Plasmodium infection in two districts of the north-eastern state of Meghalaya and evaluate risk factors that might explain these patterns.Despite declining incidence over the past decade, malaria remains an important health burden in India. This study aimed to assess the village-level temporal patterns of Plasmodium infections using PCR. A questionnaire collected socio-demographic, environmental, and behavioural data, and households were spatially mapped via GPS. Adult mosquitoes were sampled at a subset of subjects' houses, and Anopheles were identified by PCR and sequencing. Risk factors for Plasmodium infection were evaluated using bivariate and multivariate logistic regression analysis, and spatial cluster analysis was undertaken.Primary Health Centre passive malaria case data from 2014 to 2018 were analysed to characterize village-specific annual incidence and temporal trends. Active malaria case detection was undertaken in 2018 and 2019 to detect Plasmodium falciparum prevalence per 100 people varied from 0 to 4.1% in the nine villages of West Jaintia Hills, and from 0 to 10.6% in the 12 villages of West Khasi Hills. Significant clustering of P. falciparum infections  was observed in West Khasi Hills. A total of 13 Anopheles species were found at 53 houses in five villages, with Anopheles jeyporiensis being the most abundant. Risk of infection increased with presence of mosquitoes and electricity in the households [Odds Ratio (OR) = 1.19 and 1.11], respectively. Households with reported animals had reduced infection risk (OR = 0.91).The annual malaria incidence from PHC-based passive surveillance datasets in 2014–2018 was heterogenous but declining across villages in both districts. Active surveillance in 2018 enrolled 1468 individuals from 468 households (West Jaintia Hills) and 1274 individuals from 359 households (West Khasi Hills). Plasmodium infection and certain household characteristics. Since species of Plasmodium-competent mosquito vectors continue to be abundant, malaria resurgence remains a threat, and control efforts should continue.Malaria incidence during 2014–2018 declined in all study villages covered by the passive surveillance data, a period that includes the first widespread insecticide-treated net campaign. The survey data from 2018 revealed a significant association between  Plasmodium falciparum was introduced in the northeastern states of India in 2013 and has not changed since then  (Thermo Scientific™), each primer at 0.16 μM, 2 mM [MgCl2], and 0.2 mg/ml [BSA]. The PCR conditions were 95 °C for 2 min followed by 35 cycles of 95 °C for 45 s, 55 °C for 1 min, and 72 °C for 1 min, with a final extension at 72 °C for 7 min. The PCR products were purified using EXOSAP, and Sanger sequencing was subsequently performed in the reverse direction using the HCO-2198 reverse primer.DNA extracted from 161 individual adult mosquito specimens (QIAamp DNA Mini kit) was amplified using either the Internal Transcribed Spacer 2 (ITS2) region of ribosomal DNA (101 specimens) or a region of the mitochondrial cytochrome oxidase subunit I (COI) gene (60 specimens). The ITS2 primers were forward primer 5.8S (5′-TGTGAACTGCAGGACACATG-3′) and reverse primer 28S (5′-ATGCTTAAATTTAGGGGGTA-3′) . Each 25http://www.geneious.com) [http://www.boldsystems.org) [Anopheles species identity. If the query search produced two species matches, the ‘phylogenetic tree’ generated by BOLD using public and private COI sequences was referred to in order to confirm the closest match. The sequences generated in this study were deposited in GenBank with Accession Numbers as follows: Anopheles jeyporiensis: MT863705 (COI), MT872791 (ITS2); Anopheles maculatus: MT863706 (COI), MT862759 (ITS2); Anopheles pseudowillmori: MT871948 (COI), MT872797 (ITS2); Anopheles nivipes: MT863711 (COI), MT872792 (ITS2); Anopheles philippinensis: MT872793 (ITS2); Anopheles barbirostris: MT863707 (COI)i; Anopheles dissidens: MT872790 (ITS2); Anopheles nitidus: MT863712 (COI), MT872789 (ITS2); Anopheles peditaeniatus: MT872794 (ITS2); Anopheles xui: MT871949 (COI); Anopheles vagus: MT872798 (ITS2); Anopheles splendidus: MT872799 (ITS2); Anopheles jamesii: MT871938 (COI).Sequences were edited as necessary for accurate base calling, and primer sequences were removed using Geneious Prime® 2020.1.2 (ous.com) . ITS2 seems.org)  to deterPlasmodium infection prevalence per 100 people was calculated for each village included in the survey. Associations between demographic, environmental, and behavioral risk factors and village-level Plasmodium infection prevalence were determined using logistic regression analysis and odds ratios (OR) ). Risk factors with significant associations were analysed using multivariate logistic regression to adjust for confounders. All data were analysed using Statistical Package of Social Sciences (SPSS) version 15.0  and Stata version 14.4 .https://www.satscan.org/) was used to identify significant spatial clustering of Plasmodium infection prevalence among the study villages. The statistical test employs a circular window to systematically search for significant spatial clusters over a defined geographic area. The radius of the window may vary from zero to a user defined upper limit, here defined as the geographic area that included 50% of the study region to allow for the detection of both small and large clusters [Plasmodium infection in each village was defined as ‘cases.’ The total number of people sampled per village was defined as the ‘population.’SaTScan 9.6 software  between 2014 and 2015 (2016 disaggregated village level data were not available), and declined thereafter, except in Barato A and Barato B, where case numbers continued to increase until 2017 , except in Nonglang, Langja, and Khylleimsangrin, where incidence was still increasing until 2017  in the nine villages of West Jaintia Hills and from 0 to 10.6% P. falciparum  in the 12 villages of West Khasi Hills , and of the 12 villages in West Khasi Hills, P. falciparum infections were identified in six villages, with Siangra showing the highest prevalence , where five species were identified . Interestingly, An. jeyporiensis was the most abundant in both West Khasi Hills (1.9 per trap-night) and West Jaintia Hills (1.0 per trap-nights), even though it has not been frequently reported in recent studies in the region. Anopheles maculatus and An. pseudowillmori were more abundant in West Khasi Hills as compared to West Jaintia Hills. Additionally, many of the species associated with rice paddies, including those of the An. annularis, An. hyrcanus and An. barbirostris groups, were only found in West Jaintia Hills, whereas, An. splendidus was captured solely in West Khasi Hills. The relative abundances of each species  with village-level prevalence of each covariate, here defined as percentage of households in the village reporting the covariate, showed significant positive associations of Plasmodium infection with presence of mosquitoes, presence of electricity, and absence of domestic animals  are shown in Fig. The geographic locations of each village, and the proportion of residents with Plasmodium infections among villages in two districts of Meghalaya state in northeast India in the context of recently declining malaria incidence. This is the first active surveillance-based report on malaria in the region using PCR-based methods to determine infection status. These community surveys occurred after a widespread distribution of LLINs throughout the region in 2016 that is believed to have contributed to declining malaria incidence recorded through PHC-based passive surveillance. Even with declining incidence, inter-annual variation among villages in Plasmodium infection prevalence was observed, along with risk factors that partly explain the patterns identified.This study investigated the spatial and temporal prevalence of . [PHC data for the period 2014–2018 showed a general decline in the number of malaria cases, but this varied among villages. These results expand on the recent district-level decline described by Kessler et al.  have dem.  results . .An. culicifacies densities and decreasing human malaria incidence [Another study during 2009 in Uttar Pradesh indicated that people considered LLINs to be safe, effective, and socially acceptable . Investincidence . Althougncidence .Plasmodium transmission, microscopy and RDTs may not be sensitive enough to detect all cases of parasitaemia [Plasmodium infections compared to microscopy and RDTs [Plasmodium transmission. Indeed, elimination efforts in India will depend on improved diagnosis and treatment of asymptomatic infections combined with enhanced vector control and more focused disease surveillance [Malaria diagnosis at the study PHCs, and throughout Meghalaya, is mainly performed using slide microscopy and RDTs. Due to declining sitaemia  resultinsitaemia . PCR detand RDTs  and is vand RDTs . In thisand RDTs  to betteeillance , 27.Plasmodium infection prevalence. Similar spatial clustering of malaria village-level prevalence has been used in other regions, such as Ethiopia [As a component of surveillance, analysis of spatial patterns can serve many purposes, including data exploration, visualizing configurations, defining spatial resolution, determining clusters, developing causal hypotheses, and more . In thiEthiopia , SolomonEthiopia , and BanEthiopia , but rarEthiopia . Here, aEthiopia .Local environmental risk factors such as proximity to water bodies, elevation, topography, and land use/land cover have been shown to explain spatial clustering of higher prevalence locations , 34. ThePlasmodium infection, which is similar to findings in a study conducted in Zambia [Villages with more households that kept domestic animals at the house had lower odds of n Zambia . This fin Zambia  that sugn Zambia . Howevern Zambia , possibln Zambia .Plasmodium infection prevalence was higher in villages with a greater proportion of households that had access to electricity. In some developing country settings, electricity availability is an indicator of higher SES, which is often related to lower risk of malaria. Most studies recognize malaria as a disease of poverty, as the malaria burden is often concentrated in the poorest continents and countries. In the study area, perhaps presence of electricity in these rural households attracts Anopheles vectors [Village-level  vectors , thereby vectors  showed tMany other studies have revealed how different types of housing material used in the construction of roofs and walls are associated with malaria incidence . HoweverPlasmodium infection prevalence. Perhaps there was insufficient variation among villages in use of these prevention measures [Studies conducted in India  have shomeasures  or possimeasures ) are neemeasures .Anopheles species were captured despite limited sampling. This is consistent with previous studies in the region that have identified a high diversity of anophelines, some of which were involved in malaria transmission [Anopheles species diversity was greater in the West Jaintia Hills than in the West Khasi Hills. This may be because the former is relatively warmer and at generally lower elevation, but further studies in other villages and seasons are needed to determine if this is a general pattern. The predominance of An. jeyporiensis, which has not often been reported in the region in recent years, is noteworthy.A total of 13 smission –48. ThesAn. baimaii and An. minimus, were not identified in the samples, which corroborates more extensive recent studies in northeast India that these forest-associated mosquito species are in decline [An. jeyporiensis and An. maculatus are of particular concern as they were the most abundant species in this study and both have been reported to be infected with P. falciparum in this region, specifically: An. jeyporiensis in Assam [An. maculatus from Assam and Meghalaya [Two species historically considered as primary vectors in the region,  decline , 48. The decline . In thisin Assam  and Bangin Assam  and in seghalaya , 51. In Plasmodium infection risk and the presence of disease clustering, such as Anopheles mosquito abundance and distribution, distances from houses to water bodies or rice paddies, or elevation. Second, no data on weather variables such as temperature, precipitation, and/or humidity were included, which could alter vector-human contact and impact malaria parasite prevalence [Although this study offers important findings, it also has some limitations. First, it was not possible to evaluate all possible risk factors that may contribute to the geographic variation in evalence . Finallyevalence .This study demonstrated that malaria incidence has recently declined perhaps in part due to the widespread introduction of control strategies, especially LLINs. Nevertheless, temporal and spatial variation has left some villages at higher risk, making efforts aimed at regional elimination more difficult. Therefore, anti-malarial interventions should be continued, even expanded, if the government goal of malaria elimination by 2030 is to be achieved. The second round of state-wide LLIN distribution in Meghalaya was scheduled for 2020 but paused due to the COVID-19 pandemic. Malaria-prevention education for people of all ages in these communities should be pursued through different educational mediums. Because of cross-border movement of people and parasites , 54, the


Plasmodium falciparum remain challenging despite the significant progress has been made in last few decades. Conventional vaccinology  approaches are time consuming, laborious and expensive; therefore, the use of computational vaccinology tools are imperative, which can facilitate the design of new and promising vaccine candidates.In the current scenario, designing of world-wide effective malaria vaccine against P. falciparum genome were carefully chosen for the incidence of signal peptide/ anchor using SignalP4.0 tool that resulted into 640 surface linked proteins (SLP). Out of these SLP, only 17 were predicted to contain GPI-anchors using PredGPI tool in which further 5 proteins were considered as malarial antigenic adhesins by MAAP and VaxiJen programs, respectively. In the subsequent step, T cell epitopes of 5 genome derived predicted antigenic adhesins (GDPAA) and 5 randomly selected known malarial adhesins (RSKMA) were analysed employing MHC class I and II tools of IEDB analysis resource. Finally, VaxiJen scored T cell epitopes from each antigen were considered for prediction of population coverage (PPC) analysis in the world-wide population including malaria endemic regions. The validation of the present in silico strategy was carried out by comparing the PPC of combined (MHC class I and II) predicted epitope ensemble among GDPAA (99.97%), RSKMA (99.90%) and experimentally known epitopes (EKE) of P. falciparum (97.72%) pertaining to world-wide human population.In current investigation, initially 5548 proteins of P. falciparum genome using bioinformatics tools. Interestingly, these GDPAA, RSKMA and EKE of P. falciparum epitope ensembles forecasted to contain highly promiscuous T cell epitopes, which are potentially effective for most of the world-wide human population with malaria endemic regions. Therefore, these epitope ensembles could be considered in near future for novel and significantly effective vaccine candidate against malaria.The present study systematically screened 5 potential protective antigens from The online version of this article (10.1186/s12859-018-2482-x) contains supplementary material, which is available to authorized users. Plasmodium protozoan parasite . However, P. falciparum is one of the most deadly species that responsible towards > 90% of overall malaria mediated deaths. On the basis of 2016 data, WHO evaluated that almost 445,000 deaths were due to malaria with a total of 216 million cases from 91 countries. The African region (15 countries), continues to account for about 91% of malaria deaths worldwide including 80% by sub-Saharan Africa as well as virtually all of the remaining incidence reported from South-East Asia and the Indian sub-continent with South America. Thus, if we want to get the global response of malaria vaccine, these heavily affected countries in African region must be our primary focus [The human malaria is triggered as a result of 5 species of Human infection with the malaria parasite is developed following the inoculation of the sporozoite stage of the protozoan parasite by female anopheles mosquitoes. Infants and young children in malaria-endemic countries of African region naturally come across numerous clinical episodes of malaria before they build up partial immunity that defends towards severe disease as well as malaria mediated death. The mechanisms related to naturally acquired immunity are not completely explored; nevertheless, there are two foremost theories. First is the gradual attainment of strain-specific immunity, while second is the recurring antigenic exposure, possibly in combination with an age-linked immune maturation . Althoug+ T cells during phase-III clinical trials [+ T cell response remain missing from the RTS,S vaccine. Moreover, the attainment of naturally acquired partial immunity against malaria infection and some correlation to protection against experimental malaria vaccines, provide a positive perception towards the design of globally efficient malaria vaccines. Nonetheless, the large size of the Plasmodium genome (23.3 Mb) with more than 5000 genes pose significant challenges in experimental identification of immunodominant epitopes for activating both CD4+ and CD8+ T cells.Although, there are more than 30 vaccine candidates of malaria that were involved in different clinical phase trail, but they did not make a globally effective vaccine , 6. The l trials . However+ and CD8+ T cell epitopes turn out to be more straightforward approach along with less laborious and low cost [+ and CD8+ T cell responses [P. falciparum [In past two decades, as a result of development of reverse vaccinology and immunoinformatics, identification of antigen-specific CD4low cost –14. Concesponses , 16. Henesponses . Criticalciparum .P. falciparum-resulting epitopes specific for CD4+ and CD8+ T cells involved in world-wide protection towards malaria. This study focus on genome-wide in silico screening of putative antigens of P. falciparum 3D7 genome and further prediction of human leukocyte antigen (HLA) class I and II binding epitopes covered by major world-wide population including malaria endemic regions through immune epitope database (IEDB) based prediction of population coverage (PPC). Ultimately, the predicted epitope ensemble of genome derived predicted antigenic adhesins (GDPAA) showed considerably higher world-wide population coverage (99.97%) compared to randomly selected known malarial adhesins (RSKMA) (99.90%) and EKE of P. falciparum (97.72%). These predicted epitope ensembles could be considered as promising candidate for effective nano vaccine design against malaria.In the present work, we hypothesized that application of reverse vaccinology along with immunological bioinformatics tools might uncover P. falciparum 3D7 genome were retrieved from PlasmoDB database (http://plasmodb.org) into the FASTA format, accessed on October 3, 2017 together with 5 RSKMA. Similarly, 151 non redundant EKE of P. falciparum 3D7 including 121 MHC class I and 30 MHC class II were retrieved from IEDB database (www.iedb.org) accessed on February 20, 2018 using MHC biding assay as search criteria  of P. falciparum genome involved three major steps: i) prediction of probable antigens ii) prediction of T cell epitopes iii) screening of minimal epitope ensemble for maximum human population coverage. The methodology flow chart is given in Fig. This investigation facilitates the screening of vaccine candidates from entire protein sequences (5548) of P. falciparum were reflected as high-grade antigen candidates if they have forecasted features of a surface protein, i.e., a signal peptide, a glycophosphatidylinositol (GPI) anchor and transmembrane domain(s). Currently, SignalP4.0 is the most advanced and widely used tool for prediction of signal peptides from amino acid sequences of protein. The protein with signal peptide is targeted to the secretory route, nevertheless it is not necessarily to be secreted [Plasmodium parasites is mediated by proteins called adhesins. The parasite can be inhibited by immunizing the host with adhesins. Therefore, these GPI-anchored surface proteins were screened as malarial adhesins and adhesin-like proteins by MAAP tool [P. falciparum) were predicted using TMHMM2.0 tool [The first step involved hypothetical confirmation for occurrence of surface protein and antigenic features assessed from protein primary sequences employing well-known tools: SignalP4.0, PredGPI, MAAP, VaxiJen and TMHMM in sequential manner to identify probable antigens , 20. Thesecreted . Numerousecreted . The possecreted . SurfaceAAP tool –26. The AAP tool , 28. Fur2.0 tool –31. The 2.0 tool –34. More50 ≤ 500 nM) and 54 HLA class II alleles (cut off threshold ≤ 3 percentile rank) [The subsequent step predicts the presence of immunogenic T cell epitopes in all the 5 GDPAA and 5 RSKMA by employing consensus technique of MHC class I and II tools accessible at IEDB web server. The prediction of T cell epitopes were performed for 51 HLA class I (cut off threshold ICle rank) . These Tle rank) .P. falciparum [In the following step, PPC analysis of aforesaid combined (MHC class I and II) epitopes for all 5 GDPAA were conducted by utilizing IEDB based PPC tool against world-wide population. Furthermore, the minimal combined epitope set for each antigen was formed along with the inclusion of only those epitope which have highest PPC value and restricted by different set of MHC alleles. In case, PPC value of epitopes are equal then epitope with highest VaxiJen score was included in the minimal epitope set. Then, after employing the same protocol of minimal epitope selection, an ultimate epitope ensemble was designed with the joint screening of minimal epitope set of all the 5 GDPAA. Finally, the PPC of 5 GDPAA minimal epitope set and epitope ensemble were executed for the selected malaria endemic regions in the present study . So as to compare the predictive efficiency of PPC analysis for GDPAA epitope ensemble selection, the same protocol was also applied to predict epitope ensemble of 5 RSKMA and 151 EKE of lciparum , 36.The immunogenicity of the MHC class I epitope ensemble designed from GDPAA was estimated by CTLPred and TAPPred , 37, 38 P. falciparum as positive controls in docking studies. The structural information about the test peptides of GDPAA epitope ensemble  and positive control peptides were modelled using the PEP-FOLD 3 web server. The test peptides of GDPAA epitope ensemble were then docked by employing PatchDock web server to HLA–A*0201 and HLA–DRB1*0101 interacting residues as input. The best 10 HLA-peptide complexes were further refined by the FireDock web server. In order to compare the study of molecular docking, ClusPro tool was also used for the same test and control peptides along with target MHC molecule of interest [A structure-based docking approach was further carried out so as to improve the predictive capability of peptide-MHC binding. The combination of sequence and structure-based approaches not merely enhances the probability of MHC binding prediction but also calculates the docked epitope orientation. The complex crystal structures of HLA–A*02:01 (PDB ID: 1I4F) and HLA–DRB1*01:01 (PDB ID: 1AQD) were retrieved from the protein data bank (PDB) for MHC class I and class II, respectively. We used peptides NQMIFVSSI (C1) and LKELIKVGLPSFENL (C2) from EKE of interest . The detP. falciparum genome were screened for the presence of signal peptide/ anchor using SignalP4.0 and found 640 surface accompanying/screatory proteins. Out of 640, only 17 are predicted to contain GPI-anchors using PredGPI tool and further 5 proteins are predicted as malarial adhesins and adhesin-like proteins. These 5 malarial adhesin proteins viz. CSP, surface protein P113 (P113), merozoite surface protein 1 (MSP1), 28 kDa ookinete surface protein (P28) and 25 kDa ookinete surface antigen precursor (P25), were also predicted as antigenic using VaxiJen, which contain ≤ 1 transmembrane helix as predicted by TMHMM2.0 server  for the world-wide population . However, an exception was recorded for South African population (0.43%) that could be due to limited HLA typing data as detailed in Table The immunogens based on population coverage analysis enable us towards the screening of best possible minimal epitope combination, i.e., epitope ensemble so as to maximize vaccine effectiveness. In this background, the PPC strategy was followed for developing the epitope ensemble of chosen antigens towards improving the vaccine efficacy in the world-wide as well as endemic population of malaria. Primarily, the PPC of predicted T cell epitopes  from 5 GDPAA with VaxiJen score ≥ 0.5 were performed for the world-wide, South East Asia, India, South-America and Central Africa, East Africa, North Africa and West Africa populations  and 3 MHC class II epitopes  as presented in Table The interaction between the peptide and MHC is an important characteristic of T cell epitope and the accessibility of the crystal structures facilitates us to determine the mode of interaction by computational methodology . The doc+ T cell epitopes  from antigens expressed during malaria pathogen life-cycle, the universal CD4+ T cell epitope, and flagellin as a scaffold and TLR5 agonist. On the other hand, used de novo designed amino acid domains to fuel the development of the coiled-coil scaffolds that present the antigenic epitopes on the nanoparticles surface [The reverse vaccinology and immunoinformatics strategies are still under progressive phase, however reasonable triumphs may result in substantial advancement on epitope-based ensemble vaccine efficacy against malaria pathogens for example, by enhancing coverage in the target populations through reasonably bearing in mind the specificity as well as occurrence of the HLA molecules , 62. The surface . As the  surface .P. falciparum malaria endemic population. The present study screened 5 GDPAA as potential vaccine targets due to their extreme conservancy amongst Plasmodium species including isolates of different geographical region. The PPC analysis with respect to epitope ensemble of 5 GDPAA and 5 RSKMA as well as 151 EKE of P. falciparum showed more than 81% population coverage in the world-wide along with malarial endemic regions except South Africa. These promiscuous T cell epitope ensembles will significantly aid towards the fast development of more efficacious malaria vaccine against P. falciparum. Therefore, this promising strategy could be extended to other infectious diseases as well. Overall, the computational tools used here are not yet ready to substitute the wet laboratory experimentation, rather they are assisting in experimental design and reducing the time and cost of the vaccine development process.The design of epitope ensemble using computational vaccinology is one of the promising alternatives, which enables finding of new epitope based vaccine candidate in a cost-effective manner for global as well as Additional file 1:P. falciparum 3D7. (XLSX 11 kb)Experimentally known epitopes of Additional file 2:(a) The PPC of predicted MHC class I binding T cell epitopes from 5 genome derived predicted antigenic adhesions (GDPAA) with VaxiJen score ≥ 0.5, (b) The PPC of predicted MHC class II binding T cell epitopes from 5 genome derived predicted antigenic adhesions (GDPAA) with VaxiJen score ≥ 0.5. (XLSX 88 kb)Additional file 3:(a) The PPC of predicted T cell epitopes  from 5 randomly selected known malarial adhesions (RSKMA) with VaxiJen score ≥ 0.5, (b) The PPC of predicted T cell epitopes  from 5 RSKMA, (c) Predicted set of T cell epitopes of 5 RSKMA, (d) The PPC of minimal T cell epitopes  from 5 RKSMA, (e) The predicted ensemble epitope (MHC class I and MHC class II) from 5 RKSMA. (XLSX 145 kb)Additional file 4:The PPC of predicted T cell epitopes for 5 genome derived predicted antigenic adhesions (GDPAA). (XLSX 9 kb)Additional file 5:The PPC of individual epitope found in minimal epitope set of 5 genome derived predicted antigenic adhesions (GDPAA). (XLSX 13 kb)Additional file 6:The PPC of minimal combined epitope set for 5 GDPAA and jointly screened epitope ensemble. (XLSX 10 kb)Additional file 7:P. falciparum. (XLSX 11 kb)The PPC value of individual epitope of epitope ensemble designed from GDPAA and experimentally known epitope (EKE) of


Plasmodium malaria infections can lead to severe malaria. This systematic review and meta-analysis aimed to explore the prevalence of severe mixed Plasmodium malaria infection and to compare it with the prevalence of severe P. falciparum malaria mono-infection across the included studies. Original English-language research articles from PubMed, Scopus, and ISI Web of Science were identified and screened. Articles reporting the number of mixed infections and the number of severe mixed infections were used to determine the main outcome of this study, while the number of P. falciparum infections and the number of severe P. falciparum infections were used to determine the secondary outcome of this study. For the main outcome, the pooled prevalence and 95% confidence interval (CI) of severe mixed infections was analysed using STATA software version 15.0 . For the secondary outcome, the rate of severe mixed infections compared to severe P. falciparum infections was analysed using the meta-analysis approach, and summary odds ratios (ORs) and 95% CIs were calculated. Random-effects models were used to produce the summary ORs. The Mantel–Haenszel method and calculated I2 were also reported to test whether there was heterogeneity among the included studies. Publication bias was also assessed using funnel plots. The meta-analysis of secondary outcomes was conducted using Review Manager 5.3 software (Cochrane Community). A total of 894,561 malaria patients were reported in all 16 included studies. Overall, a pooled analysis showed that 9%  of patients with mixed Plasmodium infection had severe mixed infection. A meta-analysis of 14 studies demonstrated that patients with mixed Plasmodium infection  and patients with P. falciparum malaria  had an equal risk of developing severe malaria . Both mixed infection and P. falciparum mono-infection showed a similar trend of complications in which severe anaemia, pulmonary failure, and renal impairment were the three most common complications found. However, patients with mixed infection had a higher proportion of severe anaemia and pulmonary complications than those with P. falciparum infection. Moreover, patients with mixed infection had a higher proportion of multiple organ failure than those with P. falciparum mono-infection. Mixed Plasmodium spp. infections were common but often unrecognized or underestimated, leading to severe complications among these malaria patients. Therefore, in routine clinical laboratories, using an accurate combination of diagnostic procedures to identify suspected patients with mixed infections is crucial for therapeutic decisions, prompt treatment, and effective patient management.Mixed  Plasmodium spp. that include P. falciparum, P. vivax, P. malariae, P. ovale, and P. knowlesi1. Molecular methods have demonstrated the existence of two distinct species of P. ovale: P. ovale curtisi and P. ovale wallikeri2. P. knowlesi naturally occurs in macaques inhabiting forested areas of Southeast Asia and is the fifth species of Plasmodium causing human malaria3. In some areas where more than one species of Plasmodium is endemic, mixed Plasmodium spp. infections can frequently occur4.Mixed Plasmodium spp. infections are often unrecognized or underestimated, as a low proportion (2%) is detected by microscopy6. This might be due to observer error, technical difficulties, and low parasite densities4. If the mixed infection is misdiagnosed as a P. vivax mono-infection, treatment of P. vivax will increase the risk of P. falciparum parasitaemia, leading to anti-malarial drug resistance and, eventually, development of severe P. falciparum malaria7. Therefore, in routine clinical laboratories, the use of the most accurate diagnostic procedures to identify Plasmodium species in cases of suspected mixed malaria infection is crucial for therapeutic decisions and management among those patients8. A research study indicated that mixed P. falciparum and P. vivax infection led to an increase in the disease severity among children10. Another study demonstrated that mixed infection with P. falciparum and P. vivax led to suppression of the severity of P. falciparum infection11. Although mixed P. falciparum and P. vivax malaria is common, systematic review and meta-analysis of severe mixed infection has been limited. No recent study has demonstrated the prevalence and differences between mixed Plasmodium infection and P. falciparum malaria infection. This is very important for physicians to plan therapeutic options and determine the prognostic signs of severity during drug treatment. Therefore, this systematic review and meta-analysis aimed to explore the prevalence of severe Plasmodium mixed infection and to compare it with that of severe P. falciparum malaria infection across the included studies.Human malaria is caused by five species of The protocol for this systematic review and meta-analysis followed the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines  and included respiratory distress or acidosis , pulmonary oedema , impaired consciousness (a Glasgow Coma Score < 11 in adults or a Blantyre coma score < 3 in children), convulsions (more than two episodes within 24 h), prostration , hypotension/shock (systolic blood pressure < 70 mmHg in children or < 80 mmHg in adults), jaundice [plasma bilirubin > 50 µM/L (3 mg/dL)], severe anaemia (A haemoglobin concentration < 5 g/dL), bleeding/Disseminated Intravascular Coagulation (DIC) , hyperparasitemia , and hypoglycaemia [blood or plasma glucose < 2.2 mM (< 40 mg/dL)]12. Cerebral malaria, one criterion of severe P. falciparum malaria in the former version of the WHO definition, was assigned to the group “impaired consciousness” and described as “impaired consciousness/cerebral malaria” for further analysis and demonstration in the results section.The major complications of severe mixed malaria were considered to be the same as those defined for Plasmodium with other agents were excluded from the present study.Original research articles published in the English language were included in the current analysis if they met the following criteria: (1) malaria positivity confirmed by any combination of rapid diagnostic tests (RDTs), microscopy, or polymerase chain reaction (PCR); (2) enrolled both uncomplicated and complicated malaria; (3) the numbers of mixed infections and severe mixed infections were reported, and (4) all complications in the patients with severe mixed infections were reported. Case reports, animal studies, experimental studies, clinical trials, book or book chapters, letters to the editor, editorials, reviews or systematic reviews, conference papers, short surveys, and studies of co-infection of P. falciparum infections, number of P. falciparum infections, complications of severe mixed infections, and complications of P. falciparum infections. The number of mixed infections and the number of severe mixed infections was used to determine the main outcome of this study, while the number of P. falciparum infections and the number of severe P. falciparum infections were used to determine the secondary outcome of this study.For all articles included in the analysis, the following information was extracted: name of the authors, year of publication, country of the participants, duration of the study, the total number of malaria patients, number of severe mixed infections, number of mixed infections, number of severe P. falciparum infection was analysed using the meta-analysis approach and summary odds ratios (ORs) and 95% CI were calculated. Random-effects models were used to produce summary ORs as described previously13. The Mantel–Haenszel method and the calculated I2 were also reported to determine whether there was heterogeneity among the included studies. Publication bias was also assessed using funnel plots and Egger's test as described elsewhere14. The meta-analysis of the secondary outcomes was conducted using Review Manager 5.3 software (Cochrane Community).For the main outcome, the pooled prevalence and 95% confidence interval (CI) of severe mixed infection was analysed using STATA software version 15.0 . For the secondary outcome, the rate of severe mixed infection compared to severe 30. Most of the included studies  were descriptive studies or cross-sectional observational designs30. Six studies  were prospective studies or prospective cohort studies29. One study was a retrospective observational study25. The majority of patients in all included studies were infected with P. vivax , followed by P. falciparum . Almost all of the included studies reported that P. falciparum/P. vivax mixed infection was frequently found among those with mixed Plasmodium spp. infection. One study reported patients with P. vivax/P. malariae mixed infection and other types of mixed infection24. The majority of malaria patients were identified from the SIVIGILA study conducted in Colombia 16. Half of the studies were conducted in India , followed by Colombia . Half of the included studies  used combined microscopy techniques and other techniques to confirm the parasite species. Four studies used PCR to confirm the Plasmodium parasite species  among the included studies, suggesting a high level of heterogeneity between studies, so random-effects models were used to produce the summary ORs in the present meta-analysis. Among the 16 included studies, only 14 studies were used to perform the meta-analysis, as two studies by Devineni et al., 2015 and Hermansyah et al., 2016 did not report the number of patients who had severe P. falciparum infections. The meta-analysis of these 14 studies demonstrated that patients with mixed Plasmodium infection  and those with P. falciparum mono-infection  had an equal risk of developing severe malaria   of patients with mixed Plasmodium spp. infection and patients with P. falciparum mono-infection had an equal risk of developing severe malaria   in this subgroup analysis, indicating that the study area (India and non-India) was one source of heterogeneity in the present study. Further stratification by diagnostic technique  also showed that patients with mixed infection had an equal risk of developing severe malaria compared to those with P. falciparum mono-infection   in this subgroup analysis, indicating that diagnostic technique  was also a source of heterogeneity in the present study.In a subgroup analysis comparing the results from India and non-India areas in 13 studies, the pooled analysis showed that patients with mixed P. falciparum mono-infection were severe anaemia , pulmonary complications (14.6), and renal impairment (11.4%). For all complications, the proportions of severe mixed malaria infection and severe P. falciparum infection are shown in Fig. P. falciparum mono-infection showed similar trends of severe complications by severe anaemia, pulmonary failure, and renal impairment, which were the three most common complications found in this study. Patients with mixed infection had a higher proportion of severe anaemia (65.8% vs 57.6%) and pulmonary complications (20.9% vs 14.6%) than those with P. falciparum mono-infection. Patients with mixed infection (13.1%) had a higher proportion of multiple organ failure than those with P. falciparum mono-infection (3.95%). The publication bias among studies was assessed by funnel plots .Common severe complications found in patients with mixed malaria infection were severe anaemia , pulmonary failure , renal impairment , jaundice , cerebral malaria/impaired consciousness 3.94%, 79), convulsions , bleeding/DIC , shock , hyperparasitemia , hypoglycaemia , and more than one complication . The mortality rate of severe mixed infection was 0.9% (18/2006). The most common severe complications of 9, convulPlasmodium infection across the included studies (9%), demonstrating for the first time, to our knowledge, that mixed infection can cause a high rate of severe malaria. Although the mixed malaria prevalence was predominantly due to P. falciparum/P. vivax infection, the prevalence of non-P. falciparum/P. vivax mixed infections, such as P. falciparum/P. malariae mixed infections was also reported in our study, but they were not shown in their literature24. This high prevalence of severe mixed malaria partly explains why malaria remains one of the leading causes of morbidity and mortality worldwide despite available interventions, public health control, and management employed. These findings suggested that there is a need for continued detection and monitoring of mixed infection using species-specific RDTs in combination with routine microscopy, or even using PCR as soon as possible, to move towards malaria elimination and to protect against severe malaria resulting in death. A previous study indicated that the severity of mixed P. falciparum/P. vivax infection occurred when P. vivax superinfection occurred over an existing P. falciparum infection. However, P. falciparum superinfection over an existing P. vivax infection results in a lower risk of severe malaria28. The present study demonstrated the equal prevalence of severe mixed malaria infection compared to severe P. falciparum mono-infections across the included studies. However, when considering individual studies, it was demonstrated that three of our included studies reported that patients with mixed malaria infection had a significantly lower risk of developing severe malaria than patients with P. falciparum mono-infection19. These results were consistent with results from a previous study conducted in Thailand, which observed that severe malaria was less common among patients with mixed infections compared to those with P. falciparum mono-infection31. However, three other included studies demonstrated that patients with mixed infections had a significantly higher risk of developing severe malaria than patients with P. falciparum mono-infections28. This could be because mixed infections are often unrecognized or underestimated by microscopists33, leading to treatment failure, anti-malarial drug resistance, and the development of severe P. falciparum malaria7. Previous studies also demonstrated that age was associated with mixed infection35. They found that children under two years of age had a lower frequency of mixed Plasmodium malaria compared to those at an older age. They suggested that maternal antibodies could be the source of mixed infection malaria protection34.The present study showed a high prevalence of severe mixed 12. The present study demonstrated that 9% of severe malaria was caused by mixed infection, whereas a previous study showed that severe malaria accounts for approximately 5% of total malaria-infected patients36. The mortality rate of severe mixed malaria in the present study was 0.9%, which was consistent with the case fatality rate in previously reported P. falciparum malaria mono-infection (0.6–3.8%)4. The present study also indicated that both mixed infection and P. falciparum mono-infection showed similar trends of complications in which severe anaemia, pulmonary failure, and renal impairment were the three most common complications. However, patients with mixed infection had a higher proportion of severe anaemia and pulmonary complications than those with P. falciparum mono-infection. Moreover, patients with mixed infection had a higher proportion of multiple organ failure than those with P. falciparum mono-infection. A study in Thailand indicated that mixed P. falciparum/P. vivax infection could reduce the risk of severe anaemia among patients with falciparum malaria by cross-species immunity37. In Southeast Asia, other possible reasons behind the reduction of the risk for severe anaemia among patients with malaria infections were haemoglobinopathies and enzymatic deficiencies38. Haemoglobinopathies related to the reduced risk of malaria infections or reducing the risk of severe malaria included sickle cell traits39, haemoglobin C40, haemoglobin E41, and thalassemia40. Enzymatic deficiencies related to the reduced risk of malaria infections include glucose-6-phosphate dehydrogenase (G6PD) deficiency42 and pyruvate kinase deficiency43. In addition, individuals with blood type O were less susceptible to severe malaria than individuals who were not blood type O44. The expression of the host RBC surface protein called Duffy antigen receptor for chemokines (DARC) has been shown to protect against malaria infections38. Moreover, altered RBC morphologies such as Southeast Asian ovalocytosis (SAO) could reduce the risk of malaria infection or severe malaria46.The major complications of severe malaria defined by the World Health Organization (WHO) included respiratory distress, acidosis, pulmonary oedema, death, impaired consciousness, convulsions, prostration, hypotension/shock, jaundice, severe anaemia, bleeding/DIC, hyperparasitemia, and hypoglycaemiaPlasmodium mono-infection alone19. The results of our study were also consistent with the results of studies in India9 and Indonesia47 that reported a high prevalence of severe anaemia among patients with mixed infections. The higher proportion of severe mixed infection than that of P. falciparum and P. vivax mono-infection was due to mixed infection having higher parasite densities19.The included studies conducted in Papua New Guinea (1997–2004) demonstrated that mixed infection caused more severe anaemia than did the Plasmodium mixed infections. The analysis of mixed-species infections compared with P. falciparum mono-infections needs to be carefully interpreted as it is highly likely to be confounded by a proportion of undiagnosed mixed infections in the P. falciparum mono-infection groups. Fifth, a large number of additional factors related to transmission intensity, host immunity, and vectors that likely influenced the large variance seen in the mixed-Plasmodium species infections could not be taken into account because of the inherent data limitations from each study. Lastly, the present Review submits analysis of data which is relevant for the asexual blood stages of Plasmodium spp. infections resulting to severe manifestation and does not take into account hypnozoites and/or submicroscopic co-infections.The present study had limitations. First, there was a high level of heterogeneity across the included studies. Second, except for the area of the study (India and non-India) and diagnostic method, the source(s) of heterogeneity could not be explored due to the incomplete data among the included studies. Third, a limited number of studies met the criteria for inclusion because many publications included patients with severe complications and infections with etiologic agents other than malaria. Fourth, most of the included studies used microscopy for malaria detection, which might have led to missed detection of Plasmodium spp. infections are common but often unrecognized or underestimated, leading to severe complications among malaria patients. Therefore, in routine clinical laboratories, using an accurate combination of diagnostic procedures or repeat blood film examinations by microscopists to identify mixed infection in suspected patients is crucial for therapeutic decisions, prompt treatment, and effective management among those patients.Mixed Supplementary information


Malaria transmission depends on infected mosquitoes and can be controlled by transmission-blocking drugs. The recently discovered FREP1-mediated malaria transmission pathway is an excellent target to screen drugs for limiting transmission.To identify candidate small molecules, we used an ELISA-based approach to analyze extracts from a fungal library for inhibition of the FREP1–parasite interaction. We isolated and determined one active compound by chromatography and crystallography, respectively. We measured the effects of the bioactive compound on malaria transmission to mosquitoes through standard membrane-feeding assays (SMFA) and on parasite proliferation in blood by culturing.Purpureocillium lilacinum that inhibited Plasmodium falciparum transmission to mosquitoes. Pre-exposure to the extract rendered Anopheles gambiae resistant to Plasmodium infection. Furthermore, we isolated one novel active compound from the extract and identified it as 3-amino-7,9-dihydroxy-1-methyl-6H-benzo[c]chromen-6-one, or “pulixin.” Pulixin prevented FREP1 from binding to P. falciparum-infected cell lysate. Pulixin blocked the transmission of the parasite to mosquitoes with an EC50  of 11 µM based on SMFA. Notably, pulixin also inhibited the proliferation of asexual-stage P. falciparum with an EC50 of 47 nM. The compound did not show cytotoxic effects at a concentration of 116 µM or lower.We discovered the ethyl acetate extract of the fungus Plasmodium interaction, we discovered that Purpureocillium lilacinum extract blocked malaria transmission. We isolated and identified the bioactive agent pulixin as a new compound capable of stopping malaria transmission to mosquitoes and inhibiting parasite proliferation in blood culture.By targeting the FREP1–The online version contains supplementary material available at 10.1186/s13071-021-04677-7. Plasmodium parasites transmitted by anopheline mosquitoes caused approximately 200 million clinical malaria cases and half a million deaths in 2019, according to a recent report by the World Health Organization (WHO) chromen-6-one , an amino group (δ = 10.61 ppm), and a methyl group (δ = 2.67 ppm). The 13C-NMR data , two hydroxyl groups bearing aromatic carbon atoms (δ = 164.6 and 164.0 ppm), one amino group attached to an aromatic carbon atom (δ = 152.6 ppm), and a methyl carbon atom attached to the aromatic ring (δ = 25.2 ppm). Table H-benzo[c]chromen-6-one.We also determined the molecular mass of pulixin using HR-ESI-mass spectrometry to confirm its identity in the bulk extract. An [M+H]405 values differed among wells with different concentrations of pulixin , in experimental mosquitoes compared to that of the control group was 11 μM, calculated using a serial dilution of samples with an LC50 calculator [Next, we analyzed the effects of pulixin on lculator . We analp > 0.2; Fig. Pulixin at a level of 40 μM completely blocked malaria transmission. Therefore, we examined whether 40 μM of pulixin inhibited conversion of gametocytes to ookinetes. Results showed no significant difference in gametocyte transformation rates between the control and pulixin-treated samples  than that on day 1, and almost all infected cells were at the asexual stage. This result is consistent with the P. falciparum 48-h asexual replication cycle. Based on this result, we examined the activity of pulixin in inhibiting the development of asexual-stage P. falciparum on the fourth day after inoculation. The results showed that the inhibitory effect of pulixin on asexual-stage P. falciparum development was dose-dependent, and inhibition increased as the pulixin concentration increased  were incubated with pulixin at different concentrations. No significant difference in the density of living cells was observed in cultures with a pulixin level ranging from 0 to 30 µg/mL , consistent with the hypothesis of targeting the FREP1-mediated transmission pathway to block malaria transmission. Since this pathway is conserved across Plasmodium species [Purpureocillium lilacinum extract and pulixin are expected to inhibit other malaria pathogens such as P. vivax carried by Anopheles mosquitoes. The structure of pulixin suggests that it is stable, which is consistent with our observation that pulixin did not lose any activity after storing it in a laboratory at RT for more than 6 months. To develop a commercial spray product from the pulixin, measurements of its stability in the environment and toxicity to plants, birds, and fish are needed.We also isolated and identified pulixin, a novel compound that inhibited n nature , 27. Alt, 1.5 mM . Our res species , PurpureP. falciparum at an EC50 of 47 nM; this activity is comparable to that of one of the most powerful antimalarial drugs, artemisinin, whose EC50 is 17.3 nM [P. falciparum transmission at a dose of 0.5–0.75 mg/kg (2–3 μM) [It was a surprise that pulixin also inhibited the reproduction of asexual  17.3 nM . While a(2–3 μM) . HoweverPurpureocillium lilacinum, that produces small molecules to block malaria transmission. Moreover, we isolated and identified the structure of the bioactive compound, pulixin, in the fungal extract. It is a novel natural compound that inhibits asexual Plasmodium replication and sexual Plasmodium transmission. Purpureocillium lilacinum extract and pulixin are excellent new tools for malaria control.We screened a sizable fungal extract library and discovered a fungus, Additional file 1: Figure S1. Identification of the candidate compound through the mass spectrometric profile of pulixin showing a mass of 258.0764, which matched the calculated mass. Figure S2. The H-NMR profile of pulixin confirmed its structure. Figure S3. The C-NMR profile of pulixin was consistent with the proposed structure.


Plasmodium vivax or P. ovale, relapse from the dormant liver stage of the parasite. The aim of this study was to quantify the impact of recurrent malaria episodes on morbidity and mortality in Papua, Indonesia.An acute episode of malaria can be followed by multiple recurrent episodes either due to re-infection, recrudescence of a partially treated parasite or, in the case of We undertook a retrospective analysis of routinely collected data from malaria patients attending the primary referral hospital in Papua, Indonesia, between April 2004 and December 2013. Multi-state modelling was used to estimate the effect of recurring malaria episodes on re-presentation and admission to hospital and death. The risks of early (≤ 14 days) and late (15 to 365 days) hospital admission and death were estimated separately in our study to distinguish between the direct and indirect effects of malaria recurrence on adverse outcomes.P. falciparum, 22,209 (32.5%) with P. vivax, and 8984 (13.1%) with other species. During 12 months of follow-up after each of the first four malaria episodes, 10,868 (15.9%) patients were admitted to hospital and 897 (1.3%) died. The risk of re-presenting to the hospital with malaria increased from 34.7%  at first episode to 58.6%  following the third episode of malaria. After adjusting for co-factors, infection with P. vivax was a significant risk factor for re-presentation (hazard ratio (HR) = 1.48 ) and late admission to hospital ). Patients infected with P. falciparum had a greater overall rate of mortality within 14 days ), but after multiple episodes of malaria, there was a trend towards a higher rate of early death in patients infected with P. vivax compared to P. falciparum ).A total of 68,361 patients were included in the analysis, of whom 37,168 (54.4%) presented initially with P. falciparum, those infected with P. vivax had significantly more re-presentations to hospital with malaria, and this contributed to a high risk of inpatient admission and death. These findings highlight the importance of radical cure of P. vivax to eliminate the dormant liver stages that trigger relapses.Compared to patients initially infected with The online version of this article (10.1186/s12916-020-1497-0) contains supplementary material, which is available to authorized users. P. falciparum, P. vivax can form dormant liver stages (hypnozoites) which can reactivate weeks to months after the initial infection to cause further blood stage infections and clinical illness known as relapses.Malaria remains a major threat to health in malaria-endemic countries, where it is associated with significant morbidity, mortality and a high socioeconomic burden . MalariaP. vivax relapses; however, poor adherence to the standard 14-day regimen limits its effectiveness [The acute illness of malaria is attributable to the asexual blood stages of the parasite, which are treated by administration of blood schizonticides, such as chloroquine- or artemisinin-based therapies. Each recurrence of malaria, irrespective of whether it is a recrudescence, re-infection or a relapse, is associated with haemolysis due to rupture of both infected and uninfected red blood cells, compounded by dyserythropoesis, both of which lead to an increased risk of severe anaemia and associated morbidity and mortality , 4. Primtiveness  and its tiveness , 6.P. falciparum and P. vivax malaria. In this region, antimalarial drug resistance has emerged in both species, and the local population is at significant risk of recurrent malaria [Papua, the most easterly province of Indonesia, is co-endemic for both  malaria –9. The aP. falciparum, 39% P. vivax, 4% P. malariae and 11% mixed infections [Plasmodium ovale infections are rare. Local P. vivax strains have a typical equatorial relapse periodicity of 3–4 weeks.The study was undertaken in Timika, the capital of Mimika District located in the southern part of Papua province in eastern Indonesia. The region includes forested lowlands, coastal areas and subalpine and alpine regions. Malaria transmission is restricted to lowland areas where rainfall is perennial and the temperature is relatively consistent and warm . In 2013The population of Mimika District was estimated at 120,457 in 2004 rising to 189,447 in 2013, mostly comprising Highland and Lowland Papuans of Melanesian ancestry as well as Indonesians from elsewhere in the country.The RSMM is a hospital with a busy outpatient department, emergency department and 110 inpatient beds. Until January 2010, it was the only public referral hospital in Mimika District. Hospital administrators collect demographic data, clinical information (including ICD10 codes assigned by the attending physician) and vital status information for each patient presentation and link each record to a unique hospital identification number. Data from the hospital pharmacy and the hospital’s full blood coulter counter are also collected and linked to the same individual identification number. Hospital protocol dictates that all inpatients and any outpatient with symptoms potentially consistent with malaria have a blood sample taken for malaria microscopy and/or a rapid diagnostic test.Plasmodium species was changed to dihydroartemisinin-piperaquine (DHP) plus 14 days of unsupervised primaquine  for patients with vivax malaria. At the same time, the first-line treatment of severe malaria was changed from intravenous quinine to intravenous artesunate [Before 2006, oral quinine was the first-line treatment for falciparum malaria in the hospital with a 14-day course of primaquine added for patients with vivax malaria . After atesunate .Plasmodium species. Patients younger than 1 month were excluded to avoid the confounding effects of congenital infection and perinatal mortality [Plasmodium species at each malaria episode. Other potential confounders included in the analysis were age, ethnicity  and sex.This was a retrospective analysis of routinely collected data including all patients older than 1 month who presented to RSMM between April 2004 and December 2013 at least once with malaria due to any The impact of multiple recurrences on adverse outcomes  was addressed using multi-state modelling –16 to quearly hospital admissions or death. Patients who survived the initial 14-day period were considered to be at risk of late hospital admission and death. To determine the overall effect of the risk factors, the model was fitted initially to the data assuming that the risk factors for re-presentation and admission/death had the same effect across the multiple re-presentations. The transition-specific effects of risk factors are presented in Section B of Additional file The cumulative probability of re-presentation, hospital admission and death following each of the four episodes was estimated from the multi-state models and, throughout this article, is referred to as the risk of these outcomes. Cox proportional hazard modelling was used to estimate the rates of transitioning from each malaria episode to another re-presentation or admission/death and distinct baseline hazards were considered for each transition. All of the reported HRs in this manuscript are estimated using the multivariable model, adjusting for the effect of potential confounders. The results of the univariable analyses are presented in Section C of Additional file mstate [survival [The proportional hazards assumption was tested by visual inspection of the cumulative hazards. The Cox regression analyses were stratified by year to account for effect modification over the study period, due to the changing efficacy of prescribed antimalarial treatment. Since all episodes of malaria within 15 days of an initial infection were likely to reflect the acute illness rather than recurrence, the dataset for Model (1) was concatenated so that all outpatient re-presentations with malaria within this period were counted as a single event. For Model (2), the 15-day concatenation was performed on any re-presentation with malaria due to either the outpatient or inpatient departments. All statistical analyses were performed using R, version 3.5.2 , and themstate  and survsurvival  packagesP. falciparum, 22,209 (32.5%) to P. vivax and 7234 (10.6%) to mixed infections. Plasmodium malariae and P. ovale accounted for 1712 (2.5%) and 38 (0.1%) episodes respectively; Table During the study period, there were a total of 1,054,674 clinical presentations to the RSMM hospital, generated by 162,966 individuals. In total, 68,361 patients older than 1 month presented at least once with malaria. At the first presentation, 37,168 (54.4%) of the infections were attributable to admission); Model (2) where the terminal event is death due to any cause. The re-presentations in Model (1) are only those hospital attendances in which outpatients received antimalarial treatments. However, in Model (2), re-presentation could be either patients receiving antimalarial treatment in the outpatient clinic or hospital wards. Figure The schematic of the multi-state model and the number (and percentage) of patients re-presenting with malaria, requiring hospital admission (due to any cause) and dying within 12 months of each malaria episode are given in Fig. The times to re-presentation with malaria and all-cause admission to hospital in Model (1) and the times to re-presentation with malaria and all-cause deaths in Model 2) are presented in Fig.  are presPlasmodium falciparum was the most prevalent species at the first presentation in patients who re-presented to hospital, comprising 46.4% ) of transitions over episode 1 → episode 2. Thereafter, mono-infection with P. vivax was the main cause of re-presentation  and 44.0% (885/2009), respectively). However, after the third and fourth episodes of malaria, P. vivax prevailed over other species (48.5% (415/855) and 50.4% (200/397), respectively) , for re-presentation and death presentations with P. falciparum; for P. vivax, the percentage was 56.4%. By counting the mixed infections at episodes 1–3 as either P. falciparum or P. vivax, these percentages rise to 63.0% and 70.3% for P. falciparum and P. vivax, respectively.Following the first and second episode of malaria, P. vivax was significantly higher compared to P. falciparum across all of the episodes  following the first episode to 58.6%  following the 3rd episode ). Patients with mixed infection also had a higher rate of re-presentation to hospital than those infected with P. falciparum ) . PatieA total of 10,868 patients were admitted to hospital following either their initial or subsequent episodes of malaria. Overall, 5381 (49.5%) of admissions were due to malaria, and 1696 (15.6%) admissions occurred within 14 days of the initial malaria episode.P. falciparum, the risk of early admission decreased significantly with malaria recurrence, but this was not apparent for patients with P. vivax. By the third episode of re-presentation with any malaria species, the risk of early hospital admission following P. vivax infection was 2.45%  compared to 1.64%  following P. falciparum  after the first presentation with malaria, but fell to 1.53%  after the 4th episode  were similar, 9.19% 95%CI 8.96, 9.42) following the first episode and 8.98%  following the second episode, but declined thereafter, falling to 7.02%  after the fourth episode ); both rates of early and late admission  and 1.21  respectively) were greater after mixed infections compared with P. falciparum. Furthermore, the hazard ratio for the rate of late admissions following P. vivax infection compared to P. falciparum was highest in young children, with an HR of 1.25  in infants less than 1 year old and 1.36  in children aged 1 to 5 years. The corresponding HRs were 1.06  in older children (5 to 15 years old), and 1.09  in adults older than 15 years ) and late ) hospital admission  P. falciparum, the risk of early death was 0.76%  following the first episode of malaria and 0.36%  following the second episode. These risks were significantly lower in patients initially infected with P. vivax infection  and 0.14%  respectively). However, for subsequent episodes, this trend was inverted, the risk of death rising to 0.24% ) after the third episode of P. vivax compared to 0.14%  following the third episode of P. falciparum ). Similarly, mixed infections were associated with a higher rate of early death after three episodes of malaria, compared to P. falciparum )  patients died with 75.7% (679) of deaths occurring following the first episode of malaria. The risk of early death (within 14 days) was 0.65%  after the first episode of malaria, but fell thereafter  that of P. falciparum  compared to P. vivax), whereas for late death this rose to 1.16-fold   and 1.84 , respectively, and those for P. vivax were 10.26  and 2.58 , respectively.Severity of the initial episode significantly impacted the rates of early and late death . Patients with P. vivax were also offered radical cure with 14 days of primaquine to eradicate the dormant liver stages, but previous studies in this population have shown that when unsupervised this regimen is associated with very poor effectiveness [P. falciparum will also have been attributable to P. vivax, since in co-endemic areas, there is a high risk of heterologous P. vivax relapse following falciparum malaria [Patients initially presenting with nfection . The blotiveness . Further malaria –27.P. vivax infection compared to a P. falciparum infection, and this remained apparent after controlling for baseline characteristics such as age. We hypothesise that the cumulative risk of anaemia attributable to recurrent bouts of malarial haemolysis and dyserythropoiesis underlies the higher risks of adverse outcomes after repeated P. vivax infections. Indeed, in this region the haematological morbidity due to P. vivax malaria is particularly severe [P. falciparum results in a more severe acute disease than P. vivax, multiple re-presentations with vivax malaria may cause either a debilitating illness [P. vivax. In a cohort of children from Vanuatu, infection with P. vivax, but not P. falciparum, was a major predictor of acute malnutrition [P. vivax are at high risk of both acute and delayed mortality [Overall, patients were significantly more likely to have a late admission or late death respectively following a y severe –30. Alth illness  or may autrition , and ourortality , 9, 21.P. falciparum malaria was the major species causing early admission and death following the first two malaria episodes. However, after two re-presentations with malaria, this trend was inverted such that the cumulative risks of early admission and death with P. vivax infection rose to 1.5- and 1.7-fold, respectively, higher than the risks following P. falciparum malaria. Similar higher risks were observed following more than two re-presentations with P. vivax.In our current analysis, the species causing malaria exerted varying effects on morbidity and mortality across multiple malaria episodes. P. vivax infection has increased over the last 20 years [P. vivax at RSMM rose from 32% in 2004 to 54% by 2009. Whereas the risk of death due to P. falciparum over the same time period halved, the proportion of deaths due to P. vivax remained steady. The differential impact of malaria control activities on the two species is likely due to the inadequate radical cure of P. vivax and prevention of multiple relapses [In southern Papua, Indonesia, the proportion of malaria morbidity and mortality attributable to 20 years . The proP. falciparum and P. vivax; hence, the comparative hazards presented are likely to be valid and our estimates of mortality conservative. Third, our study focused on the effect of malaria species at an episode of interest, although consideration of the history of the species during the preceding malaria infections may have been more relevant to the outcome of the disease. However, accommodating the history of infections would require non-Markovian multi-state modelling and a much larger number of deaths to estimate precisely the risks associated with each species distribution of prior infections. Finally, the increased risk of all-cause mortality following the initial infection cannot be attributed solely to malaria. Our study does not address causality, but rather quantifies the difference between P. vivax and P. falciparum in the risk of early/late morbidity and mortality following recurrent episodes of malaria.Our study has some important limitations. First, the data used in this work are left-truncated; hence, the patients’ history of malaria prior to the start of data collection in 2004 is unknown. This has resulted in some disparities between the risk of hospital admissions and death for the first and the subsequent episodes. For instance, the acute condition of patients (characterised by the large number of early deaths) at the first episode may denote possible frequent malaria recurrences prior to the first presentation. Distinguishing between the early and late mortality helped to mitigate this issue. Second, due to the passive follow-up of the patients, episodes of malaria treated in the community will have been missed and therefore the true number of clinical malaria recurrences experienced by the individuals in this study will have been higher than that reported. However, the attrition bias in the detection of severe episodes of malaria requiring admission to hospital or resulting in death is likely to be low, since RSMM was the primary facility providing inpatient care in the region during the study period; this was confirmed by a community household survey of treatment-seeking behaviour in 2005, in which 82% 9/11) of children who died in the preceding year were reported to have had done so at the RSMM hospital [ of childP. falciparum is associated with a greater acute risk of severe and fatal outcome than infection with P. vivax. In absolute terms, most deaths and admissions related to malaria occur after the first clinical episode. However, malaria recurrence is also associated with increasingly poor outcomes particularly in infants and young children. Compared to patients initially presenting with P. falciparum malaria, those presenting with P. vivax were at a significantly greater risk of recurrent malaria, and this was associated with a higher risk of mortality. Whilst the acute management of malaria is paramount to prevent early death, our analysis highlights the importance of preventing recurrent malaria. The latter can be achieved either through bed-net distribution, chemoprophylaxis or, in the case of P. vivax, the delivery of safe and effective radical cure of the hypnozoite reservoir of infection.Our results highlight that infection with Additional file 1.The file contains details of the developed multi-state model and some additional statistical analyses under the following sections: A. Multi-state modelling of malaria recurrence; B. Episode-specific effects of risk factors; C. Results of univariable analysis; D. Effect modification of species on the rate of events by patients’ age; E. Effect of severity of the initial episode on mortality; F. Re-admission and death multi-state model.


A previously healthy Japanese man in his fifties was admitted to our hospital because of a recurrent fever after returning from Kenya and Madagascar. He was ambulant with a body temperature of 36.6 °C. His physical examination revealed normal except for tender hepatomegaly. The blood test results showed no apparent abnormality except thrombocytopenia and mild liver dysfunction. The rapid diagnostic test and Giemsa-stained blood film were repeatedly negative for malaria. Computed tomography scans of the chest, abdomen, and pelvis revealed no significantly abnormal findings. A previously healthy Japanese man in his fifties was admitted to the Department of Infectious Disease at Nagasaki University Hospital in Japan because of a fever after returning from a 6-month stay in Kenya. The patient stayed in Madagascar from November 20YY-1 to January 20YY prior to his stay a rural city in Kenya from January to late June 20YY to conduct behavioral research at a community hospital. According to the patient, the hospital ward consisted of many febrile patients, including malaria patients, but his research work did not involve direct contact with any patients. He also reported having many mosquito bites and walking on muddy roads since it was the rainy season. He drank tea with unpasteurized milk from water buffalo for breakfast, ate local food for lunch, and cooked supper for himself. He drank bottled water throughout the stay. He was vaccinated against hepatitis A and B, tetanus, rabies, typhoid fever, and yellow fever prior to his travel, but he had not taken anti-malarial chemoprophylaxis. He denied having sexual contact.Twenty days (day x-8) after returning to Japan, he experienced episodes of fever with headache, fatigue, and back pain. Five days before admission, his body temperature increased to more than 38 °C, and he had frequent urination. On the following day, he visited a local clinic where he was prescribed levofloxacin and acetaminophen and diagnosed with urinary tract infection. Two days before admission, his temperature increased to 39.6 °C at night and was accompanied by chills; thus, he visited our outpatient clinic on the next day (day x-1).4/μL, total bilirubin 0.8 mg/dL, AST 42 U/L, ALT 50 U/L, BUN 13 mg/dL, and creatinine 0.77 mg/dL. The urinalysis showed proteinuria (±), occult blood in the urine (−), and WBC (−). The rapid diagnostic test (BinaxNOW®) and Giemsa-stained blood film were negative for malaria. Computed tomography (CT) scans of the chest, abdomen, and pelvis revealed no significantly abnormal findings.Upon examination at the first visit, he was ambulant with a body temperature of 36.6 °C, a blood pressure of 108/85 mmHg, a pulse of 85 bpm, and a respiratory rate of 14 per minute. His abdominal examination revealed tender hepatomegaly. There was no rash, no lymphadenopathy, no bleeding tendency, and no joint pain. The remainder of the physical examination results was normal. The blood test results showed no apparent abnormality except moderate thrombocytopenia and mild liver dysfunction: WBC 6500/μL , hemoglobin 14.0 g/dL, platelets 7.4 × 10On day x, the patient was admitted for further investigation. Intravenous ceftriaxone 2 g per day was initiated after two sets of blood cultures were taken. On the second hospital day day x+1), the rapid diagnostic test and Giemsa-stained blood test were repeated, and the results were negative. However, his body temperature increased again at night. On the third hospital day, the blood culture results were negative. Ceftriaxone was discontinued, and oral minocycline 100 mg every 12 h was started. On the 5th hospital day (day x+4), his body temperature increased again at night  and PCR assays for rickettsia and Borrelia at the national reference laboratory, the National Institute of Infectious Diseases in Tokyo; and PCR assays for malaria at the Institute of Tropical Medicine at Nagasaki University.cytb was positive, and the sequence analysis showed that it matched with Plasmodium malariae. The Giemsa-stained films of the blood samples were re-examined by an experienced malariologist, and they revealed a low level of malaria parasites with the proportion of erythrocytes ranging from < 0.006 to 0.018%. Thus, the patient was diagnosed with quartan malaria and treated with artemether/lumefantrine for 3 days. On the 9th hospital day, defervescence was achieved, and the patient was discharged. The results of the Brucella agglutination reaction (B. abortus and B. canis) were negative. Anti-rickettsia antibodies (Orientia species and Rickettsia japonica) and PCR assays for rickettsia and Borrelia species were not performed eventually because malaria was diagnosed, and the fever did not respond to minocycline. One and a half years after discharge, the patient remained free from recrudescence.On the 6th hospital day (day x+5), it was reported that PCR amplification targeting malaria In evaluating febrile patients who travelled to malaria-endemic countries, it is imperative to pay special attention to exclude falciparum malaria, as it can rapidly progress and cause death unless the patient is promptly treated with anti-malaria drugs. Therefore, if there is any clinical suspicion of malaria, malaria tests should be repeated at least three times with 12- to 24-h interval to exclude malaria [P. falciparum is significantly lower (median 74.1%) if the parasitemia level was below 100/μL compared with above 100/μL (median 94.3%) [Plasmodium malariae infection, the parasitemia level remains very low; thus, the presence of parasitemia is often missed by malaria blood film tests [P. falciparum-specific histidine-rich-protein-2 (PfHRP2) and a pan-malarial antigen, Plasmodium aldolase, but the sensitivity of this test is lower for non-falciparum malaria, especially in the detection of P. ovale and P. malariae, probably due to the lower affinity of some monoclonal antibodies to these species [P. malariae infection, as the current case exemplifies. Would the patient have been treated empirically with antimalarial drugs if PCR assays had not been available? The answer highly depends on other factors, such as clinical severity, access to antimalarial drugs, and in this case, we would less likely have started empirical malaria treatment without knowledge of P. malariae disease.In the current case study, malaria was the number one suspected disease among the probable differentials, and malaria tests, including the rapid diagnosis test and blood smear examination, were repeated and showed negative results. It is worth knowing that in the setting of travel medicine, sensitivity of rapid diagnosis tests for n 94.3%)  and that species . PCR assHe also had moderate thrombocytopenia, but we considered it rather non-specific reaction and did not investigate further, because his general condition was good, and DIC was excluded. It returned to normal rapidly following malaria treatment.P. malariae is much lower than that of P. falciparum but not uncommon. According to recent reports from Kenya [P. malariae among symptomatic patients was 3.3% and 1.2%, respectively, while that of P. falciparum was 77% and 52%, respectively. Moreover, the vast majority of P. malariae infections were coinfected with P. falciparum. Therefore, P. malariae infection is often overlooked and neglected in the local population.Treating a fever of an individual who has returned from traveling abroad, especially in tropical countries, is challenging for physicians working in high-income countries because (a) they have to diagnose tropical infectious diseases, for which clinicians often have little experience; (b) some tropical infectious diseases, such as falciparum malaria, are fatal; and (c) other diseases, such as typhoid fever, lead to significant public health threats to the local population. In approaching the fever of a returned traveller, thorough and directional evaluations are essential: comprehensive history taking, examinations to differentiate critical and curable diseases, and timely consultations with experts should be conducted when necessary. Collecting the latest epidemiological information of endemic diseases is also crucial. The prevalence of om Kenya  and Madaom Kenya , the preFinally, the fever pattern should also be carefully observed. It was noted that the patient in this case study had a fever that was periodic with an exact 72-h interval, which could have been a clue for the diagnosis of quartan malaria Fig. .P. malariae infection)Quartan malaria (


Plasmodium falciparum malaria increases plasma levels of the cytokine Fms-like tyrosine kinase 3 ligand (Flt3L), a haematopoietic factor associated with dendritic cell (DC) expansion. It is unknown if the zoonotic parasite Plasmodium knowlesi impacts Flt3L or DC in human malaria. This study investigated circulating DC and Flt3L associations in adult malaria and in submicroscopic experimental infection.+ DC, CD1c+ DC and plasmacytoid DC (pDC) numbers were assessed in (i) volunteers experimentally infected with P. falciparum and in Malaysian patients with uncomplicated (ii) P. falciparum or (iii) P. knowlesi malaria.Plasma Flt3L concentration and blood CD141Plasmodium knowlesi caused a decline in all circulating DC subsets in adults with malaria. Plasma Flt3L was elevated in acute P. falciparum and P. knowlesi malaria with no increase in a subclinical experimental infection. Circulating CD141+ DCs, CD1c+ DCs and pDCs declined in all adults tested, for the first time extending the finding of DC subset decline in acute malaria to the zoonotic parasite P. knowlesi.Plasmodium infection causes no change in plasma Flt3L but does reduce circulating DCs. Plasma Flt3L concentrations increase in acute malaria, yet this increase is insufficient to restore or expand circulating CD141+ DCs, CD1c+ DCs or pDCs. These data imply that haematopoietic factors, yet to be identified and not Flt3L, involved in the sensing/maintenance of circulating DC are impacted by malaria and a submicroscopic infection. The zoonotic P. knowlesi is similar to other Plasmodium spp in compromising DC in adult malaria.In adults, submicroscopic  Plasma samples were collected from volunteers before infection and at peak infection, plasma was cryopreserved within 30 min of blood collection. For CD141+ DCs characterization and function fresh whole blood was processed within 2 h of collection. Volunteers were recruited from clinical trials registered with US NIH ClinicalTrails.gov .For participants in experimental infection studies (n = 33), inoculum preparation, recruitment, infection and monitoring was performed as described previously , 23. ForPlasma Flt3L levels were detected using human Flt-3 Ligand Quantikine ELISA kit , as per manufacturer’s instructions. Heparin plasma samples for each individual participant were tested in duplicate. Absorbance was measured at 450 nm and plasma Flt3L concentration was calculated using standards provided in the kit (minimum sensitivity less than 7 pg/mL). Mean concentration of duplicates tested is reported.9 cells/L), dividing the sum by 100, multiplying the percentage of DCs, and multiplying the product by 1000 to give the cell count/µL.200 µL of fresh whole blood or 3 million PBMCs were stained at RT with surface antibodies, CD3 (HIT3a), CD14 (HCD14), CD19 (HIB19), CD56 (HCD56), HLA-DR (L243), CD11c (B-Ly6), CD123 (6H6), CD303 (201A), CD1c (L161), CD141 (M80), all antibodies were purchased from BD biosciences or Biolegend. For whole blood, RBCs were lysed with FACS lysing solution (BD) and cells fixed with 1% (w/v) paraformaldehyde in phosphate-buffered saline. Absolute numbers of DCs were determined by adding automated lymphocyte and monocyte counts  and Flowjo V10.6 .FACS data was acquired using a FACSCanto™ II  or Gallios™  and data analysed using Kaluzaversus malaria patients). Tests were two-tailed and considered significant with p-values < 0.05.Statistical analyses used GraphPad Prism 6 . To compare within patient changes, the Wilcoxon matched-pairs sign rank test was used and the Mann–Whitney test was used to compare between patient changes , and remained decreased for at least 24 h post treatment  and at peak-infection. There was no change in plasma Flt3L levels Fig. c. There int Fig. d. Similaeviously , 7  and CD141+ DCs  numbers significantly decreased in both P. falciparum and P. knowlesi infection when compared to healthy uninfected controls  or knowlesi (p < 0.0001) malaria, compared to healthy uninfected local controls , and pDCs (7) CD141+ DCs are reduced during experimental P. falciparum infection. Similarly, this study shows that pDC, CD1c+ DC and CD141+ DCs subsets are significantly reduced during clinical falciparum and for the first time, also in knowlesi malaria. These data suggest that Flt3L released in adults with uncomplicated malaria may not be sufficient to expand or restore circulating DC subsets during acute infection. Rather, multiple signals, not just Flt3L, may be required to expand CD141+ DCs and other DC subsets [In summary, similar to classical CD1c subsets , 31.Additional file1: Figure S1. No significant correlation between plasma Flt3L and peripheral blood pDC or CD1c+ DC in induced blood-stage malaria. Correlations of DC subset numbers and Flt3L concentration on day 0 (left plot), correlation of DC subset numbers and Flt3L concentration at peak infection (middle plot) and correlation of DC subset numbers at peak infection and Flt3L concentration at day 0 (right plot) for A. pDC (red) and B. CD1c+ DCs (green) in volunteer infection studies. Spearman correlation was used and tests were two-tailed and considered significant if p-values <0.05. Figure S2. No significant correlation between plasma Flt3L and parasitemia in acute clinical malaria. Correlations between Flt3L concentration at acute infection and parasitemia (left plots), and correlations between Flt3L concentration at convalescence and parasitemia (right plots), for A. P. falciparum malaria and B. P. knowlesi malaria. Spearman correlation was used and tests were two-tailed and considered significant if p-values <0.05.


Plasmodium falciparum from RDTs. The knowledge of gametocyte carriage in the population is important to better assess the level of parasite transmission in elimination settings. The aim of this study was to detect P. falciparum gametocytes from used RDTs by quantitative PCR for molecular monitoring of malaria transmission.Malaria surveillance requires powerful tools and strategies to achieve malaria elimination. Rapid diagnostic tests for malaria (RDTs) are easily deployed on a large scale and are helpful sources of parasite DNA. The application of sensitive molecular techniques to these RDTs is a modern tool for improving malaria case detection and drug resistance surveillance. Several studies have made it possible to extract the DNA of DNA was extracted from 303 RDT devices  using the Chelex-100 protocol. qPCR was performed in a 20 μL reaction to detect and quantify transcripts of the pfs25 gene. The cycle threshold (Ct) was determined by the emission fluorescence corresponding to the initial amount of amplified DNA.The study found an overall prevalence of 53.47% with an average Ct of 32.12 ± 4.28 cycles. In 2018, the prevalence of gametocytes was higher in the Ranérou district (76.24%) than in the Saint-Louis district (67.33%) where an increase in the number of gametocyte carriers in 2018 was noted, in comparison with 2017.RDTs are a good source of DNA for molecular monitoring of gametocyte carriage. This method is a simple and effective tool to better understand the level of malaria transmission with a view to elimination. The presence of mature gametocytes (stage V) in the human peripheral blood is the determining factor in the maintenance and increase of malaria transmission . In SeneGametocytes are sexual forms of the parasite that are transmitted from the human host to mosquito vectors and thus perpetuate the transmission of malaria –6. The pSince mosquito infection requires the presence of gametocytes in humans, knowledge of the epidemiology of gametocyte carriage is an essential parameter for assessing malaria transmission and predicting infectiousness in vectors , 7, 8. SPlasmodium falciparum in RDT samples by PCR [Large-scale RDTs in endemic countries are now used as sources of parasite DNA , 11 and s by PCR . HoweverP. falciparum gametocyte carriage on used RDT samples by the quantitative real time PCR (qPCR) technique. This molecular technique for detecting gametocytes from collected RDTs can be an effective tool for molecular surveillance of malaria in northern Senegal.This study aimed to determine the RDTs were collected as part of malaria surveillance in northern Senegal where the incidence of malaria is low (< 5‰). Febrile patients who went to public or private health facilities were tested by RDTs and treated. All RDTs used (negative and positive) were stored in plastic bags at room temperature Fig. a.Fig. 1RIn Senegal, malaria transmission is closely related to the rate of rainfall and generally increases during the rainy season. The density of vector populations is dependent on rainfall.For this study, two (2) health districts of two (2) regions were concerned, including that of Saint-Louis in the Saint-Louis region and that of Ranérou in the Matam region. The large shares of malaria cases recorded in the two regions were reported in these two districts. In recent years, the district of Saint-Louis had recorded the majority of malaria cases in the Region. As part of malaria elimination, the programme of Senegal began in 2018 to use primaquine combined with the usual treatment of malaria cases in this area to reduce gametocyte carriage and malaria transmission. The 2018 epidemiological data showed that the number of malaria cases in this district had doubled compared to 2017 despite the intensification of control strategies. In order to better appreciate the impact of primaquine on gametocyte carriage, it is necessary to study the gametocyte prevalence in 2018 in the district of Saint-Louis, which will be compared with the prevalence in 2017 before using primaquine and the prevalence in 2018 in the district of Ranérou where primaquine is not used. At the time of testing, patients were informed about the protocol and their consent was obtained.A total of 303 positive RDTs were randomly selected from different time periods and sites. In Saint-Louis, we chose 101 positive TDRs completed in 2017 and 101 completed in 2018. In Ranérou 101 positive TDRs of 2018 were chosen.P. falciparum specific Histidine-Rich Protein II (HRP-II) antigen. DNA extraction was done by opening the TDR cassettes  different RDT samples  were made by depositing 5 μL of 0.5% and 1% diluted gametocytes cultures in the RDT wells. From the 1% diluted culture 50 μL were deposited on a filter paper. To these samples, two more positive RDTs were added whose gametocytes with different densities were identified by microscopy and another sample of filter paper whose PCR was positive for the detection of gametocytes as a positive control. qPCR-steps were done in duplicate for samples. The SYBR Green Master Mix has been standardized to detect and quantify Pfs25 gene transcripts using the BIO-RAD CFX96™ tool which showed positive results.P. falciparum collected worldwide [For the Pfs25 assay we used a specific female gametocytes primers tested by Schneider et al. . The Fororldwide .The real time-PCR was performed in a 20 µL reaction. The reaction was prepared with SYBR Green Master Mix. DNA template and Nuclease-Free Water were used. The Pfs25 primers used are specific for female gametocytes and showed limited polymorphism . Each 20The Bio-Rad CFX Manager 3.0 software was used for the post-amplification data analysis.Quantification cycle (Cq) or cycle threshold (Ct) determination mode was set to single threshold with baseline-subtracted curve fit and a user defined threshold of 50 relative fluorescence units (RFUs) for analysis of parasite. A cycle threshold value (Ct) < 35 was chosen for positive PCRs. Samples with a cycle value greater than 35 Ct were considered negative for PCR. The data was also exported to Excel and analysed with the Epi Info software.A qualitative and conventional qPCR protocol was standardized to detect pfs25 gene transcripts in RDT samples. With this method, 162 of the 303 RDT samples analysed (53.47%) were positive . When the Ct is low the initial amount of DNA is large. For the qPCR positive samples n = 162) gene amplification was shown before 35 cycles. The overall gametocyte prevalence was 53.47% (162/303)  in the Ranérou district and 67.33% (68/101) in the Saint-Louis district  used in the treatment of malaria cases promotes the production of gametocytes. More malaria cases treated entail more gametocyte carriers. The Saint-Louis region has experienced excessive rainfall in 2018 as compared to 2017 when a deficit in rainfall was recorded depressed , 22. ThiThe increase of gametocyte carriers is a real problem for the parasite reservoir control as an element of the malaria prevention strategy. The collected RDTs analysis in the Matam area (Ranérou district) showed a gametocyte prevalence of 67.33% 68/101). According to the National Malaria Control Programme, this district has for many years recorded the vast majority of reported cases in the region where the transmission of the disease is strong at the local level /101. Acc and it iThe collection of RDTs in the field is simple and systematic in Senegal for the surveillance of malaria. The use of this tool to determine the prevalence of gametocytes is practical and affordable. The development of this method with a large-scale estimation of gametocyte density, including on asymptomatic individuals, will increase the monitoring power and allow further elimination. PCR is a tool with good sensitivity for detection of gametocyte genes expression . The pre