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.

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.

Ex vivo activity of Proveblue, a methylene blue, against field isolates of Plasmodium falciparum in Dakar, Senegal from 2013-2015.

Resistance to most antimalarial drugs has spread from Southeast Asia to Africa. Accordingly, new therapies to use with artemisinin-based combination therapy (triple ACT) are urgently needed. Proveblue, a methylene blue preparation, was found to exhibit antimalarial activity against Plasmodium falciparum strains in vitro. Proveblue has synergistic effects when used in combination with dihydroartemisinin, and has been shown to significantly reduce or prevent cerebral malaria in mice. The objectives of the current study were to evaluate the in vitro baseline susceptibility of clinical field isolates to Proveblue, compare its activity with that of other standard antimalarial drugs and define the patterns of cross-susceptibility between Proveblue and conventional antimalarial drugs. The Proveblue IC50 of 76 P. falciparum isolates ranged from 0.5 nM to 135.1 nM, with a mean of 8.1 nM [95% confidence interval, 6.4-10.3]. Proveblue was found to be more active against P. falciparum parasites than chloroquine, quinine, monodesethylamodiaquine, mefloquine, piperaquine, doxycycline (P <0.001) and lumefantrine (P = 0.014). Proveblue was as active as pyronaridine (P = 0.927), but was less active than dihydroartemisinin and artesunate (P <0.001). The only significant cross-susceptibilities found were between Proveblue and dihydroartemisinin (r2 = 0.195, P = 0.0001), artesunate (r2 = 0.187, P = 0.0002) and piperaquine (r2 = 0.063, P = 0.029). The present study clearly demonstrates the potential of Proveblue as an effective therapeutic agent against P. falciparum. In this context, the use of Proveblue as part of the triple ACT treatment for multidrug-resistant malaria warrants further investigation.

Absence of association between polymorphisms in the K13 gene and the presence of Plasmodium falciparum parasites at day 3 after treatment with artemisinin derivatives in Senegal.

In 2006, the Senegalese National Malaria Control Programme recommended artemisinin-based combination therapy as first-line treatment for uncomplicated malaria. In addition, intravenous (i.v.) injection of artesunate and artemether has gradually replaced quinine for the treatment of severe malaria. Mutations in the propeller domain of the Kelch 13 gene (K13-propeller, PF3D71343700), such as Y493H, R539T, I543T and C580Y, were recently associated with in vivo and in vitro resistance to artemisinin in Southeast Asia. However, these mutations were not identified in Africa. In total, 181 isolates of Plasmodium falciparum from 161 patients from Dakar, Senegal, were collected between August 2015 and January 2016. The K13-propeller gene of the isolates was sequenced. A search for non-synonymous mutations in the propeller region of K13 was performed in the 181 isolates collected from Dakar from 2015 to 2016. Three synonymous mutations were detected (D464D, C469C and R471R). Of 119 patients treated with i.v. artesunate or intramuscular artemether followed by artemether/lumefantrine, 9 patients were still parasitaemic on Day 3. Parasites from these nine patients were wild-type for K13-propeller. None of the polymorphisms known to be involved in artemisinin resistance in Asia were detected. These results suggest that K13 is not the best predictive marker for artemisinin resistance in Africa. More isolates from clinical failure cases or patients with delayed parasite clearance after treatment with artemisinin derivatives are necessary to identify new molecular markers.

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.

Absence of Association between Polymorphisms in the RING E3 Ubiquitin Protein Ligase Gene and Ex Vivo Susceptibility to Conventional Antimalarial Drugs in Plasmodium falciparum Isolates from Dakar, Senegal.

The RING E3 ubiquitin protein ligase is crucial for facilitating the transfer of ubiquitin. The only polymorphism identified in the E3 ubiquitin protein ligase gene was the D113N mutation (62.5%) but was not significantly associated with the 50% inhibitory concentration (IC50) of conventional antimalarial drugs. However, some mutated isolates (D113N) present a trend of reduced susceptibility to piperaquine (P = 0.0938). To evaluate the association of D113N polymorphism with susceptibility to antimalarials, more isolates are necessary.

High-quality draft genome sequence and description of Haemophilus massiliensis sp. nov.

Strain FF7(T) was isolated from the peritoneal fluid of a 44-year-old woman who suffered from pelvic peritonitis. This strain exhibited a 16S rRNA sequence similarity of 94.8 % 16S rRNA sequence identity with Haemophilus parasuis, the phylogenetically closest species with a name with standing in nomenclature and a poor MALDI-TOF MS score (1.32 to 1.56) that does not allow any reliable identification. Using a polyphasic study made of phenotypic and genomic analyses, strain FF7(T) was a Gram-negative, facultatively anaerobic rod and member of the family Pasteurellaceae. It exhibited a genome of 2,442,548 bp long genome (one chromosome but no plasmid) contains 2,319 protein-coding and 67 RNA genes, including 6 rRNA operons. On the basis of these data, we propose the creation of Haemophilus massiliensis sp. nov. with strain FF7(T) (= CSUR P859 = DSM 28247) as the type strain.

Low prevalences of HIV infection and HSV genital shedding in the general adult female population in Senegal.

INTRODUCTION: Herpes simplex virus (HSV) is the main co-factor for heterosexual transmission of the human immunodeficiency virus (HIV) in sub-Saharan Africa, and could be involved in the dynamics of the HIV epidemic in Senegal. METHODOLOGY: Genital shedding of HSV was evaluated in adult females who had visited the provincial healthcare centres in Diass, Louga, and Kebemer in Senegal. Study subjects were interviewed by a healthcare worker for sociodemographic characteristics and sexual behavior, and HIV serology was offered. In addition, cervical secretion lavage samples were evaluated for HSV DNA by real-time polymerase chain reaction (PCR), the melting curve analysis of which permitted distinction between HSV type 1 (HSV-1) and HSV type 2 (HSV-2). RESULTS: Among 302 women (mean age, 40 years) enrolled, none were infected by HIV. The mean age at first sexual intercourse was 20 years, and the mean number of sexual partners in the previous year was 1.3 (range, 1-7). Only 6 of 302 (1.9%) women had cervico-vaginal secretions positive for HSV DNA. No association between HSV DNA shedding and any sociodemographic or biological variables was found. Surprisingly, genital shedding of HSV-1 was found in two (0.7%) women, representing 33% of herpes-shedding women, and HSV-2 in four (1.5%) women. CONCLUSIONS: Taken together, our observations indicate a low prevalence of HSV DNA genital shedding in adult Senegalese women.

MALDI-TOF Mass Spectrometry: A Powerful Tool for Clinical Microbiology at Hôpital Principal de Dakar, Senegal (West Africa).

Our team in Europe has developed the routine clinical laboratory identification of microorganisms by matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry (MS). To evaluate the utility of MALDI-TOF MS in tropical Africa in collaboration with local teams, we installed an apparatus in the Hôpital Principal de Dakar (Senegal), performed routine identification of isolates, and confirmed or completed their identification in France. In the case of discordance or a lack of identification, molecular biology was performed. Overall, 153/191 (80.1%) and 174/191 (91.1%) isolates yielded an accurate and concordant identification for the species and genus, respectively, with the 2 different MALDI-TOF MSs in Dakar and Marseille. The 10 most common bacteria, representing 94.2% of all bacteria routinely identified in the laboratory in Dakar (Escherichia coli, Klebsiella pneumoniae, Streptococcus agalactiae, Acinetobacter baumannii, Pseudomonas aeruginosa, Staphylococcus aureus, Staphylococcus haemolyticus, Enterobacter cloacae, Enterococcus faecalis, and Staphylococcus epidermidis) were accurately identified with the MALDI-TOF MS in Dakar. The most frequent misidentification in Dakar was at the species level for Achromobacter xylosoxidans, which was inaccurately identified as Achromobacter denitrificans, and the bacteria absent from the database, such as Exiguobacterium aurientacum or Kytococcus schroeteri, could not be identified. A few difficulties were observed with MALDI-TOF MS for Bacillus sp. or oral streptococci. 16S rRNA sequencing identified a novel bacterium, "Necropsobacter massiliensis." The robust identification of microorganisms by MALDI-TOF MS in Dakar and Marseille demonstrates that MALDI-TOF MS can be used as a first-line tool in clinical microbiology laboratories in tropical countries.

The ongoing revolution of MALDI-TOF mass spectrometry for microbiology reaches tropical Africa.

Matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) represents a revolution in routine pathogen identification in clinical microbiology laboratories. A MALDI-TOF MS was introduced to tropical Africa in the clinical microbiology laboratory of the Hôpital Principal de Dakar (Senegal) and used for routine pathogen identification. Using MS, 2,429 bacteria and fungi isolated from patients were directly assayed, leading to the identification of 2,082 bacteria (85.7%) and 206 fungi (8.5%) at the species level, 109 bacteria (4.5%) at the genus level, and 16 bacteria (0.75%) at the family level. Sixteen isolates remained unidentified (0.75%). Escherichia coli was the most prevalent species (25.8%) followed by Klebsiella pneumoniae (14.8%), Streptococcus agalactiae (6.2%), Acinetobacter baumannii (6.1%), Pseudomonas aeruginosa (5.9%), and Staphylococcus aureus (5.9%). MALDI-TOF MS has also enabled the detection of rare bacteria and fungi. MALDI-TOF MS is a powerful tool for the identification of bacterial and fungal species involved in infectious diseases in tropical Africa.

Low seroprevalence of herpes simplex virus type 2 among pregnant women in Senegal.

Herpes simplex virus type 2 (HSV-2) is considered as a major co-factor of both sexual transmission and acquisition of the human immunodeficiency virus (HIV). The HIV epidemic in Senegal is characterized by a remarkable and stable low prevalence. Whether HSV-2 may also constitute a possible co-factor favouring the spreading of HIV epidemic in Senegal is yet unknown. This prompted us to evaluate the HSV-2 seroprevalence in the sentinel population of pregnant women in Senegal. Two hundred and sixty pregnant women attending Roi Baudouin maternity in the capital city Dakar (n = 14; 135) and the antenatal clinic in Kaolack (n = 125), the third city of Senegal, were prospectively recruited between March and August 2003. Fifty-six women (22%) were positive for HSV-2 serology. The prevalence of HSV-2 seropositivity was higher in women living in Dakar (26%) than in those living in Kaolack (16%) (P < 0.01). Only two women from Dakar and two other from Kaolack were found to be HIV-1-infected. Our observations suggest a seemingly low seroprevalence of HSV-2 infection in adult women Senegal, comparable with those usually reported in Western countries. Further, epidemiological surveys are needed to confirm these results in the general population.