the treatment of uncomplicated malaria: artesunate-amodiaquine and artemether-lumefantrine. This study investigated the presence of markers associated with resistance to the current first-line artemisinin-based combination therapy (ACT) in isolates of Plasmodium falciparum from treatment failure patients in the Democratic Republic of Congo. Methods: From November 2018 to November 2019, dried blood spots were taken from patients returning to health centres for fever within 28 days after an initial malaria treatment in six sentinel sites of the National Malaria Control Programme across Democratic Republic of Congo. The new episode of malaria was first detected by a rapid diagnostic test and then confirmed by a real-time PCR assay to define treatment failure. Fragments of interest in pfk13 and pfcrt genes were amplified by conventional PCR before sequencing and the Pfmdr1 gene copy number was determined by a TaqMan real-time PCR assay. Results: Out of 474 enrolled patients, 364 (76.8%) were confirmed positive by PCR for a new episode of P. falciparum malaria, thus considered as treatment failure. Of the 325 P. falciparum isolates obtained from 364 P. falciparum-positive patients and successfully sequenced in the pfk13-propeller gene, 7 (2.2%) isolates carried non-synonymous mutations, among which 3 have been previously reported (N498I, N554K and A557S) and 4 had not yet been reported (F506L, E507V, D516E and G538S). Of the 335 isolates successfully sequenced in the pfcrt gene, 139 (41.5%) harboured the K76T mutation known to be associated with chloroquine resistance. The SVMNT haplotype associated with resistance to amodiaquine was not found. None of the isolates carried an increased copy number of the pfmdr1 gene among the 322 P. falciparum isolates successfully analysed.

BACKGROUND: The national policy for malaria treatment of the Democratic Republic of Congo recommends two first-line artemisinin-based combinations for the treatment of uncomplicated malaria: artesunate-amodiaquine and artemether-lumefantrine. This study investigated the presence of markers associated with resistance to the current first-line artemisinin-based combination therapy (ACT) in isolates of Plasmodium falciparum from treatment failure patients in the Democratic Republic of Congo. METHODS: From November 2018 to November 2019, dried blood spots were taken from patients returning to health centres for fever within 28 days after an initial malaria treatment in six sentinel sites of the National Malaria Control Programme across Democratic Republic of Congo. The new episode of malaria was first detected by a rapid diagnostic test and then confirmed by a real-time PCR assay to define treatment failure. Fragments of interest in pfk13 and pfcrt genes were amplified by conventional PCR before sequencing and the Pfmdr1 gene copy number was determined by a TaqMan real-time PCR assay. RESULTS: Out of 474 enrolled patients, 364 (76.8%) were confirmed positive by PCR for a new episode of P. falciparum malaria, thus considered as treatment failure. Of the 325 P. falciparum isolates obtained from 364 P. falciparum-positive patients and successfully sequenced in the pfk13-propeller gene, 7 (2.2%) isolates carried non-synonymous mutations, among which 3 have been previously reported (N498I, N554K and A557S) and 4 had not yet been reported (F506L, E507V, D516E and G538S). Of the 335 isolates successfully sequenced in the pfcrt gene, 139 (41.5%) harboured the K76T mutation known to be associated with chloroquine resistance. The SVMNT haplotype associated with resistance to amodiaquine was not found. None of the isolates carried an increased copy number of the pfmdr1 gene among the 322 P. falciparum isolates successfully analysed. CONCLUSION: No molecular markers currently known to be associated with resistance to the first-line ACT in use were detected in isolates of P. falciparum from treatment failure patients. Regular monitoring through in vivo drug efficacy and molecular studies must continue to ensure the effectiveness of malaria treatment in Democratic Republic of Congo. ; Peer reviewed