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.

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.

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.

Allelic diversity of MSP1 and MSP2 repeat loci correlate with levels of malaria endemicity in Senegal and Nigerian populations.

BACKGROUND: Characterizing the genetic diversity of malaria parasite populations in different endemic settings (from low to high) could be helpful in determining the effectiveness of malaria interventions. This study compared Plasmodium falciparum parasite population diversity from two sites with low (pre-elimination) and high transmission in Senegal and Nigeria, respectively. METHODS: Parasite genomic DNA was extracted from 187 dried blood spot collected from confirmed uncomplicated P. falciparum malaria infected patients in Senegal (94) and Nigeria (93). Allelic polymorphism at merozoite surface protein 1 (msp1) and merozoite surface protein- 2 (msp2) genes were assessed by nested PCR. RESULTS: The most frequent msp1 and msp2 allelic families are the K1 and IC3D7 allelotypes in both Senegal and Nigeria. Multiplicity of infection (MOI) of greater that 1 and thus complex infections was common in both study sites in Senegal (Thies:1.51/2.53; Kedougou:2.2/2.0 for msp1/2) than in Nigeria (Gbagada: 1.39/1.96; Oredo: 1.35/1.75]). The heterozygosity of msp1 gene was higher in P. falciparum isolates from Senegal (Thies: 0.62; Kedougou: 0.53) than isolates from Nigeria (Gbagada: 0.55; Oredo: 0.50). In Senegal, K1 alleles was associated with heavy than with moderate parasite density. Meanwhile, equal proportions of K1 were observed in both heavy and moderate infection types in Nigeria. The IC3D7 subtype allele of the msp2 family was the most frequent in heavily parasitaemic individuals from both countries than in the moderately infected participants. CONCLUSION: The unexpectedly low genetic diversity of infections high endemic Nigerian setting compared to the low endemic settings in Senegal is suggestive of possible epidemic outbreak in Nigeria.

High prevalence of asymptomatic Plasmodium infection in Bandafassi, South-East Senegal.

BACKGROUND: Malaria control and elimination strategies are based on levels of transmission that are usually determined by data collected from health facilities. In endemic areas, asymptomatic Plasmodium infection is thought to represent the majority of infections, though they are not diagnosed nor treated. Therefore, there might be an underestimation of the malaria reservoir, resulting in inadequate control strategies. In addition, these untreated asymptomatic Plasmodium infections maintain transmission, making it difficult or impossible to reach malaria elimination goals. Thus, the aim of this study was to determine the prevalence of asymptomatic Plasmodium infections in southeastern Senegal. METHODS: A cross sectional study was conducted among asymptomatic individuals (N = 122) living in the village of Andiel located in Bandafassi, Kédougou, which consisted of about 200 inhabitants during the malaria transmission season in late October 2019. For each individual without malaria-related symptoms and who consented to participate, a rapid diagnostic test (RDT) was performed in the field. Results were confirmed in the laboratory with photo-induced electron transfer (PET-PCR). RESULTS: Malaria prevalence was 70.3% by PET-PCR and 41.8% by RDT. During the same period, the health post of the area reported 49. 1% test positivity rate by RDT. The majority of the infected study population, 92.9%, was infected with a single species and 7.1% had two or three species of Plasmodium. Plasmodium falciparum was predominant and represented 90.2% of the infections, while 6.5% were due to Plasmodium ovale and 3.3% to Plasmodium malariae. 59.4% of children targeted for SMC (zero to ten years old) were infected. CONCLUSION: In southeastern Senegal, where the transmission is the highest, malaria control strategies should address asymptomatic Plasmodium infections at the community level. The results suggest that this area could be eligible for mass drug administration. Moreover, non-falciparum species could be more common and its prevalence should be determined countrywide.

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.

Analysis of anti-Plasmodium IgG profiles among Fulani nomadic pastoralists in northern Senegal to assess malaria exposure.

BACKGROUND: Northern Senegal is a zone of very low malaria transmission, with an annual incidence of < 5/1000 inhabitants. This area, where the Senegal National Malaria Control Programme has initiated elimination activities, hosts Fulani, nomadic, pastoralists that spend the dry season in the south where malaria incidence is higher (150-450/1000 inhabitants) and return to the north with the first rains. Previous research demonstrated parasite prevalence of < 1% in this Fulani population upon return from the south, similar to that documented in the north in cross-sectional surveys. METHODS: A modified snowball sampling survey of nomadic pastoralists was conducted in five districts in northern Senegal during September and October 2014. Demographic information and dried blood spots were collected. Multiplex bead-based assays were used to assess antibody responses to merozoite surface protein (MSP-119) antigen of the four primary Plasmodium species, as well as circumsporozoite protein (CSP) and liver stage antigen (LSA-1) of Plasmodium falciparum. RESULTS: In the five study districts, 1472 individuals were enrolled, with a median age of 22 years (range 1 to 80 years). Thirty-two percent of subjects were under 14 years and 57% were male. The overall seroprevalence of P. falciparum MSP-119, CSP and LSA-1 antibodies were 45, 12 and 5%, respectively. Plasmodium falciparum MSP-119 antibody responses increased significantly with age in all study areas, and were significantly higher among males. The highest seroprevalence to P. falciparum antigens was observed in the Kanel district (63%) and the lowest observed in Podor (28%). Low seroprevalence was observed for non-falciparum species in all the study sites: 0.4, 0.7 and 1.8%, respectively, for Plasmodium ovale, Plasmodium vivax and Plasmodium malariae MSP-1. Antibody responses to P. vivax were observed in all study sites except Kanel. CONCLUSION: Prevalence of P. falciparum MSP-119 antibodies and increases by study participant age provided data for low levels of exposure among this transient nomadic population. In addition, antibody responses to P. falciparum short half-life markers (CSP and LSA-1) and non-falciparum species were low. Further investigations are needed to understand the exposure of the Fulani population to P. vivax.

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.

Quality control of malaria microscopy reveals misdiagnosed non-falciparum species and other microscopically detectable pathogens in Senegal.

BACKGROUND: In developing countries, malaria diagnosis relies on microscopy and rapid diagnostic tests. In Senegal, national malaria control program (NMCP) regularly conducts supervisory visits in health services where malaria microscopy is performed. In this study, expert microscopists assessed the performance of laboratory technicians in malaria microscopy. METHODS: The present external quality assessment (EQA) was conducted in three different areas of malaria transmission. Participants were laboratory technicians previously trained by NMCP on malaria microscopy. Stored read slides were randomly collected for blinded re-checking by expert microscopists. At the same time a set of 8 slides (3 positive P. falciparum and 5 negative slides) were submitted to participants for proficiency testing. Microscopists performance were evaluated on the basis of the errors rates on slide reading-high false positive (HFP), high false negative (HFN), low false positive (LFP) and low false negative (LFN)-and the calculation of their sensitivities and specificities relative to expert microscopy. Data were entered and analysed using Microsoft Excel software. RESULTS: A total of 450 stored slides were collected from 17 laboratories for re-checking. Eight laboratories scored 100% of correct reading. Only one major error was recorded (HFP). Six laboratories recorded LFN results: Borrelia, P. ovale, and low parasite densities (95 and 155 p/µl) were missed. Two P. falciparum slides were misidentified as P. malariae and one P. ovale slide as P. vivax. The overall sensitivities and specificities for all participants against expert microscopists were 97.8 and 98.2% respectively; Sensitivities and specificities of hospital microscopists (96.7 and 98.9%) were statistically similar to those of health centre microscopists (98.5 and 97.8% respectively) (p = 0.3993 and p = 0.9412 respectively). Overall, a very good agreement was noted with kappa value of 0.96 (CI95% 93.4-98.6%) relative to expert microscopy. Proficiency testing showed that among the 17 participants, 11 laboratories scored 100% of correct reading. Three LFN and four LFP results were recorded respectively. The P. falciparum slide with Maurer dots was misidentified as P. ovale in 1 centre and the same slide was misread as P. vivax in another centre; No major error (HFP or HFN) was noted. CONCLUSION: EQA of malaria microscopy showed an overall good performance especially regarding P. falciparum detection. However, efforts need to be made addressing the ability to detect non-falciparum species and others endemic blood pathogens such as Borrelia. The further NMCP training sessions and evaluations should consider those aspects to expect high quality-assured capacity for malaria microscopy.

Ex vivo susceptibility and genotyping of Plasmodium falciparum isolates from Pikine, Senegal.

BACKGROUND: The monitoring of Plasmodium falciparum sensitivity to anti-malarial drugs is a necessity for effective case management of malaria. This species is characterized by a strong resistance to anti-malarial drugs. In Senegal, the first cases of chloroquine resistance were reported in the Dakar region in 1988 with nearly 7% population prevalence, reaching 47% by 1990. It is in this context that sulfadoxine-pyrimethamine temporarily replaced chloroquine as first line treatment in 2003, pending the introduction of artemisinin-based combination therapy in 2006. The purpose of this study is to assess the ex vivo sensitivity to different anti-malarial drugs of the P. falciparum population from Pikine. METHODS: Fifty-four samples were collected from patients with non-complicated malaria and aged between 2 and 20 years in the Deggo health centre in Pikine in 2014. An assay in which parasites are stained with 4', 6-di-amidino-2-phenylindole (DAPI), was used to study the ex vivo sensitivity of isolates to chloroquine, amodiaquine, piperaquine, pyrimethamine, and dihydroartemisinin. High resolution melting was used for genotyping of pfdhps, pfdhfr, pfmdr1, and pfcrt genes. RESULTS: The mean IC50s of chloroquine, amodiaquine, piperaquine, dihydroartemisinin, and pyrimethamine were, respectively, 39.44, 54.02, 15.28, 2.23, and 64.70 nM. Resistance mutations in pfdhfr gene, in codon 437 of pfdhps gene, and an absence of mutation at position 540 of pfdhps were observed. Mutations in codons K76T of pfcrt and N86Y of pfmdr1 were observed at 51 and 11% population prevalence, respectively. A relationship was found between the K76T and N86Y mutations and ex vivo resistance to chloroquine. CONCLUSION: An increase in sensitivity of isolates to chloroquine was observed. A high sensitivity to dihydroartemisinin was observed; whereas, a decrease in sensitivity to pyrimethamine was observed in the parasite population from Pikine.

High resolution melting: a useful field-deployable method to measure dhfr and dhps drug resistance in both highly and lowly endemic Plasmodium populations.

BACKGROUND: Emergence and spread of drug resistance to every anti-malarial used to date, creates an urgent need for development of sensitive, specific and field-deployable molecular tools for detection and surveillance of validated drug resistance markers. Such tools would allow early detection of mutations in resistance loci. The aim of this study was to compare common population signatures and drug resistance marker frequencies between two populations with different levels of malaria endemicity and history of anti-malarial drug use: Tanzania and Sénégal. This was accomplished by implementing a high resolution melting assay to study molecular markers of drug resistance as compared to polymerase chain reaction-restriction fragment length polymorphism (PCR/RFLP) methodology. METHODS: Fifty blood samples were collected each from a lowly malaria endemic site (Sénégal), and a highly malaria endemic site (Tanzania) from patients presenting with uncomplicated Plasmodium falciparum malaria at clinic. Data representing the DHFR were derived using both PCR-RFLP and HRM assay; while genotyping data representing the DHPS were evaluated in Senegal and Tanzania using HRM. Msp genotyping analysis was used to characterize the multiplicity of infection in both countries. RESULTS: A high prevalence of samples harbouring mutant DHFR alleles was observed in both population using both genotyping techniques. HRM was better able to detect mixed alleles compared to PCR/RFLP for DHFR codon 51 in Tanzania; and only HRM was able to detect mixed infections from Senegal. A high prevalence of mutant alleles in DHFR (codons 51, 59, 108) and DHPS (codon 437) were found among samples from Sénégal while no mutations were observed at DHPS codons 540 and 581, from both countries. Overall, the frequency of samples harbouring either a single DHFR mutation (S108N) or double mutation in DHFR (C59R/S108N) was greater in Sénégal compared to Tanzania. CONCLUSION: Here the results demonstrate that HRM is a rapid, sensitive, and field-deployable alternative technique to PCR-RFLP genotyping that is useful in populations harbouring more than one parasite genome (polygenomic infections). In this study, a high levels of resistance polymorphisms was observed in both dhfr and dhps, among samples from Tanzania and Sénégal. A routine monitoring by molecular markers can be a way to detect emergence of resistance involving a change in the treatment policy.

Non-falciparum malaria in Dakar: a confirmed case of Plasmodium ovale wallikeri infection.

BACKGROUND: Plasmodium ovale is rarely described in Senegal. A case of clinical malaria due to P. ovale wallikeri in West Central of Senegal is reported. CASE: A 34-year-old male baker in Dakar, with no significant previous medical history, was admitted to a health clinic with fever and vomiting. Fever had been lasting for 4 days with peaks every 48 h. As monospecific Plasmodium falciparum HRP-2 RDT was negative, he was treated with antibiotics. However, owing to persisting symptoms, he was referred to the emergency unit of the Youssou Mbargane Diop Hospital, Dakar, Senegal. Clinical examination found impaired general condition. All other physical examinations were normal. Laboratory tests showed anaemia (haemoglobin 11.4 g/dl), severe thrombocytopaenia (platelets 30 × 10(9)/mm(3)), leukopenia (3650/mm(3)), lymphocytopenia (650/mm(3)). Renal function was normal as indicated by creatininaemia and uraemia (11 mg/l and 0.25 g/l, respectively) and liver enzymes were slightly elevated (aspartate aminotransferase 77 UI/l and alanine aminotransferase 82 UI/l). Blood smear evaluations in Parasitology Laboratory of Aristide Le Dantec Hospital showed malaria parasites of the species P. ovale with a 0.08 % parasitaemia. Molecular confirmation was done by real time PCR targeting the 18S rRNA gene. The P. ovale infection was further analysed to species level targeting the potra gene and was identified as P. ovale wallikeri. According to the hospital's malaria treatment guidelines for severe malaria, treatment consisted of intravenous quinine at hour 0 (start of treatment) and 24 h after initial treatment, followed by artemether-lumefantrine 24 h later. A negative microscopy was noted on day 3 post-treatment and the patient reported no further symptoms. CONCLUSION: Malaria due to non-falciparum species is probably underestimated in Senegal. RDTs specific to non-falciparum species and/or pan specific RDTs should be included as tools of diagnosis to fight against malaria in Senegal. In addition, a field-deployable molecular tool such as the loop-mediated isothermal amplification can be considered as an additional useful tool to detect low malaria parasite infections and for speciation. In addition, national malaria control policies should consider other non-falciparum species in treatment guidelines, including the provision of primaquine for the treatment of relapsing parasites.

Selection of N86F184D1246 haplotype of Pfmrd1 gene by artemether-lumefantrine drug pressure on Plasmodium falciparum populations in Senegal.

BACKGROUND: The use of artemisinin as a monotherapy resulted in the emergence of artemisinin resistance in 2005 in Southeast Asia. Monitoring of artemisinin combination therapy (ACT) is critical in order to detect and prevent the spread of resistance in endemic areas. Ex vivo studies and genotyping of molecular markers of resistance can be used as part of this routine monitoring strategy. One gene that has been associated in some ACT partner drug resistance is the Plasmodium falciparum multidrug resistance protein 1 (pfmdr1) gene. The purpose of this study was to assess the drug susceptibility of P. falciparum populations from Thiès, Senegal by ex vivo assay and typing molecular markers of resistance to drug components of ACT currently used for treatment. METHODS: The ex vivo susceptibility of 170 P. falciparum isolates to chloroquine, amodiaquine, lumefantrine, artesunate, and artemether was determined using the DAPI ex vivo assay. The high resolution melting technique was used to genotype the pfmdr1 gene at codons 86, 184 and 1246. RESULTS: A significant decrease in IC50 values was observed between 2012 and 2013: from 13.84 to 6.484 for amodiaquine, 173.4 to 113.2 for lumefantrine, and 39.72 to 18.29 for chloroquine, respectively. Increase of the wild haplotype NYD and the decrease of the mutant haplotype NFD (79 and 62.26 %) was also observed. A correlation was observed between the wild type allele Y184 in pfmdr1 and higher IC50 for all drugs, except amodiaquine. CONCLUSION: This study has shown an increase in sensitivity over the span of two transmission seasons, marked by an increase in the WT alleles at pfmdr1. Continuous the monitoring of the ACT used for treatment of uncomplicated malaria will be helpful.

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.

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.

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.

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.

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.

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.