Circulation of Non-falciparum Species in Niger: Implications for Malaria Diagnosis.

Background: Niger's National Malaria Control Programme and its partners use histidine-rich protein 2-based RDTs, which are specific to Plasmodium falciparum diagnosis. This study aimed to screen for the circulation of non-falciparum species in Zinder, a region of Niger, West Africa. Methods: A cross-sectional study was carried out from July to December 2022 at the district hospital of the Zinder region of Niger. P falciparum histidine-rich protein 2-based rapid diagnostic tests were performed, and dried blood spot samples were collected for further laboratory multiplexed photo-induced electron transfer-polymerase chain reaction (PET-PCR) analysis on positive light microscopy from all patients with fever who attended the Zinder district hospital during the study period. Results: In total, 340 dried blood spots were collected and analyzed by PET-PCR. Overall, 73.2% (95% CI, 68.2%-77.9%; 249/340) were positive for Plasmodium genus and species and represented the study population. Plasmodium species proportions were 89.5% (95% CI, 85.1%-93.1%; 223/249) for P falciparum, 38.5% (95% CI, 32.5%-44.9%; 96/249) for P malariae, 10.8% (95% CI, 7.3%-15.4%; 27/249) for P vivax, and 1.6% (95% CI, .4%-4.1%; 4/249) for P ovale. Single infection with Plasmodium species counted for 61.8% (95% CI, 55.5%-67.9%; 154/249), and the mixed infections rate, with at least 2 Plasmodium species, was 38.1% (95% CI, 32.1%-44.5%; 95/249). Single non-falciparum infections represented a rate of 10.0% (95% CI, 6.6%-14.5%; 25/249). Conclusion: This study confirms the first evidence of Plasmodium vivax by PET-PCR in Niger in addition to the other 3 Plasmodium species. These findings underline the need to adapt malaria diagnostic tools and therapeutic management, as well as the training of microscopists, for recognition of non-falciparum plasmodial species circulating in the country. This will better inform the strategies toward malaria control and elimination, as well as the decision making of the health authorities of Niger.

Direct 16S/ITS rRNA Gene PCR Followed by Sanger Sequencing for Detection of Mycetoma Causative Agents in Dakar, Senegal: A Pilot Study Among Patients with Mycetoma Attending Aristide Le Dantec University Hospital.

Mycetoma can be caused either by fungi or aerobic Actinomycetes. A precise identification of the causal agents is critical for the therapeutic outcome. Thus, this study aimed to identify the pathogens of mycetoma using 16S/ITS rRNA gene polymerase chain reaction (PCR) followed by Sanger sequencing directly on grains. In sum, 32 samples including 15 black grains, 12 red grains, and five white/yellow grains collected from patients with mycetoma at the Aristide Le Dantec University Hospital in Dakar, Senegal, between October 2014 and September 2020 were submitted to PCR/sequencing. For black grain eumycetoma, the ITS rRNA region was targeted. Similarly, the 16S rRNA gene was targeted for red grain actinomycetoma. These two regions were targeted in parallel for white/yellow grains, which could be of either bacterial or fungal origin. The age of the patients ranged from 14 to 72 years with a mean age of 36 ± 14 years. Thirteen (86%) of the 15 samples with black grains, were successfully sequenced with only one established eumycetoma pathogen, Madurella mycetomatis identified in 11 (73%). Cladosporium sphaerospermum was identified in one sample. For the 16S rRNA sequencing of red grains, a 58.3% (7/12) success rate was obtained with Actinomadura pelletieri identified in six samples. Among the five samples sequenced twice, the 16S rRNA allowed us to identify the causative agent in 2 cases, A. madurae in one, and A. geliboluensis in the other. The ITS rRNA identified 3 fungi, of which none was a mycetoma agent. Overall, direct 16S/ITS rRNA sequencing of the grains for detecting and identifying mycetoma pathogens was successful in 59.4% of cases. Fungi, led by M. mycetomatis, were the predominant pathogens identified. Two probable new mycetoma agents, C. sphaerospermum, and A. geliboluensis were identified and both deserve to be confirmed in further studies.

Two decades of molecular surveillance in Senegal reveal rapid changes in known drug resistance mutations over time.

BACKGROUND: Drug resistance in Plasmodium falciparum is a major threat to malaria control efforts. Pathogen genomic surveillance could be invaluable for monitoring current and emerging parasite drug resistance. METHODS: Data from two decades (2000-2020) of continuous molecular surveillance of P. falciparum parasites from Senegal were retrospectively examined to assess historical changes in malaria drug resistance mutations. Several known drug resistance markers and their surrounding haplotypes were profiled using a combination of single nucleotide polymorphism (SNP) molecular surveillance and whole genome sequence based population genomics. RESULTS: This dataset was used to track temporal changes in drug resistance markers whose timing correspond to historically significant events such as the withdrawal of chloroquine (CQ) and the introduction of sulfadoxine-pyrimethamine (SP) in 2003. Changes in the mutation frequency at Pfcrt K76T and Pfdhps A437G coinciding with the 2014 introduction of seasonal malaria chemoprevention (SMC) in Senegal were observed. In 2014, the frequency of Pfcrt K76T increased while the frequency of Pfdhps A437G declined. Haplotype-based analyses of Pfcrt K76T showed that this rapid increase was due to a recent selective sweep that started after 2014. DISCUSSION (CONCLUSION): The rapid increase in Pfcrt K76T is troubling and could be a sign of emerging amodiaquine (AQ) resistance in Senegal. Emerging AQ resistance may threaten the future clinical efficacy of artesunate-amodiaquine (ASAQ) and AQ-dependent SMC chemoprevention. These results highlight the potential of molecular surveillance for detecting rapid changes in parasite populations and stress the need to monitor the effectiveness of AQ as a partner drug for artemisinin-based combination therapy (ACT) and for chemoprevention.

Efficient cytosolic delivery of luminescent lanthanide bioprobes in live cells for two-photon microscopy.

Lanthanide(iii) (Ln3+) complexes have desirable photophysical properties for optical bioimaging. However, despite their advantages over organic dyes, their use for microscopy imaging is limited by the high-energy UV excitation they require and their poor ability to cross the cell membrane and reach the cytosol. Here we describe a novel family of lanthanide-based luminescent probes, termed dTAT[Ln·L], based on (i) a DOTA-like chelator with a picolinate moiety, (ii) a two-photon absorbing antenna to shift the excitation to the near infrared and (ii) a dimeric TAT cell-penetrating peptide for cytosolic delivery. Several Tb3+ and Eu3+ probes were prepared and characterized. Two-photon microscopy of live cells was attempted using a commercial microscope with the three probes showing the highest quantum yields (>0.15). A diffuse Ln3+ emission was detected in most cells, which is characteristic of cytosolic delivery of the Ln3+ complex. The cytotoxicity of these three probes was evaluated and the IC50 ranged from 7 µM to >50 µM. The addition of a single positive or negative charge to the antenna of the most cytotoxic compound was sufficient to lower significantly or suppress its toxicity under the conditions used for two-photon microscopy. Therefore, the design reported here provides excellent lanthanide-based probes for two-photon microscopy of living cells.

Two mosquito salivary antigens demonstrate promise as biomarkers of recent exposure to P. falciparum infected mosquito bites.

Background: Measuring malaria transmission intensity using the traditional entomological inoculation rate is difficult. Antibody responses to mosquito salivary proteins such as SG6 have previously been used as biomarkers of exposure to Anopheles mosquito bites. Here, we investigate four mosquito salivary proteins as potential biomarkers of human exposure to mosquitoes infected with P. falciparum: mosGILT, SAMSP1, AgSAP, and AgTRIO. Methods: We tested population-level human immune responses in longitudinal and cross-sectional plasma samples from individuals with known P. falciparum infection from low and moderate transmission areas in Senegal using a multiplexed magnetic bead-based assay. Results: AgSAP and AgTRIO were the best indicators of recent exposure to infected mosquitoes. Antibody responses to AgSAP, in a moderate endemic area, and to AgTRIO in both low and moderate endemic areas, were significantly higher than responses in a healthy non-endemic control cohort (p-values = 0.0245, 0.0064, and <0.0001 respectively). No antibody responses significantly differed between the low and moderate transmission area, or between equivalent groups during and outside the malaria transmission seasons. For AgSAP and AgTRIO, reactivity peaked 2-4 weeks after clinical P. falciparum infection and declined 3 months after infection. Discussion: Reactivity to both AgSAP and AgTRIO peaked after infection and did not differ seasonally nor between areas of low and moderate transmission, suggesting reactivity is likely reflective of exposure to infectious mosquitos or recent biting rather than general mosquito exposure. Kinetics suggest reactivity is relatively short-lived. AgSAP and AgTRIO are promising candidates to incorporate into multiplexed assays for serosurveillance of population-level changes in P. falciparum-infected mosquito exposure.

Evidence of Plasmodium vivax circulation in western and eastern regions of Senegal: implications for malaria control.

BACKGROUND: Malaria elimination in Senegal requires accurate diagnosis of all Plasmodium species. Plasmodium falciparum is the most prevalent species in Senegal, although Plasmodium malariae, Plasmodium ovale, and recently Plasmodium vivax have also been reported. Nonetheless, most malaria control tools, such as Histidine Rich Protein 2 rapid diagnosis test (PfHRP2-RDT,) can only diagnose P. falciparum. Thus, PfHRP2-RDT misses non-falciparum species and P. falciparum infections that fall below the limit of detection. These limitations can be addressed using highly sensitive Next Generation Sequencing (NGS). This study assesses the burden of the four different Plasmodium species in western and eastern regions of Senegal using targeted PCR amplicon sequencing. METHODS: Three thousand samples from symptomatic and asymptomatic individuals in 2021 from three sites in Senegal (Sessene, Diourbel region; Parcelles Assainies, Kaolack region; Gabou, Tambacounda region) were collected. All samples were tested using PfHRP2-RDT and photoinduced electron transfer polymerase chain reaction (PET-PCR), which detects all Plasmodium species. Targeted sequencing of the nuclear 18S rRNA and the mitochondrial cytochrome B genes was performed on PET-PCR positive samples. RESULTS: Malaria prevalence by PfHRP2-RDT showed 9.4% (94/1000) and 0.2% (2/1000) in Diourbel (DBL) and Kaolack (KL), respectively. In Tambacounda (TAM) patients who had malaria symptoms and had a negative PfHRP2-RDT were enrolled. The PET-PCR had a positivity rate of 23.5% (295/1255) overall. The PET-PCR positivity rate was 37.6%, 12.3%, and 22.8% in Diourbel, Kaolack, and Tambacounda, respectively. Successful sequencing of 121/295 positive samples detected P. falciparum (93%), P. vivax (2.6%), P. malariae (4.4%), and P. ovale wallikeri (0.9%). Plasmodium vivax was co-identified with P. falciparum in thirteen samples. Sequencing also detected two PfHRP2-RDT-negative mono-infections of P. vivax in Tambacounda and Kaolack. CONCLUSION: The findings demonstrate the circulation of P. vivax in western and eastern Senegal, highlighting the need for improved malaria control strategies and accurate diagnostic tools to better understand the prevalence of non-falciparum species countrywide.

Evaluating the performance of Plasmodium falciparum genetic metrics for inferring National Malaria Control Programme reported incidence in Senegal.

BACKGROUND: Genetic surveillance of the Plasmodium falciparum parasite shows great promise for helping National Malaria Control Programmes (NMCPs) assess parasite transmission. Genetic metrics such as the frequency of polygenomic (multiple strain) infections, genetic clones, and the complexity of infection (COI, number of strains per infection) are correlated with transmission intensity. However, despite these correlations, it is unclear whether genetic metrics alone are sufficient to estimate clinical incidence. METHODS: This study examined parasites from 3147 clinical infections sampled between the years 2012-2020 through passive case detection (PCD) across 16 clinic sites spread throughout Senegal. Samples were genotyped with a 24 single nucleotide polymorphism (SNP) molecular barcode that detects parasite strains, distinguishes polygenomic (multiple strain) from monogenomic (single strain) infections, and identifies clonal infections. To determine whether genetic signals can predict incidence, a series of Poisson generalized linear mixed-effects models were constructed to predict the incidence level at each clinical site from a set of genetic metrics designed to measure parasite clonality, superinfection, and co-transmission rates. RESULTS: Model-predicted incidence was compared with the reported standard incidence data determined by the NMCP for each clinic and found that parasite genetic metrics generally correlated with reported incidence, with departures from expected values at very low annual incidence (< 10/1000/annual [‰]). CONCLUSIONS: When transmission is greater than 10 cases per 1000 annual parasite incidence (annual incidence > 10‰), parasite genetics can be used to accurately infer incidence and is consistent with superinfection-based hypotheses of malaria transmission. When transmission was < 10‰, many of the correlations between parasite genetics and incidence were reversed, which may reflect the disproportionate impact of importation and focal transmission on parasite genetics when local transmission levels are low.

Quality assessment of malaria microscopic diagnosis at the Aristide Le Dantec University Hospital of Dakar, Senegal, in 2020.

BACKGROUND: Following WHO guidelines, microscopy is the gold standard for malaria diagnosis in endemic countries. The Parasitology-Mycology laboratory (LPM) is the National Reference Laboratory and is currently undergoing ISO 15189 accreditation. In this context, we assessed the performance of the laboratory by confirming the reliability and the accuracy of results obtained in accordance with the requirements of the ISO 15189 standards. This study aimed to verify the method of microscopic diagnosis of malaria at the LPM, in the Aristide Le Dantec hospital (HALD) in Dakar, Senegal. METHODS: This is a validation/verification study conducted from June to August 2020. Twenty (20) microscopic slides of thick/thin blood smear with known parasite densities (PD) selected from the Cheick Anta Diop University malaria slide bank in Dakar were used for this assessment. Six (6) were used to assess microscopists' ability to determine PD and fourteen (14) slides were used for detection (positive vs negative) and identification of parasites. Four (4) LPM-HALD microscopists read and recorded their results on prepared sheets. Data analysis was done with Microsoft Excel 2010 software. RESULTS: A minimum threshold of 50% concordance was used for comparison. Of the twenty (20) slides read, 100% concordance was obtained on eight (8) detection (positive vs negative) slides. Four (4) out of the six (6) parasite density evaluation slides obtained a concordance of less than 50%. Thirteen (13) out of the fourteen (14) identification slides obtained a concordance greater than 50%. Only one (1) identification slide obtained zero agreement from the microscopists. For species identification a concordance greater than 80% was noted and the microscopists obtained scores between 0.20 and 0.4 on a scale of 0 to 1 for parasite density reading. The microscopists obtained 100% precision, sensitivity, specificity and both negative and positive predictive values. CONCLUSION: This work demonstrated that the microscopic method of malaria diagnosis used in the LPM/HALD is in accordance with the requirements of WHO and ISO 15189. Further training of microscopists may be needed to maintain competency.

Investigating the etiologies of non-malarial febrile illness in Senegal using metagenomic sequencing.

The worldwide decline in malaria incidence is revealing the extensive burden of non-malarial febrile illness (NMFI), which remains poorly understood and difficult to diagnose. To characterize NMFI in Senegal, we collected venous blood and clinical metadata in a cross-sectional study of febrile patients and healthy controls in a low malaria burden area. Using 16S and untargeted sequencing, we detected viral, bacterial, or eukaryotic pathogens in 23% (38/163) of NMFI cases. Bacteria were the most common, with relapsing fever Borrelia and spotted fever Rickettsia found in 15.5% and 3.8% of cases, respectively. Four viral pathogens were found in a total of 7 febrile cases (3.5%). Sequencing also detected undiagnosed Plasmodium, including one putative P. ovale infection. We developed a logistic regression model that can distinguish Borrelia from NMFIs with similar presentation based on symptoms and vital signs (F1 score: 0.823). These results highlight the challenge and importance of improved diagnostics, especially for Borrelia, to support diagnosis and surveillance.

SEROPREVALENCE TO SCHISTOSOMA SOLUBLE EGG ANTIGEN AMONG NOMADIC PASTORALISTS RESIDING IN NORTHERN SENEGAL.

Urinary and intestinal schistosomiasis are endemic in Senegal, with prevalence heterogeneous throughout the country. Because of their way of life, nomadic pastoralists are not typically included in epidemiological surveys, and data on the prevalence of schistosomiasis in Senegalese nomadic populations are largely non-existent. The purpose of this study was to determine the seroprevalence of schistosomiasis in Senegalese nomadic pastoralists. A modified snowball sampling survey was conducted among 1,467 nomadic pastoralists aged 6 mo and older in 5 districts in northern Senegal. Dried blood spots from participants of all ages and data regarding demographics were collected to assess IgG antibody responses against Schistosoma mansoni soluble egg antigen (SEA) using a bead-based multiplex assay. Out of 1,467 study subjects, 1,464 (99.8%) provided IgG serological data that cleared quality assurance. Of the participants with appropriate data, 56.6% were male, the median age was 22 yr, and 31.6% were under 15 yr of age. The overall anti-SEA IgG seroprevalence was 19.1% (95% confidence interval [CI]: 17.1-21.1%) with the highest estimates observed in Dagana (35.9%) and the lowest observed in Podor nomadic groups (3.4%). Antibody responses increased significantly with age except for the oldest age groups (>40 yr of age), which saw lower levels of antibody response compared to younger adults. When controlling for age and location by multivariate regression, the male sex was associated with a 2-fold greater odds of anti-SEA IgG seropositivity (aPOR: 2.0; 95% CI: 1.5-2.7). Serosurveys for anti-SEA IgG among nomadic peoples in northern Senegal found a substantial percentage of individuals with evidence for current or previous Schistosoma spp. infection with the highest levels of exposure in the district adjacent to the Diama dam along the Senegal River. With IgG prevalence increased by age except in the older adults, and the male sex significantly associated with seropositivity, these data point toward sex-associated behavioral practices and human environmental modification as risk factors for Schistosoma exposure.

Evaluating the performance of Plasmodium falciparum genetics for inferring National Malaria Control Program reported incidence in Senegal.

Genetic surveillance of the Plasmodium falciparum parasite shows great promise for helping National Malaria Control Programs (NMCPs) assess parasite transmission. Genetic metrics such as the frequency of polygenomic (multiple strain) infections, genetic clones, and the complexity of infection (COI, number of strains per infection) are correlated with transmission intensity. However, despite these correlations, it is unclear whether genetic metrics alone are sufficient to estimate clinical incidence. Here, we examined parasites from 3,147 clinical infections sampled between the years 2012-2020 through passive case detection (PCD) across 16 clinic sites spread throughout Senegal. Samples were genotyped with a 24 single nucleotide polymorphism (SNP) molecular barcode that detects parasite strains, distinguishes polygenomic (multiple strain) from monogenomic (single strain) infections, and identifies clonal infections. To determine whether genetic signals can predict incidence, we constructed a series of Poisson generalized linear mixed-effects models to predict the incidence level at each clinical site from a set of genetic metrics designed to measure parasite clonality, superinfection, and co-transmission rates. We compared the model-predicted incidence with the reported standard incidence data determined by the NMCP for each clinic and found that parasite genetic metrics generally correlated with reported incidence, with departures from expected values at very low annual incidence (<10/1000/annual [‰]). When transmission is greater than 10 cases per 1000 annual parasite incidence (annual incidence >10 ‰), parasite genetics can be used to accurately infer incidence and is consistent with superinfection-based hypotheses of malaria transmission. When transmission was <10 ‰, we found that many of the correlations between parasite genetics and incidence were reversed, which we hypothesize reflects the disproportionate impact of importation and focal transmission on parasite genetics when local transmission levels are low.

Malaria surveillance reveals parasite relatedness, signatures of selection, and correlates of transmission across Senegal.

We here analyze data from the first year of an ongoing nationwide program of genetic surveillance of Plasmodium falciparum parasites in Senegal. The analysis is based on 1097 samples collected at health facilities during passive malaria case detection in 2019; it provides a baseline for analyzing parasite genetic metrics as they vary over time and geographic space. The study's goal was to identify genetic metrics that were informative about transmission intensity and other aspects of transmission dynamics, focusing on measures of genetic relatedness between parasites. We found the best genetic proxy for local malaria incidence to be the proportion of polygenomic infections (those with multiple genetically distinct parasites), although this relationship broke down at low incidence. The proportion of related parasites was less correlated with incidence while local genetic diversity was uninformative. The type of relatedness could discriminate local transmission patterns: two nearby areas had similarly high fractions of relatives, but one was dominated by clones and the other by outcrossed relatives. Throughout Senegal, 58% of related parasites belonged to a single network of relatives, within which parasites were enriched for shared haplotypes at known and suspected drug resistance loci and at one novel locus, reflective of ongoing selection pressure.

Improving diagnosis of non-malarial fevers in Senegal: Borrelia and the contribution of tick-borne bacteria.

The worldwide decline in malaria incidence is revealing the extensive burden of non-malarial febrile illness (NMFI), which remains poorly understood and difficult to diagnose. To characterize NMFI in Senegal, we collected venous blood and clinical metadata from febrile patients and healthy controls in a low malaria burden area. Using 16S and unbiased sequencing, we detected viral, bacterial, or eukaryotic pathogens in 29% of NMFI cases. Bacteria were the most common, with relapsing fever Borrelia and spotted fever Rickettsia found in 15% and 3.7% of cases, respectively. Four viral pathogens were found in a total of 7 febrile cases (3.5%). Sequencing also detected undiagnosed Plasmodium, including one putative P. ovale infection. We developed a logistic regression model to distinguish Borrelia from NMFIs with similar presentation based on symptoms and vital signs. These results highlight the challenge and importance of improved diagnostics, especially for Borrelia, to support diagnosis and surveillance.

Structural Variations in Carboxylated Bispidine Ligands: Influence of Positional Isomerism and Rigidity on the Conformation, Stability, Inertness and Relaxivity of their Mn2+ Complexes.

Mn2+ complexes of 2,4-pyridyl-disubstituted bispidine ligands have emerged as more biocompatible alternatives to Gd3+ -based MRI probes. They display relaxivities comparable to that of commercial contrast agents and high kinetic inertness, unprecedented for Mn2+ complexes. The chemical structure, in particular the substituents on the two macrocyclic nitrogens N3 and N7, are decisive for the conformation of the Mn2+ complexes, and this will in turn determine their thermodynamic, kinetic and relaxation properties. We describe the synthesis of four ligands with acetate substituents in positions N3, N7 or both. We evidence that the bispidine conformation is dependent on N3 substitution, with direct impact on the thermodynamic stability, kinetic inertness, hydration state and relaxivity of the Mn2+ complexes. These results unambiguously show that (i) solely a chair-chair conformation allows for favorable inertness and relaxivity, and (ii) in this family such chair-chair conformation is accessible only for ligands without N3-appended carboxylates.

Simultaneous seroprevalence of Toxoplasma gondii and rubella virus infections in pregnant women in Dakar (Senegal).

Context: Toxoplasma gondii and rubella virus are microorganisms that can cause intrauterine infections and congenital anomalies in the fetus. Data regarding the simultaneous seroprevalence of these infections are not available in Senegal. Aims: This study aimed to determine for the first time the simultaneous seroprevalence of toxoplasmosis and rubella among pregnant women in Dakar. Materials and Methods: In this retrospective study, anti-Toxoplasma and anti-rubella antibodies were analyzed in the serum samples obtained from pregnant women receiving prenatal care at Military Hospital of Ouakam between 2016 and 2021 using a chemiluminescent microparticle immunoassay for the quantitative determination of immunoglobulin G (IgG) and IgM antibodies to Toxoplasma gondii and rubella in human serum. Results: Overall, data from 2589 women were analyzed. The median age was 29 years (interquartile range: 23.14-34.86). Serum IgG and IgM were positive for T. gondii with 35.84% and 1.66%, respectively. Rubella seroprevalence was 87.14% and 0.35%, respectively, for IgG and IgM. Seroprevalence of toxoplasmosis increases significantly with age and study period. For rubella infection, the highest seroprevalence rates were noted in the youngest age group and at the end of the study period. Conclusions: Data from this first-time study regarding simultaneous seroprevalence of toxoplasmosis and rubella among pregnant women in Senegal indicate a continuing high risk of congenital toxoplasmosis and congenital rubella syndrome in Dakar. Further studies are needed to fully assess the efficacy of rubella vaccination in women of childbearing age.

Evaluation of knowledge and experience of fungal infections (mycoses) among clinical doctors in Senegal.

In order to assess the knowledge and experience of fungal infections (FIs) among clinicians in Senegal, a cross-sectional survey was carried out among medical practitioners in Senegal via a questionnaire designed with "Google Forms" between 24 January and 24 April 2022. A total of 100 clinicians responded to the questionnaire. Clinicians in the 31- 40-year-old age group formed the majority of respondents (51%). Male respondents were predominant (72%). Forty-one percent of respondents were general practitioners, 40% were specialist doctors, and the rest were residents. Dermatologists were the most common at 15% (6/40). In terms of clinicians' general knowledge of fungi, FIs and their therapeutic management, an average of 70% correct answers was recorded. The majority (70%) of respondents cared for between two to four different categories of patients at risk of invasive FIs (IFIs) at a time, with diabetes predominating. Eighty percent confirmed that they had been confronted with FIs, including 43% with superficial FIs, 3% with subcutaneous FIs and 5% with IFIs. Thirty-four percent of doctors stated that they had never suspected an IFI. Candidiasis was the most commonly mentioned mycosis by doctors. To support the diagnosis of these FIs, 22% of the clinicians said that they had recourse only to the clinical diagnosis. In total, 79% of clinicians responded that they had never used an antifungal chemoprophylaxis. In addition, 28% and 22% of practicing physicians chose a combination of antifungals for the chemoprophylaxis of invasive candidiasis and invasive aspergillosis, respectively. This survey shows that both clinicians' knowledge and experience of fungi, antifungals, FIs and their therapeutic management, as well as chemoprophylaxis, need to be improved. Indeed, half of the clinicians seem to be unaware of the incidence of FIs, in particular IFIs, which, nevertheless, represent some of the deadliest infectious diseases in the world.

Two decades of molecular surveillance in Senegal reveal changes in known drug resistance mutations associated with historical drug use and seasonal malaria chemoprevention.

Drug resistance in Plasmodium falciparum is a major threat to malaria control efforts. We analyzed data from two decades (2000-2020) of continuous molecular surveillance of P. falciparum parasite strains in Senegal to determine how historical changes in drug administration policy may have affected parasite evolution. We profiled several known drug resistance markers and their surrounding haplotypes using a combination of single nucleotide polymorphism (SNP) molecular surveillance and whole-genome sequence (WGS) based population genomics. We observed rapid changes in drug resistance markers associated with the withdrawal of chloroquine and introduction of sulfadoxine-pyrimethamine in 2003. We also observed a rapid increase in Pfcrt K76T and decline in Pfdhps A437G starting in 2014, which we hypothesize may reflect changes in resistance or fitness caused by seasonal malaria chemoprevention (SMC). Parasite populations evolve rapidly in response to drug use, and SMC preventive efficacy should be closely monitored.

A seven-coordinate Mn(II) complex with a pyridine-based 15-membered macrocyclic ligand containing one acetate pendant arm: structure, stability and relaxation properties.

A new 15-membered pyridine-based macrocyclic ligand containing one acetate pendant arm (N-carboxymethyl-3,12,18-triaza-6,9-dioxabicyclo[12.3.1]octadeca-1(18),14,16-triene, L1) was synthesized and its Mn(II) complex MnL1 was investigated in the context of MRI contrast agent development. The X-ray molecular structure of MnL1 confirmed a coordination number of seven with an axially compressed pentagonal bipyramidal geometry and one coordination site available for an inner-sphere water molecule. The protonation constants of L1 and the stability constants of Mn(II), Zn(II), Cu(II) and Ca(II) complexes were determined by potentiometry, and revealed higher thermodynamic stabilities in comparison with complexes of 15-pyN3O2, the parent macrocycle without an acetate pendant arm. The MnL1 complex is fully formed at physiological pH 7.4, but it shows fast dissociation kinetics, as followed by relaxometry in the presence of an excess of Zn(II). The short dissociation half-life estimated for physiological pH (ca. 3 minutes) is related to fast spontaneous dissociation of the non-protonated complex. At lower pH values, the proton-assisted dissociation pathway becomes important, while the Zn(II) concentration has no effect on the dissociation rate. 17O NMR and 1H NMRD data indicated the presence of one inner-sphere water molecule with a rather slow exchange (k298ex = 4.5 × 106 s-1) and provided information about other microscopic parameters governing relaxation. The relaxivity (r1 = 2.45 mM-1 s-1 at 20 MHz, 25 °C) corresponds to typical values for monohydrated Mn(II) chelates. Overall, the acetate pendant arm in L1 has a beneficial effect with respect to 15-pyN3O2 in increasing the thermodynamic stability and kinetic inertness of its Mn(II) complex, but leads to a reduced number of inner-sphere water molecules and thus lower relaxivity.

Ex vivo RSA and pfkelch13 targeted-amplicon deep sequencing reveal parasites susceptibility to artemisinin in Senegal, 2017.

BACKGROUND: Malaria control is highly dependent on the effectiveness of artemisinin-based combination therapy (ACT), the current frontline malaria curative treatment. Unfortunately, the emergence and spread of parasites resistant to artemisinin (ART) derivatives in Southeast Asia and South America, and more recently in Rwanda and Uganda (East Africa), compromise their long-term use in sub-Saharan Africa, where most malaria deaths occur. METHODS: Here, ex vivo susceptibility to dihydroartemisinin (DHA) was evaluated from 38 Plasmodium falciparum isolates collected in 2017 in Thiès (Senegal) expressed in the Ring-stage Survival Assay (RSA). Both major and minor variants were explored in the three conserved-encoding domains of the pfkelch13 gene, the main determinant of ART resistance using a targeted-amplicon deep sequencing (TADS) approach. RESULTS: All samples tested in the ex vivo RSA were found to be susceptible to DHA (parasite survival rate < 1%). The non-synonymous mutations K189T and K248R in pfkelch13 were observed each in one isolate, as major (99%) or minor (5%) variants, respectively. CONCLUSION: The results suggest that ART is still fully effective in the Thiès region of Senegal in 2017. Investigations combining ex vivo RSA and TADS are a useful approach for monitoring ART resistance in Africa.

Malaria surveillance reveals parasite relatedness, signatures of selection, and correlates of transmission across Senegal.

Parasite genetic surveillance has the potential to play an important role in malaria control. We describe here an analysis of data from the first year of an ongoing, nationwide program of genetic surveillance of Plasmodium falciparum parasites in Senegal, intended to provide actionable information for malaria control efforts. Looking for a good proxy for local malaria incidence, we found that the best predictor was the proportion of polygenomic infections (those with multiple genetically distinct parasites), although that relationship broke down in very low incidence settings (r = 0.77 overall). The proportion of closely related parasites in a site was more weakly correlated ( r = -0.44) with incidence while the local genetic diversity was uninformative. Study of related parasites indicated their potential for discriminating local transmission patterns: two nearby study areas had similarly high fractions of relatives, but one area was dominated by clones and the other by outcrossed relatives. Throughout the country, 58% of related parasites proved to belong to a single network of relatives, within which parasites were enriched for shared haplotypes at known and suspected drug resistance loci as well as at one novel locus, reflective of ongoing selection pressure.