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.

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.

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.

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.

R H: a genetic metric for measuring intrahost Plasmodium falciparum relatedness and distinguishing cotransmission from superinfection.

Multiple-strain (polygenomic) infections are a ubiquitous feature of Plasmodium falciparum parasite population genetics. Under simple assumptions of superinfection, polygenomic infections are hypothesized to be the result of multiple infectious bites. As a result, polygenomic infections have been used as evidence of repeat exposure and used to derive genetic metrics associated with high transmission intensity. However, not all polygenomic infections are the result of multiple infectious bites. Some result from the transmission of multiple, genetically related strains during a single infectious bite (cotransmission). Superinfection and cotransmission represent two distinct transmission processes, and distinguishing between the two could improve inferences regarding parasite transmission intensity. Here, we describe a new metric, R H, that utilizes the correlation in allelic state (heterozygosity) within polygenomic infections to estimate the likelihood that the observed complexity resulted from either superinfection or cotransmission. R H is flexible and can be applied to any type of genetic data. As a proof of concept, we used R H to quantify polygenomic relatedness and estimate cotransmission and superinfection rates from a set of 1,758 malaria infections genotyped with a 24 single nucleotide polymorphism (SNP) molecular barcode. Contrary to expectation, we found that cotransmission was responsible for a significant fraction of 43% to 53% of the polygenomic infections collected in three distinct epidemiological regions in Senegal. The prediction that polygenomic infections frequently result from cotransmission stresses the need to incorporate estimates of relatedness within polygenomic infections to ensure the accuracy of genomic epidemiology surveillance data for informing public health activities.

Genetic surveillance for monitoring the impact of drug use on Plasmodium falciparum populations.

The use of antimalarial drugs is an effective strategy in the fight against malaria. However, selection of drug resistant parasites is a constant threat to the continued use of this approach. Antimalarial drugs are used not only to treat infections but also as part of population-level strategies to reduce malaria transmission toward elimination. While there is strong evidence that the ongoing use of antimalarial drugs increases the risk of the emergence and spread of drug-resistant parasites, it is less clear how population-level use of drug-based interventions like seasonal malaria chemoprevention (SMC) or mass drug administration (MDA) may contribute to drug resistance or loss of drug efficacy. Critical to sustained use of drug-based strategies for reducing the burden of malaria is the surveillance of population-level signals related to transmission reduction and resistance selection. Here we focus on Plasmodium falciparum and discuss the genetic signatures of a parasite population that are correlated with changes in transmission and related to drug pressure and resistance as a result of drug use. We review the evidence for MDA and SMC contributing to malaria burden reduction and drug resistance selection and examine the use and impact of these interventions in Senegal. Throughout we consider best strategies for ongoing surveillance of both population and resistance signals in the context of different parasite population parameters. Finally, we propose a roadmap for ongoing surveillance during population-level drug-based interventions to reduce the global malaria burden.

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.

Efficacy and safety of artemisinin-based combination therapy and the implications of Pfkelch13 and Pfcoronin molecular markers in treatment failure in Senegal.

In 2006, Senegal adopted artemisinin-based combination therapy (ACT) as first-line treatment in the management of uncomplicated malaria. This study aimed to update the status of antimalarial efficacy more than ten years after their first introduction. This was a randomized, three-arm, open-label study to evaluate the efficacy and safety of artemether-lumefantrine (AL), artesunate-amodiaquine (ASAQ) and dihydroartemisinin-piperaquine (DP) in Senegal. Malaria suspected patients were screened, enrolled, treated, and followed for 28 days for AL and ASAQ arms or 42 days for DP arm. Clinical and parasitological responses were assessed following antimalarial treatment. Genotyping (msp1, msp2 and 24 SNP-based barcode) were done to differentiate recrudescence from re-infection; in case of PCR-confirmed treatment failure, Pfk13 propeller and Pfcoronin genes were sequenced. Data was entered and analyzed using the WHO Excel-based application. A total of 496 patients were enrolled. In Diourbel, PCR non-corrected/corrected adequate clinical and parasitological responses (ACPR) was 100.0% in both the AL and ASAQ arms. In Kedougou, PCR corrected ACPR values were 98.8%, 100% and 97.6% in AL, ASAQ and DP arms respectively. No Pfk13 or Pfcoronin mutations associated with artemisinin resistance were found. This study showed that AL, ASAQ and DP remain efficacious and well-tolerated in the treatment of uncomplicated P. falciparum malaria in Senegal.

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.

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.

Serological Data Shows Low Levels of Chikungunya Exposure in Senegalese Nomadic Pastoralists.

The chikungunya virus (CHIKV) is spread by Aedes aegypti and Ae. albopictus mosquitos worldwide; infection can lead to disease including joint pain, fever, and rash, with some convalescent persons experiencing chronic symptoms. Historically, CHIKV transmission has occurred in Africa and Asia, but recent outbreaks have taken place in Europe, Indonesia, and the Americas. From September to October 2014, a survey was undertaken with nomadic pastoralists residing in the northeast departments of Senegal. Blood dried on filter paper (dried blood spots; DBS) were collected from 1465 participants of all ages, and assayed for Immunoglobulin G (IgG) antibodies against CHIKV E1 antigen by a bead-based multiplex assay. The overall seroprevalence of all participants to CHIKV E1 was 2.7%, with no persons under 10 years of age found to be antibody positive. Above 10 years of age, clear increases of seroprevalence and IgG levels were observed with increasing age; 7.6% of participants older than 50 years were found to be positive for anti-CHIKV IgG. Reported net ownership, net usage, and gender were all non-significant explanatory variables of seropositivity. These data show a low-level historical exposure of this pastoralist population to CHIKV, with no evidence of recent CHIKV transmission in the past decade.

Increased Frequency of Pfdhps A581G Mutation in Plasmodium falciparum Isolates from Gabonese HIV-Infected Individuals.

BACKGROUND: Studying malaria parasites cross resistance to sulfadoxine-pyrimethamine (SP) and trimethoprim-sulfamethoxazole (cotrimoxazole, CTX) is necessary in areas coendemic for malaria and HIV. Polymorphism and frequency of drug resistance molecular markers, Pfdhfr and Pfdhps genes have been assessed in Plasmodium falciparum isolates from HIV-infected adults, in Gabon. MATERIEL AND METHODS: A cross-sectional study was conducted in three HIV care and treatment centers, at Libreville, the capital city of Gabon and at Oyem and Koulamoutou, two rural cities between March 2015 and June 2016. P. falciparum-infected HIV adults were selected. Analysis of Pfdhfr and Pfdhps genes was performed using high resolution melting (HRM) technique. RESULTS: Pfdhps A581G mutation was found in 23.5% (8/34) of the isolates. Triple Pfdhfr mutation (51I-59R-108N) was predominant (29.4%; n=10) while 17.6% (n=6) of the isolates carried a quadruple mutation (Pfdhfr 51I-59R-108N + Pfdhps 437G; Pfdhfr 51I-108N + Pfdhps 437G-Pfdhps581G; Pfdhfr 51I-59R-108N + Pfdhps 581G). Highly resistant genotype was detected in around 10% (n=3) of the isolates. The quintuple mutation (triple Pfdhfr 51I-59R-108N and double Pfdhps437-581) was only found in isolates from two patients who did not use CTX. The most frequent haplotypes were those with a single mutation (NCNIAKA) (36%) and a quadruple mutation (NCIIGKG, NRIIGKA, and NRIIAKG). Mixed unknown genotypes were found at codon 164 in three isolates. Mixed genotypes were more frequent at codons 51 (23.5%; n=8) and 59 (20.5%; n=7) (p<0.01). CONCLUSION: Pfdhps A581G mutation as well as new combination of quintuple mutations is found for the first time in isolates from HIV-infected patients in Gabon in comparison to a previous study. The detection of these genotypes at a nonnegligible frequency underlines the need of a regular surveillance of antifolates drug resistance.

Molecular identification of Plasmodium species responsible for malaria reveals Plasmodium vivax isolates in Duffy negative individuals from southwestern Nigeria.

BACKGROUND: Malaria in Nigeria is principally due to Plasmodium falciparum and, to a lesser extent to Plasmodium malariae and Plasmodium ovale. Plasmodium vivax is thought to be absent in Nigeria in particular and sub-Saharan Africa in general, due to the near fixation of the Duffy negative gene in this population. Nevertheless, there are frequent reports of P. vivax infection in Duffy negative individuals in the sub-region, including reports from two countries sharing border with Nigeria to the west (Republic of Benin) and east (Cameroon). Additionally, there were two cases of microscopic vivax-like malaria from Nigerian indigenous population. Hence molecular surveillance of the circulating Plasmodium species in two states (Lagos and Edo) of southwestern Nigeria was carried out. METHODS: A cross-sectional survey between September 2016 and March 2017 was conducted. 436 febrile patients were included for the present work. Venous blood of these patients was subjected to RDT as well as microscopy. Further, parasite DNA was isolated from positive samples and PCR diagnostic was employed followed by direct sequencing of the 18S rRNA of Plasmodium species as well as sequencing of a portion of the promoter region of the Duffy antigen receptor for chemokines. Samples positive for P. vivax were re-amplified several times and finally using the High Fidelity Taq to rule out any bias introduced. RESULTS: Of the 256 (58.7%) amplifiable malaria parasite DNA, P. falciparum was, as expected, the major cause of infection, either alone 85.5% (219/256; 97 from Edo and 122 from Lagos), or mixed with P. malariae 6.3% (16/256) or with P. vivax 1.6% (4/256). Only one of the five P. vivax isolates was found to be a single infection. DNA sequencing and subsequent alignment of the 18S rRNA of P. vivax with the reference strains displayed very high similarities (100%). Remarkably, the T-33C was identified in all P. vivax samples, thus confirming that all vivax-infected patients in the current study are Duffy negative. CONCLUSION: The present study gave the first molecular evidence of P. vivax in Nigeria in Duffy negative individuals. Though restricted to two states; Edo in South-South and Lagos in South-west Nigeria, the real burden of this species in Nigeria and sub-Saharan Africa might have been underestimated, hence there is need to put in place a country-wide, as well as a sub-Saharan Africa-wide surveillance and appropriate control measures.

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.

Malaria prevalence, prevention and treatment seeking practices among nomadic pastoralists in northern Senegal.

BACKGROUND: Malaria transmission in Senegal is highly stratified, from low in the dry north to moderately high in the moist south. In northern Senegal, along the Senegal River Valley and in the Ferlo semi-desert region, annual incidence is less than five cases per 1000 inhabitants. Many nomadic pastoralists have permanent dwellings in the Ferlo Desert and Senegal River Valley, but spend dry season in the south with their herds, returning north when the rains start, leading to a concern that this population could contribute to ongoing transmission in the north. METHODS: A modified snowball sampling survey was conducted at six sites in northern Senegal to determine the malaria prevention and treatment seeking practices and parasite prevalence among nomadic pastoralists in the Senegal River Valley and the Ferlo Desert. Nomadic pastoralists aged 6 months and older were surveyed during September and October 2014, and data regarding demographics, access to care and preventive measures were collected. Parasite infection was detected using rapid diagnostic tests (RDTs), microscopy (thin and thick smears) and polymerase chain reaction (PCR). Molecular barcodes were determined by high resolution melting (HRM). RESULTS: Of 1800 participants, 61% were male. Sixty-four percent had at least one bed net in the household, and 53% reported using a net the night before. Only 29% had received a net from a mass distribution campaign. Of the 8% (142) who reported having had fever in the last month, 55% sought care, 20% of whom received a diagnostic test, one-third of which (n = 5) were reported to be positive. Parasite prevalence was 0.44% by thick smear and 0.50% by PCR. None of the molecular barcodes identified among the nomadic pastoralists had been previously identified in Senegal. CONCLUSIONS: While access to and utilization of malaria control interventions among nomadic pastoralists was lower than the general population, parasite prevalence was lower than expected and sheds doubt on the perception that they are a source of ongoing transmission in the north. The National Malaria Control Program is making efforts to improve access to malaria prevention and case management for nomadic populations.

Distribution of Parasites Detected in Stool Samples of Patients in Le Dantec University Hospital of Dakar, Senegal, from 2011 to 2015.

To identify the parasites responsible for intestinal parasitic infections diagnosed at Le Dantec University Hospital of Dakar, distribution of parasites detected in stool samples of patients was studied. From 2011 to 2015, 2578 patients were included in the study. A direct examination and Ritchie technique were performed as parasite search techniques. In total, 408 samples were positive showing 440 intestinal parasites; this corresponds to prevalence of 15.8%. Parasites were detected in monoparasitism (85.7%) and multiparasitism (14.3%). The most common species found in monoparasitism were Entamoeba coli (38.9%), E. histolytica/dispar (12.7%), Giardia intestinalis (8%), and Ascaris lumbricoides (7.3%). The most common associations were A. lumbricoides-Trichuris trichiura (3.6%) and E. coli-G. intestinalis (2.7%). Nonhospitalized patients were significantly more affected with 65.4% compared to hospitalized counterparts; and also there were more men (50.7%) than women. With 67.4%, adults were the most affected age group, while the elderly were less affected with only 7% (p = 0.5). This study shows increasing prevalence of intestinal parasitic infections over the years. So health education should be promoted in addition to the already begun mass treatment program. This would help to limit or even halt the spread of these diseases.

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.

Evidence of non-Plasmodium falciparum malaria infection in Kédougou, Sénégal.

BACKGROUND: Expanded malaria control efforts in Sénégal have resulted in increased use of rapid diagnostic tests (RDT) to identify the primary disease-causing Plasmodium species, Plasmodium falciparum. However, the type of RDT utilized in Sénégal does not detect other malaria-causing species such as Plasmodium ovale spp., Plasmodium malariae, or Plasmodium vivax. Consequently, there is a lack of information about the frequency and types of malaria infections occurring in Sénégal. This study set out to better determine whether species other than P. falciparum were evident among patients evaluated for possible malaria infection in Kédougou, Sénégal. METHODS: Real-time polymerase chain reaction speciation assays for P. vivax, P. ovale spp., and P. malariae were developed and validated by sequencing and DNA extracted from 475 Plasmodium falciparum-specific HRP2-based RDT collected between 2013 and 2014 from a facility-based sample of symptomatic patients from two health clinics in Kédougou, a hyper-endemic region in southeastern Sénégal, were analysed. RESULTS: Plasmodium malariae (n = 3) and P. ovale wallikeri (n = 2) were observed as co-infections with P. falciparum among patients with positive RDT results (n = 187), including one patient positive for all three species. Among 288 negative RDT samples, samples positive for P. falciparum (n = 24), P. ovale curtisi (n = 3), P. ovale wallikeri (n = 1), and P. malariae (n = 3) were identified, corresponding to a non-falciparum positivity rate of 2.5%. CONCLUSIONS: These findings emphasize the limitations of the RDT used for malaria diagnosis and demonstrate that non-P. falciparum malaria infections occur in Sénégal. Current RDT used for routine clinical diagnosis do not necessarily provide an accurate reflection of malaria transmission in Kédougou, Sénégal, and more sensitive and specific methods are required for diagnosis and patient care, as well as surveillance and elimination activities. These findings have implications for other malaria endemic settings where species besides P. falciparum may be transmitted and overlooked by control or elimination activities.