Comparison of SD Bioline Malaria Ag Pf/Pan and Acro Malaria P.f./P.v./Pan with Microscopy and Real Time PCR for the Diagnosis of Human Plasmodium Species.

The early diagnosis of malaria is crucial to controlling morbidity and mortality. The World Health Organization (WHO) recommends diagnosing malaria either using light microscopy or a malaria rapid diagnostic test (RDT). Most RDTs use antibodies to detect two P. falciparum histidine-rich proteins named PfHRP2 and PfHRP3. However, false-negative results are known to occur due to the poor performance of RDTs depending on the species and the deletion of the Pfhrp2 and Pfhrp3 genes. This study evaluated new malaria RDTs for the detection of the human Plasmodium species. The Acro Malaria P.f./P.v./Pan Rapid Test Cassette allows the qualitative detection of parasite antigens, such as PfHRP2 specific to Plasmodium falciparum, PvLDH specific to Plasmodium vivax, and/or panLDH Plasmodium genus lactate dehydrogenase, in the blood of infected individuals. This RDT was assessed against 229 samples collected from imported malaria cases, mainly from Africa. The samples were previously diagnosed using light microscopy and RDT (SD Malaria Ag P.f./Pan, SD Bioline Alere Abbott), then confirmed using real time PCR. The two RDTs were evaluated using a comparison with real time PCR as the reference method, and their performances were compared with each other. Compared to SD RDT, the Acro RDT showed a better sensitivity to P. falciparum (96.8% vs. 89.8%), P. vivax (78.6% vs. 64.3%), P. ovale (73.7% vs. 5.3%), and P. malariae (20.0% vs. 0%). The respective specificities of the Acro RDT and SD RDT are 90.7% vs. 95.3% to P. falciparum, 100% to P. vivax, and 100% vs. 100% to Plasmodium genus. Therefore, Acro RDT showed better performance in the identification of P. ovale and low parasitaemia of P. falciparum. In addition, Acro RDT has the advantage of detecting PvLDH-specific antigens. The Acro Malaria RDT presents the benefits of detecting a P. falciparum antigen (PfHRP2) and a P. vivax antigen (PvLDH) with high sensitivity (96.8% and 73.7%, respectively) and specificity (90.7% and 100%, respectively). Acro Malaria P.f./P.v./Pan rapid diagnostic tests could be effectively used in endemic areas, especially when microscopic examination cannot be performed.

Assessment of a Commercial Real-Time PCR Assay (Vitassay qPCR Malaria 5 Test) to Detect Human Malaria Infection in Travelers Returning to France.

Malaria is the most common human parasitic disease in the world with the highest morbidity and mortality. Due to the severity of malaria caused by Plasmodium falciparum and the urgency of therapeutic management, quick and reliable diagnosis is required for early detection. Blood smear microscopy remains the gold standard for malaria diagnosis. Molecular diagnosis techniques are the most sensitive and specific in cases of low parasitaemia and in the detection of mixed infections. The purpose of this study was to evaluate a new commercial test involving the molecular diagnostic technique to detect the five human Plasmodium species. The Vitassay qPCR Malaria 5 test is based on the multiplex real-time PCR of a conserved target region of the 18S rRNA gene for the five human Plasmodium species. A total of 190 samples collected from imported cases of malaria were diagnosed using this test and compared against a homemade reference real-time PCR. The sensitivities of the Vitassay qPCR Malaria 5 test for all Plasmodium species ranged from 93.8% to 100% and specificity ranged from 97.7% to 100%. Based on these criteria, this test is recommended for the diagnosis of the human Plasmodium species.

Methylene Blue-Based Combination Therapy with Amodiaquine Prevents Severe Malaria in an Experimental Rodent Model.

Untreated malaria can progress rapidly to severe forms (<24 h). Moreover, resistance to antimalarial drugs is a threat to global efforts to protect people from malaria. Given this, it is clear that new chemotherapy must be developed. We contribute new data about using methylene blue (MB) to cure malaria and cerebral malaria in a combined therapy with common antimalarial drugs, including mefloquine (MQ) and amodiaquine (AQ). A C57BL6/J mouse model was used in an experimental cerebral malaria model. Mice were infected with Plasmodium berghei ANKA on Day 0 (D0) and the treatment started on D3 (nearly 1% parasitaemia) with AQ, MQ or MB alone or in combination with AQ or MQ. AQ, MQ and MB alone were unable to prevent cerebral malaria as part of a late chemotherapy. MB-based combination therapies were efficient even if treatment began at a late stage. We found a significant difference in survival rate (p < 0.0001) between MBAQ and the untreated group, but also with the AQ (p = 0.0024) and MB groups (p < 0.0001). All the infected mice treated with MB in combination with AQ were protected from cerebral malaria. Partial protection was demonstrated with MB associated with MQ. In this group, a significant difference was found between MBMQ and the untreated group (p < 0.0001), MQ (p = 0.0079) and MB (p = 0.0039). MB associated with AQ would be a good candidate for preventing cerebral malaria.

Comparative Assessment of the Sensitivity of Ten Commercial Rapid Diagnostic Test Kits for the Detection of Plasmodium.

Malaria is one of the most common tropical diseases encountered by members of the French military who are deployed in operations under constrained conditions in malaria-endemic areas. Blood smear microscopy-the gold standard for malaria diagnosis-is often not available in such settings, where the detection of malaria relies on rapid diagnostic tests (RDTs). Ten RDTs (from Biosynex, Carestart, Humasis, SD Bioline, and CTK Biotech), based on the detection of the Plasmodium falciparum histidine-rich protein 2 (HRP2) or lactate dehydrogenase (pLDH, PfLDH, or PvLDH), were assessed against 159 samples collected from imported malaria cases, including 79 P. falciparum, 37 P. vivax, 22 P. ovale, and 21 P. malariae parasites. Samples had been previously characterised using microscopy and real-time PCR. The overall sensitivities for the Plasmodium test ranged from 69.8% (111/159) to 95% (151/159). There was no significant difference for the specific detection of P. falciparum (96.2% to 98.7%, p = 0.845). No significant difference was found between sensitivities to P. vivax by pan LDH or pvLDH (81.1% (30/37) to 94.6% (35/37) (p = 0.845)). Some of the RDTs missed most of P. ovale and P. malariae, with sensitivities for all RDTs ranging respectively from 4.5% (1/22) to 81.8% (18/22) and 14.3% (3/21) to 95.2% (20/21). Carestart Malaria Pf/Pan (pLDH) Ag G0121, a pLDH-based RDT (PfLDH and pLDH), showed the highest sensitivities to P. falciparum (98.7%, 78/79), P. vivax (94.6%, 35/37), P. ovale (81.8%, 18/22), and P. malariae (95.2%, 20/21) and meets the requirements for military deployments in malaria-endemic areas.

Copper (I)-Chloroquine Complexes: Interactions with DNA and Ferriprotoporphyrin, Inhibition of ß-Hematin Formation and Relation to Antimalarial Activity.

A new Cu(I)-chloroquine (CQ) complex [Cu(CQ)(PPh3)2]NO3 (1) was synthesized and characterized, and its mechanism of action studied concomitant with the previously reported complex [Cu(CQ)2]Cl (2). These copper (I) coordination compounds can be considered as potential antimalarial agents because they show better inhibition of the CQ-resistant strain in in vitro studies than CQ alone. In comparison with other metal-CQ complexes, only the gold complex was similar to (1), i.e., more active than CQ against both CQ-susceptible (3D7) and CQ-resistant strains (W2). These two copper (I)-compounds also demonstrated higher antiplasmodial activity against W2 than other copper complexes reported to date. This suggests that the incorporation of the copper metal center enhanced the biological activity of CQ. To better understand their significant growth inhibition of the Plasmodium falciparum parasite, the interaction with two essential molecular targets for the survival and proliferation of the malarial parasite were studied. These were the ferriprotoporphyrin group and the DNA, both important targets for current antimalarial drugs at the asexual erythrocytic stages. Both compounds (1,2) exhibited significant interactions with these targets. In particular, interactions with the DNA were dominated by the intercalator properties of the CQ ligand but may have also been affected by the presence of copper. Overall, these compounds were better parasitic inhibitors than chloroquine diphosphate (CQDP) alone or other previously reported metal-CQ complexes such as platinum, ruthenium and gold.

Prevalence of Mutations in the pfcoronin Gene and Association with Ex Vivo Susceptibility to Common Quinoline Drugs against Plasmodium falciparum.

BACKGROUND: Artemisinin-based combination therapy (ACT) was recommended to treat uncomplicated falciparum malaria. Unlike the situation in Asia where resistance to ACT has been reported, artemisinin resistance has not yet emerged in Africa. However, some rare failures with ACT or patients continuing to be parasitaemic on day 3 after ACT treatment have been reported in Africa or in travellers returning from Africa. Three mutations (G50E, R100K, and E107V) in the pfcoronin gene could be responsible for artemisinin resistance in Africa. METHODS: The aims of this study were first to determine the prevalence of mutations in the pfcoronin gene in African P. falciparum isolates by Sanger sequencing, by targeting the 874 samples collected from patients hospitalised in France after returning from endemic areas in Africa between 2018 and 2019, and secondly to evaluate their association with in vitro reduced susceptibility to standard quinoline antimalarial drugs, including chloroquine, quinine, mefloquine, desethylamodiaquine, lumefantrine, piperaquine, and pyronaridine. RESULTS: The three mutations in the pfcoronin gene (50E, 100K, and 107V) were not detected in the 874 P. falciparum isolates. Current data show that another polymorphism (P76S) is present in many countries of West Africa (mean prevalence of 20.7%) and Central Africa (11.9%) and, rarely, in East Africa (4.2%). This mutation does not appear to be predictive of in vitro reduced susceptibility to quinolines, including artemisinin derivative partners in ACT such as amodiaquine, lumefantrine, piperaquine, pyronaridine, and mefloquine. Another mutation (V62M) was identified at low prevalence (overall prevalence of 1%). CONCLUSIONS: The 76S mutation is present in many African countries with a prevalence above 10%. It is reassuring that this mutation does not confer in vitro resistance to ACT partners.

Salivette, a relevant saliva sampling device for SARS-CoV-2 detection.

Background: The gold standard for COVID-19 diagnosis relies on quantitative reverse-transcriptase polymerase-chain reaction (RT-qPCR) from nasopharyngeal swab (NPS) specimens, but NPSs present several limitations. The simplicity, low invasive and possibility of self-collection of saliva imposed these specimens as a relevant alternative for SARS-CoV-2 detection. However, the discrepancy of saliva test results compared to NPSs made of its use controversial. Here, we assessed Salivettes®, as a standardized saliva collection device, and compared SARS-CoV-2 positivity on paired NPS and saliva specimens. Methods: A total of 303 individuals randomly selected among those investigated for SARS-CoV-2 were enrolled, including 30 (9.9%) patients previously positively tested using NPS (follow-up group), 90 (29.7%) mildly symptomatic and 183 (60.4%) asymptomatic. Results: The RT-qPCR revealed a positive rate of 11.6% (n = 35) and 17.2% (n = 52) for NPSs and saliva samples, respectively. The sensitivity and specificity of saliva samples were 82.9% and 91.4%, respectively, using NPS as reference. The highest proportion of discordant results concerned the follow-up group (33.3%). Although the agreement exceeded 90.0% in the symptomatic and asymptomatic groups, 17 individuals were detected positive only in saliva samples, with consistent medical arguments. Conclusion Saliva collected with Salivette® was more sensitive for detecting symptomatic and pre-symptomatic infections.

Absence of Association between Methylene Blue Reduced Susceptibility and Polymorphisms in 12 Genes Involved in Antimalarial Drug Resistance in African Plasmodium falciparum.

Half the human population is exposed to malaria. Plasmodium falciparum antimalarial drug resistance monitoring and development of new drugs are major issues related to the control of malaria. Methylene blue (MB), the oldest synthetic antimalarial, is again a promising drug after the break of its use as an antimalarial drug for more than 80 years and a potential partner for triple combination. Very few data are available on the involvement of polymorphisms on genes known to be associated with standard antimalarial drugs and parasite in vitro susceptibility to MB (cross-resistance). In this context, MB susceptibility was evaluated against 482 isolates of imported malaria from Africa by HRP2-based ELISA chemosusceptibility assay. A total of 12 genes involved in antimalarial drug resistance (Pfcrt, Pfdhfr, Pfmdr1, Pfmdr5, Pfmdr6, PfK13, Pfubq, Pfcarl, Pfugt, Pfact, Pfcoronin, and copy number of Pfpm2) were sequenced by Sanger method and quantitative PCR. On the Pfmdr1 gene, the mutation 86Y combined with 184F led to more susceptible isolates to MB (8.0 nM vs. 11.6 nM, p = 0.03). Concerning Pfmdr6, the isolates bearing 12 Asn repetitions were more susceptible to MB (4.6 nM vs. 11.6 nM, p = 0.005). None of the polymorphisms previously described as involved in antimalarial drug resistance was shown to be associated with reduced susceptibility to MB. Some genes (particularly PfK13, Pfugt, Pfact, Pfpm2) did not present enough genetic variability to draw conclusions about their involvement in reduced susceptibility to MB. None of the polymorphisms analyzed by multiple correspondence analysis (MCA) had an impact on the MB susceptibility of the samples successfully included in the analysis. It seems that there is no in vitro cross-resistance between MB and commonly used antimalarial drugs.

Absence of association between polymorphisms in the pfcoronin and pfk13 genes and the presence of Plasmodium falciparum parasites after treatment with artemisinin derivatives in Senegal.

Due to resistance to chloroquine and sulfadoxine/pyrimethamine, treatment for uncomplicated Plasmodium falciparum malaria switched to artemisinin-based combination therapy (ACT) in 2006 in Senegal. Several mutations in the gene encoding the kelch13 helix (pfk13-propeller) have been identified as associated with in vitro and in vivo artemisinin resistance in Southeast Asia. Additionally, three mutations in the pfcoronin gene (G50E, R100K and E107V) have been identified in two culture-adapted Senegalese field isolates that became resistant in vitro to artemisinin after 4 years of intermittent selection with dihydroartemisinin. The aims of this study were to assess the prevalence of pfcoronin and pfk13 mutations in Senegalese field isolates from Dakar and to investigate their association with artemisinin derivative clinical failures. A total of 348 samples of P. falciparum from 327 patients, collected from 2015-2019 in Dakar, were successfully analysed. All sequences had wild-type pfk13 allele. The three mutations (G50E, R100K and E107V), previously identified in parasites with reduced susceptibility to artemisinin, were not found in this study, but a new mutation (P76S) was detected (mean prevalence 16.2%). The P76S mutation was identified in 5 (31.3%) of 16 isolates collected from patients still parasitaemic on Day 3 after ACT treatment and in 31 samples (15.3%) among 203 patients considered successfully cured. There was no significant association between in vivo reduced efficacy to artemisinin derivatives and the P76S mutation (P = 0.151, Fisher's exact test). These data suggest that polymorphisms in pfk13 and pfcoronin are not the best predictive markers for artemisinin resistance in Senegal.

Prevalence of mutations in the Plasmodium falciparum chloroquine resistance transporter, PfCRT, and association with ex vivo susceptibility to common anti-malarial drugs against African Plasmodium falciparum isolates.

BACKGROUND: The Plasmodium falciparum chloroquine transporter gene (pfcrt) is known to be involved in chloroquine and amodiaquine resistance, and more particularly the mutations on the loci 72 to 76 localized within the second exon. Additionally, new mutations (T93S, H97Y, C101F, F145I, M343L, C350R and G353V) were recently shown to be associated with in vitro reduced susceptibility to piperaquine in Asian or South American P. falciparum strains. However, very few data are available on the prevalence of these mutations and their effect on parasite susceptibility to anti-malarial drugs, and more particularly piperaquine in Africa. METHODS: A molecular investigation of these mutations was performed in 602 African P. falciparum parasites collected between 2017 and 2018 on malaria patients hospitalized in France after a travel in African countries. Associations between genotypes and in vitro susceptibilities to piperaquine and standard antimalarial drugs were assessed. RESULTS: None of the mutations, previously described as associated with piperaquine resistance, was found in the 602 P. falciparum African isolates. The K76T mutation is associated with resistance to chloroquine (p < 0.0002) and desethylamodiaquine (p < 0.002) in Africa. The K76T mutation is not associated with in vitro reduced susceptibility to piperaquine. The mutation I356T, identified in 54.7% (n = 326) of the African isolates, was significantly associated with reduced susceptibility to quinine (p < 0.02) and increased susceptibility to mefloquine (p < 0.04). The K76T and I356T mutations were significantly associated in West African isolates (p = 0.008). CONCLUSION: None of the mutations in pfcrt found to be associated with piperaquine reduced susceptibility in Asia or South America (T93S, H97Y, C101F, F145I, M343L C350R and G353V) were found in the 602 African isolates including the three isolates with reduced susceptibility to piperaquine. The K76T mutation, involved in resistance to chloroquine and amodiaquine, and the I356T mutation were not associated with in vitro reduced susceptibility to piperaquine. Differences in mefloquine susceptibility between I356 and 356T isolates were, while statistically different, minimal. Further analyses are needed with a more important sample size from the same geographic area to confirm the role of the I356T mutation on quinine susceptibility.

Baseline and multinormal distribution of ex vivo susceptibilities of Plasmodium falciparum to methylene blue in Africa, 2013-18.

BACKGROUND: Plasmodium falciparum resistance to most antimalarial compounds has emerged in Southeast Asia and spread to Africa. In this context, the development of new antimalarial drugs is urgent. OBJECTIVES: To determine the baseline in vitro activity of methylene blue (Proveblue®) on African isolates and to determine whether parasites have different phenotypes of susceptibility to methylene blue. METHODS: Ex vivo susceptibility to methylene blue was measured for 609 P. falciparum isolates of patients hospitalized in France for malaria imported from Africa. A Bayesian statistical analysis was designed to describe the distribution of median effective concentration (EC50) estimates. RESULTS: The EC50 ranged from 0.16 to 87.2 nM with a geometric mean of 7.17 nM (95% CI = 6.21-8.13). The 609 EC50 values were categorized into four components: A (mean = 2.5 nM; 95% CI = 2.28-2.72), B (mean = 7.44 nM; 95% CI = 7.07-7.81), C (mean = 16.29 nM; 95% CI = 15.40-17.18) and D (mean = 38.49 nM; 95% CI = 34.14-42.84). The threshold value for in vitro reduced susceptibility to methylene blue was estimated at 35 nM using the geometric mean of EC50 plus 2 SDs of the 609 isolates. This cut-off also corresponds to the lower limit of the 95% CI of the methylene blue EC50 of component D. Thirty-five isolates (5.7%) displayed EC50 values above this threshold. CONCLUSIONS: Methylene blue exerts a promising efficacy against P. falciparum and is a potential partner for triple combinations.

Late clinical failure associated with cytochrome b codon 268 mutation during treatment of falciparum malaria with atovaquone-proguanil in traveller returning from Congo.

BACKGROUND: The drug combination atovaquone-proguanil, is recommended for treatment of uncomplicated falciparum malaria in France. Despite high efficacy, atovaquone-proguanil treatment failures have been reported. Resistance to cycloguanil, the active metabolite of proguanil, is conferred by multiple mutations in the Plasmodium falciparum dihydrofolate reductase (pfdhfr) and resistance to atovaquone by single mutation on codon 268 of the cytochrome b gene (pfcytb). CASE PRESENTATION: A 47-year-old female, native from Congo and resident in France, was admitted in hospital for uncomplicated falciparum malaria with parasitaemia of 0.5%, after travelling in Congo (Brazzaville and Pointe Noire). She was treated with atovaquone-proguanil (250 mg/100 mg) 4 tablets daily for 3 consecutive days. On day 5 after admission she was released home. However, many weeks after this episode, without having left France, she again experienced fever and intense weakness. On day 39 after the beginning of treatment, she consulted for fever, arthralgia, myalgia, photophobia, and blurred vision. She was hospitalized for uncomplicated falciparum malaria with a parasitaemia of 0.375% and treated effectively by piperaquine-artenimol (320 mg/40 mg) 3 tablets daily for 3 consecutive days. Resistance to atovaquone-proguanil was suspected. The Y268C mutation was detected in all of the isolates tested (D39, D42, D47). The genotyping of the pfdhfr gene showed a triple mutation (N51I, C59R, S108N) involved in cycloguanil resistance. CONCLUSION: This is the first observation of a late clinical failure of atovaquone-proguanil treatment of P. falciparum uncomplicated malaria associated with pfcytb 268 mutation in a traveller returning from Congo. These data confirm that the Y268C mutation is associated with delayed recrudescence 4 weeks or more after initial treatment. Although atovaquone-proguanil treatment failures remain rare, an increased surveillance is required. It is essential to declare and publish all well-documented cases of treatment failures because it is the only way to evaluate the level of resistance to atovaquone.

Low polymorphisms in pfact, pfugt and pfcarl genes in African Plasmodium falciparum isolates and absence of association with susceptibility to common anti-malarial drugs.

BACKGROUND: Resistance to all available anti-malarial drugs has emerged and spread including artemisinin derivatives and their partner drugs. Several genes involved in artemisinin and partner drugs resistance, such as pfcrt, pfmdr1, pfK13 or pfpm2, have been identified. However, these genes do not properly explain anti-malarial drug resistance, and more particularly clinical failures observed in Africa. Mutations in genes encoding for Plasmodium falciparum proteins, such as P. falciparum Acetyl-CoA transporter (PfACT), P. falciparum UDP-galactose transporter (PfUGT) and P. falciparum cyclic amine resistance locus (PfCARL) have recently been associated to resistance to imidazolopiperazines and other unrelated drugs. METHODS: Mutations on pfugt, pfact and pfcarl were characterized on 86 isolates collected in Dakar, Senegal and 173 samples collected from patients hospitalized in France after a travel in African countries from 2015 and 2016 to assess their potential association with ex vivo susceptibility to chloroquine, quinine, lumefantrine, monodesethylamodiaquine, mefloquine, dihydroartemisinin, artesunate, doxycycline, pyronaridine and piperaquine. RESULTS: No mutations were found on the genes pfugt and pfact. None of the pfcarl described mutations were identified in these samples from Africa. The K784N mutation was found in one sample and the K734M mutation was identified on 7.9% of all samples for pfcarl. The only significant differences in ex vivo susceptibility according to the K734M mutation were observed for pyronaridine for African isolates from imported malaria and for doxycycline for Senegalese parasites. CONCLUSION: No evidence was found of involvement of these genes in reduced susceptibility to standard anti-malarial drugs in African P. falciparum isolates.

Modulation of in vitro antimalarial responses by polymorphisms in Plasmodium falciparum ABC transporters (pfmdr1 and pfmdr5).

The emergence of resistance to artemisinin-based combination therapies (ACT) was described in Southeast Asia. In this context, the identification of molecular markers of ACT resistance partner drugs is urgently needed for monitoring the emergence and spread of resistance. Polymorphisms in transporter genes, especially of the ATP-binding cassette (ABC) superfamily, have been involved in anti-malarial drug resistance. In this study, the association between the mutations in the P. falciparum multidrug resistance 1 gene (pfmdr1, N86Y, Y184 F, S1034C, N1042D and D1246Y) or repetitive amino acid motifs in pfmdr5 and the ex vivo susceptibility to anti-malarial drugs was evaluated. Susceptibility to chloroquine, quinine, monodesethylamodiaquine, lumefantrine, piperaquine, pyronaridine, mefloquine and dihydroartemisinin was assessed in 67 Senegalese isolates. The shorter DNNN motif ranged from to 2 to 11 copy repeats, and the longer DHHNDHNNDNNN motif ranged from 0 to 2 in pfmdr5. The present study showed the association between repetitive amino acid motifs (DNNN-DHHNDDHNNDNNN) in pfmdr5 and in vitro susceptibility to 4-aminoquinoline-based antimalarial drugs. The parasites with 8 and more copy repeats of DNNN in pfmdr5 were significantly more susceptible to piperaquine. There was a significant association between parasites whose DHHNDHNNDNNN motif was absent and replaced by DHHNDNNN, DHHNDHNNDHNNDNNN or DHHNDHNNDHNNDHNNDNNN and increased susceptibility to chloroquine, monodesethylamodiaquine and pyronaridine. A significant association between both the wild-type allele N86 in pfmdr1 and the N86-184 F haplotype and reduced susceptibility to lumefantrine was confirmed. Further studies with a large number of samples are required to validate the association between these pfmdr5 alleles and the modulation of 4-aminoquinoline-based antimalarial drug susceptibility.

Baseline Ex Vivo and Molecular Responses of Plasmodium falciparum Isolates to Piperaquine before Implementation of Dihydroartemisinin-Piperaquine in Senegal.

Dihydroartemisinin-piperaquine, which was registered in 2017 in Senegal, is not currently used as the first-line treatment against uncomplicated malaria. A total of 6.6% to 17.1% of P. falciparum isolates collected in Dakar in 2013 to 2015 showed ex vivo-reduced susceptibility to piperaquine. Neither the exonuclease E415G mutation nor the copy number variation of the plasmepsin II gene (Pfpm2), associated with piperaquine resistance in Cambodia, was detected in Senegalese parasites.

Absence of a High Level of Duplication of the Plasmepsin II Gene in Africa.

Resistance to piperaquine has been associated with the amplification of the plasmepsin II gene in Cambodia. None of the 175 African isolates that we analyzed had plasmepsin II gene amplification (piperaquine 50% inhibitory concentration ranged from 0.94 to 137.5 nM), suggesting there is a low prevalence of piperaquine reduced susceptibility in Africa. Additionally, the few isolates with reduced susceptibility to piperaquine did not harbor amplification of the plasmepsin II gene.

The D113N mutation in the RING E3 ubiquitin protein ligase gene is not associated with ex vivo susceptibility to common anti-malarial drugs in African Plasmodium falciparum isolates.

BACKGROUND: Plasmodium falciparum resistance to artemisinin-based combination therapy has emerged and spread in Southeast Asia. In areas where artemisinin resistance is emerging, the efficacy of combination is now based on partner drugs. In this context, the identification of novel markers of resistance is essential to monitor the emergence and spread of resistance to these partner drugs. The ubiquitylation pathway could be a possible target for anti-malarial compounds and might be involved in resistance. Polymorphisms in the E3 ubiquitin-protein ligase (PF3D7_0627300) gene could be associated with decreased in vitro susceptibility to anti-malarial drugs. METHODS: Plasmodium falciparum isolates were collected from patients hospitalized in France with imported malaria from a malaria-endemic country from January 2015 to December 2016 and, more particularly, from African French-speaking countries. In total, 215 isolates were successfully sequenced for the E3 ubiquitin-protein ligase gene and assessed for ex vivo susceptibility to anti-malarial drugs. RESULTS: The D113N mutation in the RING E3 ubiquitin-protein ligase gene was present in 147 out of the 215 samples (68.4%). The IC50 values for the ten anti-malarial drugs were not significantly different between the wild-type and mutant parasites (p values between 0.225 and 0.933). There was no significant difference in terms of the percentage of parasites with decreased susceptibility between the D113 wild-type and the 133N mutated P. falciparum strains (p values between 0.541 and 1). CONCLUSION: The present data confirmed the absence of the association between polymorphisms in the RING E3 ubiquitin-protein ligase gene and the ex vivo susceptibility to common anti-malarial drugs in African P. falciparum isolates.

Confirmation of Plasmodium falciparum in vitro resistance to monodesethylamodiaquine and chloroquine in Dakar, Senegal, in 2015.

BACKGROUND: In response to increasing resistance to anti-malarial drugs, Senegal adopted artemisinin-based combination therapy (ACT) as the first-line treatment for uncomplicated malaria in 2006. However, resistance of Plasmodium falciparum parasites to artemisinin derivatives, characterized by delayed parasite clearance after treatment with ACT or artesunate monotherapy, has recently emerged and rapidly spread in Southeast Asia. After 10 years of stability with rates ranging from 5.6 to 11.8%, the prevalence of parasites with reduced susceptibility in vitro to monodesethylamodiaquine, the active metabolite of an ACT partner drug, increased to 30.6% in 2014 in Dakar. Additionally, after a decrease of the in vitro chloroquine resistance in Dakar in 2009-2011, the prevalence of parasites that showed in vitro chloroquine resistance increased again to approximately 50% in Dakar since 2013. The aim of this study was to follow the evolution of the susceptibility to ACT partners and other anti-malarial drugs in 2015 in Dakar. An in vitro test is the only method currently available to provide an early indication of resistance to ACT partners. RESULTS: Thirty-two P. falciparum isolates collected in 2015 in Dakar were analysed using a standard ex vivo assay based on an HRP2 ELISA. The prevalence of P. falciparum parasites with reduced susceptibility in vitro to monodesethylamodiaquine, chloroquine, mefloquine, doxycycline and quinine was 28.1, 46.9, 45.2, 31.2 and 9.7%, respectively. None of the parasites were resistant to lumefantrine, piperaquine, pyronaridine, dihydroartemisinin and artesunate. These results confirm an increase in the reduced susceptibility to monodesethylamodiaquine observed in 2014 in Dakar and the chloroquine resistance observed in 2013. The in vitro resistance seems to be established in Dakar. Additionally, the prevalence of parasites with reduced susceptibility to doxycycline has increased two-fold compared to 2014. CONCLUSIONS: The establishment of a reduced susceptibility to monodesethylamodiaquine as well as chloroquine resistance, and the emergence of a reduced susceptibility to doxycycline are disturbing. The in vitro and in vivo surveillance of anti-malarial drugs must be implemented in Senegal.

Association between Polymorphisms in the Pfmdr6 Gene and Ex Vivo Susceptibility to Quinine in Plasmodium falciparum Parasites from Dakar, Senegal.

Polymorphisms and the overexpression of transporter genes, especially of the ATP-binding cassette superfamily, have been involved in antimalarial drug resistance. The objective of this study was to use 77 Senegalese Plasmodium falciparum isolates to evaluate the association between the number of Asn residues in the polymorphic microsatellite region of the Plasmodium falciparum multidrug resistance 6 gene (Pfmdr6) and the ex vivo susceptibility to antimalarials. A significant association was observed between the presence of 7 or 9 Asn repeats and reduced susceptibility to quinine.