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.

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.

Factors associated with diabetes knowledge, attitudes and practices among people aged 18 and over in the commune of Niakhene in Senegal.

More than 422 million people worldwide have diabetes in 2016, and 1.6 million deaths are attributed to diabetes each year. Knowledge of preventive measures would enable the adjustment of preventive policies. Hence this study on knowledge and practices in rural Senegal. This was a cross-sectional, descriptive and analytical survey of subjects aged at least 18 and living in the commune of Niakhene, carried out in October 2020. A systematic random sample, stratified by sex and age group, was used. The questionnaire was based on the STEPS 2015 tool and a review of the literature. In addition to personal characteristics, the questionnaire was used to measure knowledge of symptoms, complications, risk factors, attitude to the disease and screening practices. Descriptive and analytical analyses were performed using R 4.0.2 software. A total of 300 subjects were surveyed. The average age was 35.3 years (+/-16.9), and 52.3% were women. Knowledge (62.7%) was associated with higher education (ORaj2.46{1.16-3.44}), awareness by healthcare staff (ORaj2.88{1.60-5.34}), and a family history of diabetes (ORaj3.09{1.06-11.3}). The positive attitude (53%) was associated with male sex (ORaj1.98{2.07-7.52}), awareness via audio-visual information sources (ORaj3.87{2.07-7.52}), community awareness (ORaj 3.87{2.07-7.52}), existence of a family history of hypertension and knowledge of diabetes (ORaj3.34{2.5-7.69}). Screening was carried out in 34.3% of patients. The associated risk factors were male sex (ORaj 1.95{1.12-3.34}), higher education (ORaj2.49{1.12-559}) and positive attitudes to diabetes (ORaj1.83{1.04-3.26}). One of the most effective interventions against this disease is the adoption of preventive measures which involve early detection and strengthening communication for more effective prevention.

Perceptions, facilitators and barriers to the implementation of interpersonal group therapy to treat depression among people living with HIV in Senegal: a qualitative study.

Background: Depression is highly prevalent in people living with HIV (PLWH) but remains under treated in Sub-Saharan Africa. In this context, we conducted the first study of Group Interpersonal Therapy (IPT) to treat depression in PLWH in Senegal. We assessed the perceptions and experiences of patients and group facilitators, as well as barriers to implementation. Methods: This study was conducted at the Fann National University Hospital Center in Dakar, the urban capital of Senegal. Qualitative data were collected during the implementation phase (February to June 2020 and then from January to February 2021), with a 6-month pause due to the COVID-19 pandemic. Twenty-five patients and three group facilitators were individually interviewed by a socio-anthropologist. Qualitative data were analyzed thematically. Results: Group IPT was perceived as successful and beneficial by patients and facilitators. Patients reported positive experiences with group IPT and sustained outcomes. Beyond improving depressive symptoms, patients reported improvements in their social and professional lives, and the development of skills to prevent relapse. Group facilitators noted the benefits of therapy for their patients and for their professional skills, reporting greater clinical competence and improved supportive skills. Challenges to intervention implementation included confidentiality and patient privacy concerns, healthcare accessibility issues, and time demands. Conclusion: In this first qualitative study of group IPT for depression in PLWH in Senegal, participants described both positive experiences with the intervention and challenges to its implementation. Future studies, conducted in suburban and rural communities outside of Dakar, would further inform the implementation of IPT in Senegal.

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.

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.

Management of depression in people living with HIV/AIDS in Senegal: Acceptability, feasibility and benefits of group interpersonal therapy.

Depression is highly prevalent in people living with HIV (PLWH) and has negative consequences for daily life and care. We evaluated for the first time the acceptability, feasibility and benefits of group interpersonal therapy (IPT), combined with a task-shifting approach, to treat depression in PLWH in Senegal. PLWH with depression received group IPT following the World Health Organization protocol. Acceptability and feasibility criteria were defined from the literature data. The PHQ-9, the WHODAS, and the 12-item-stigma scale were used, pre- and post-treatment, including a 3-month follow-up, to assess depressive symptom severity, functioning and stigma, respectively. General linear mixed models were used to describe changes in outcomes over time. Of 69 participants, 60 completed group IPT. Refusal to enroll and dropout rates were 6.6 and 12.7%, respectively. Ninety-seven percent of participants attended at least seven out of eight sessions. Patients and facilitators endorsed group IPT, with willingness to recommend it. Depressive symptoms and disability improved drastically and sustainably. We showed that group IPT is well accepted and feasible in Senegal as treatment for depression in PLWH. Combined with a task-shifting approach, it can narrow the gap in mental health treatment. Implementation may be enhanced by refining patient identification procedures and increasing treatment accessibility.

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.

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.

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.

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.

Ex vivo RSA and Pfkelch13 targeted-amplicon deep sequencing reveal parasites susceptibility to artemisinin in Senegal, 2017.

INTRODUCTION: Malaria control is highly dependent on the effectiveness of artemisinin-based combination therapies (ACTs), the current frontline malaria curative treatments. 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, we evaluated ex vivo susceptibility to dihydroartemisinin (DHA) from 38 P. falciparum isolates collected in 2017 in Thiès (Senegal) expressed with the Ring-stage Survival Assay (RSA). We explored major and minor variants in the full 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. Both non-synonymous mutations K189T and K248R were observed each in one isolate, as major (99%) or minor (5%) variants, respectively. CONCLUSION: Altogether, investigations combining ex vivo RSA and TADS are a useful approach for monitoring ART resistance in Africa.

Plasmodium infection is associated with cross-reactive antibodies to carbohydrate epitopes on the SARS-CoV-2 Spike protein.

Sero-surveillance can monitor and project disease burden and risk. However, SARS-CoV-2 antibody test results can produce false positive results, limiting their efficacy as a sero-surveillance tool. False positive SARS-CoV-2 antibody results are associated with malaria exposure, and understanding this association is essential to interpret sero-surveillance results from malaria-endemic countries. Here, pre-pandemic samples from eight malaria endemic and non-endemic countries and four continents were tested by ELISA to measure SARS-CoV-2 Spike S1 subunit reactivity. Individuals with acute malaria infection generated substantial SARS-CoV-2 reactivity. Cross-reactivity was not associated with reactivity to other human coronaviruses or other SARS-CoV-2 proteins, as measured by peptide and protein arrays. ELISAs with deglycosylated and desialated Spike S1 subunits revealed that cross-reactive antibodies target sialic acid on N-linked glycans of the Spike protein. The functional activity of cross-reactive antibodies measured by neutralization assays showed that cross-reactive antibodies did not neutralize SARS-CoV-2 in vitro. Since routine use of glycosylated or sialated assays could result in false positive SARS-CoV-2 antibody results in malaria endemic regions, which could overestimate exposure and population-level immunity, we explored methods to increase specificity by reducing cross-reactivity. Overestimating population-level exposure to SARS-CoV-2 could lead to underestimates of risk of continued COVID-19 transmission in sub-Saharan Africa.

Plasmodium falciparum genomic surveillance reveals spatial and temporal trends, association of genetic and physical distance, and household clustering.

Molecular epidemiology using genomic data can help identify relationships between malaria parasite population structure, malaria transmission intensity, and ultimately help generate actionable data to assess the effectiveness of malaria control strategies. Genomic data, coupled with geographic information systems data, can further identify clusters or hotspots of malaria transmission, parasite genetic and spatial connectivity, and parasite movement by human or mosquito mobility over time and space. In this study, we performed longitudinal genomic surveillance in a cohort of 70 participants over four years from different neighborhoods and households in Thiès, Senegal-a region of exceptionally low malaria transmission (entomological inoculation rate less than 1). Genetic identity (identity by state, IBS) was established using a 24-single nucleotide polymorphism molecular barcode, identity by descent was calculated from whole genome sequence data, and a hierarchical Bayesian regression model was used to establish genetic and spatial relationships. Our results show clustering of genetically similar parasites within households and a decline in genetic similarity of parasites with increasing distance. One household showed extremely high diversity and warrants further investigation as to the source of these diverse genetic types. This study illustrates the utility of genomic data with traditional epidemiological approaches for surveillance and detection of trends and patterns in malaria transmission not only by neighborhood but also by household. This approach can be implemented regionally and countrywide to strengthen and support malaria control and elimination efforts.

Plasmodium infection induces cross-reactive antibodies to carbohydrate epitopes on the SARS-CoV-2 Spike protein.

Individuals with acute malaria infection generated high levels of antibodies that cross-react with the SARS-CoV-2 Spike protein. Cross-reactive antibodies specifically recognized the sialic acid moiety on N-linked glycans of the Spike protein and do not neutralize in vitro SARS-CoV-2. Sero-surveillance is critical for monitoring and projecting disease burden and risk during the pandemic; however, routine use of Spike protein-based assays may overestimate SARS-CoV-2 exposure and population-level immunity in malaria-endemic countries.

Genomic investigation of a dengue virus outbreak in Thiès, Senegal, in 2018.

Dengue virus is a major and rapidly growing public health concern in tropic and subtropic regions across the globe. In late 2018, Senegal experienced its largest dengue virus outbreak to date, covering several regions. However, little is known about the genetic diversity of dengue virus (DENV) in Senegal. Here we report complete viral genomes from 17 previously undetected DENV cases from the city of Thiès. In total we identified 19 cases of DENV in a cohort of 198 individuals with fever collected in October and November 2018. We detected 3 co-circulating serotypes; DENV 3 was the most frequent accounting for 11/17 sequences (65%), 4 (23%) were DENV2 and 2 (12%) were DENV1. Sequences were most similar to recent sequences from West Africa, suggesting ongoing local circulation of viral populations; however, detailed inference is limited by the scarcity of available genomic data. We did not find clear associations with reported clinical signs or symptoms, highlighting the importance of testing for diagnosing febrile diseases. Overall, these findings expand the known range of DENV in Senegal, and underscore the need for better genomic characterization of DENV in West Africa.

Genomic investigation of atypical malaria cases in Kanel, northern Senegal.

BACKGROUND: The diagnosis of malaria cases in regions where the malaria burden has decreased significantly and prevalence is very low is more challenging, in part because of reduced clinical presumption of malaria. The appearance of a cluster of malaria cases with atypical symptoms in Mbounguiel, a village in northern Senegal where malaria transmission is low, in September 2018 exemplifies this scenario. The collaboration between the National Malaria Control Programme (NMCP) at the Senegal Ministry of Health and the Laboratory of Parasitology and Mycology at Cheikh Anta Diop University worked together to evaluate this cluster of malaria cases using molecular and serological tools. METHODS: Malaria cases were diagnosed primarily by rapid diagnostic test (RDT), and confirmed by photo-induced electron transfer-polymerase chain reaction (PET-PCR). 24 single nucleotide polymorphisms (SNPs) barcoding was used for Plasmodium falciparum genotyping. Unbiased metagenomic sequencing and Luminex-based multi-pathogen antibody and antigen profiling were used to assess exposure to other pathogens. RESULTS: Nine patients, of 15 suspected cases, were evaluated, and all nine samples were found to be positive for P. falciparum only. The 24 SNPs molecular barcode showed the predominance of polygenomic infections, with identifiable strains being different from one another. All patients tested positive for the P. falciparum antigens. No other pathogenic infection was detected by either the serological panel or metagenomic sequencing. CONCLUSIONS: This work, undertaken locally within Senegal as a collaboration between the NMCP and a research laboratory at University of Cheikh Anta Diop (UCAD) revealed that a cluster of malaria cases were caused by different strains of P. falciparum. The public health response in real time demonstrates the value of local molecular and genomics capacity in affected countries for disease control and elimination.

Molecular epidemiology of Plasmodium falciparum by multiplexed amplicon deep sequencing in Senegal.

BACKGROUND: Molecular epidemiology can provide important information regarding the genetic diversity and transmission of Plasmodium falciparum, which can assist in designing and monitoring elimination efforts. However, malaria molecular epidemiology including understanding the genetic diversity of the parasite and performing molecular surveillance of transmission has been poorly documented in Senegal. Next Generation Sequencing (NGS) offers a practical, fast and high-throughput approach to understand malaria population genetics. This study aims to unravel the population structure of P. falciparum and to estimate the allelic diversity, multiplicity of infection (MOI), and evolutionary patterns of the malaria parasite using the NGS platform. METHODS: Multiplex amplicon deep sequencing of merozoite surface protein 1 (PfMSP1) and merozoite surface protein 2 (PfMSP2) in fifty-three P. falciparum isolates from two epidemiologically different areas in the South and North of Senegal, was carried out. RESULTS: A total of 76 Pfmsp1 and 116 Pfmsp2 clones were identified and 135 different alleles were found, 56 and 79 belonged to the pfmsp1 and pfmsp2 genes, respectively. K1 and IC3D7 allelic families were most predominant in both sites. The local haplotype diversity (Hd) and nucleotide diversity (π) were higher in the South than in the North for both genes. For pfmsp1, a high positive Tajima's D (TD) value was observed in the South (D = 2.0453) while negative TD value was recorded in the North (D = - 1.46045) and F-Statistic (Fst) was 0.19505. For pfmsp2, non-directional selection was found with a highly positive TD test in both areas and Fst was 0.02111. The mean MOI for both genes was 3.07 and 1.76 for the South and the North, respectively, with a statistically significant difference between areas (p = 0.001). CONCLUSION: This study revealed a high genetic diversity of pfmsp1 and pfmsp2 genes and low genetic differentiation in P. falciparum population in Senegal. The MOI means were significantly different between the Southern and Northern areas. Findings also showed that multiplexed amplicon deep sequencing is a useful technique to investigate genetic diversity and molecular epidemiology of P. falciparum infections.

Amplicon deep sequencing of kelch13 in Plasmodium falciparum isolates from Senegal.

BACKGROUND: In 2006, the Senegalese National Malaria Control Programme recommended artemisinin-based combination therapy (ACT) with artemether-lumefantrine as the first-line treatment for uncomplicated Plasmodium falciparum malaria. To date, multiple mutations associated with artemisinin delayed parasite clearance have been described in Southeast Asia in the Pfk13 gene, such as Y493H, R539T, I543T and C580Y. Even though ACT remains clinically and parasitologically efficacious in Senegal, the spread of resistance is possible as shown by the earlier emergence of resistance to chloroquine in Southeast Asia that subsequently spread to Africa. Therefore, surveillance of artemisinin resistance in malaria endemic regions is crucial and requires the implementation of sensitive tools, such as next-generation sequencing (NGS) which can detect novel mutations at low frequency. METHODS: Here, an amplicon sequencing approach was used to identify mutations in the Pfk13 gene in eighty-one P. falciparum isolates collected from three different regions of Senegal. RESULTS: In total, 10 SNPs around the propeller domain were identified; one synonymous SNP and nine non-synonymous SNPs, and two insertions. Three of these SNPs (T478T, A578S and V637I) were located in the propeller domain. A578S, is the most frequent mutation observed in Africa, but has not previously been reported in Senegal. A previous study has suggested that A578S could disrupt the function of the Pfk13 propeller region. CONCLUSION: As the genetic basis of possible artemisinin resistance may be distinct in Africa and Southeast Asia, further studies are necessary to assess the new SNPs reported in this study.