In individuals living in malaria-endemic regions, parasitaemia thresholds for the onset of clinical symptoms vary with transmission intensity. The mechanisms that mediate this relationship are however, unclear. Since inflammatory responses to parasite infection contribute to the clinical manifestation of malaria, this study investigated inflammatory cytokine responses in children with malaria from areas of different transmission intensities (ranging from low to high).®-based magnetic bead array system, and levels were compared across sites using appropriate statistical tests. The relative contributions of age, gender, parasitaemia and transmission intensity on cytokine levels were investigated using multivariate regression analysis.Blood samples were obtained from children confirmed with malaria at community hospitals in three areas with differing transmission intensities. Cytokine levels were assessed using the LuminexParasite density increased with increasing transmission intensity in children presenting to hospital with symptomatic malaria, indicating that the parasitaemia threshold for clinical malaria increases with increasing transmission intensity. Furthermore, levels of pro-inflammatory cytokines, including tumour necrosis factor alpha (TNF-α), interferon-gamma (IFN-γ), interleukin (IL)-1β, IL-2, IL-6, IL-8, and IL-12, decreased with increasing transmission intensity, and correlated significantly with parasitaemia levels in the low transmission area but not in high transmission areas. Similarly, levels of anti-inflammatory cytokines, including IL-4, IL-7, IL-10 and IL-13, decreased with increasing transmission intensity, with IL-10 showing strong correlation with parasitaemia levels in the low transmission area. Multiple linear regression analyses revealed that transmission intensity was a stronger predictor of cytokine levels than age, gender and parasitaemia.Taken together, the data demonstrate a strong relationship between the prevailing transmission intensity, parasitaemia levels and the magnitude of inflammatory responses induced during clinical malaria.The online version of this article (doi:10.1186/s12936-017-1796-x) contains supplementary material, which is available to authorized users. Plasmodium falciparum parasites [In endemic areas, protection against clinical malaria results from repeated exposure to arasites , 2, sucharasites , suggestarasites –6. TheseIncrease in the breadth and magnitude of parasite-specific antibody responses following repeated parasite exposures  is expecP. falciparum infection [Plasmodium falciparum infection causes paroxysmal fever that is triggered by strong pro-inflammatory responses involving pyrogenic cytokines such as interleukin (IL)-1β and tumour necrosis factor alpha (TNF-α) [Clues to the mechanisms of parasite tolerance may lie in the role of inflammatory cytokines, which have been shown to correlate with the onset of symptomatic disease during nfection –15. Plas (TNF-α) . Althoug (TNF-α) , 17, 18, (TNF-α) , 19–23.  (TNF-α) , 25, org (TNF-α) , 27, and (TNF-α) . To prev (TNF-α) , 30.The intensity of transmission has been shown to be a major predictor of clinical manifestations and outcomes of malaria in endemic areas , 31. In Three outpatient hospitals at locations  representing distinct malaria transmission intensities in Ghana were selected for this study. Kintampo is holo-endemic for malaria with year-round transmission, and an entomological inoculation rate (EIR) of >250 infective bites/person/year . NavrongEthical approvals were obtained from the ethics committees of the Ghana Health Service, Navrongo Health Research Centre, Kintampo Health Research Centre and Noguchi Memorial Institute for Medical Research, University of Ghana, Accra, Ghana. Participation was voluntary, and written informed consent was obtained from parents/guardians of the children. Study participants were children aged 2–14 years who were showing signs of clinical malaria, and had been referred for malaria tests by the attending physician. Parasitaemia was detected by malaria rapid diagnostic tests (RDTs) and confirmed by microscopic examination of thick and thin blood smears. Parasite density was estimated by counting the number of parasites per 200 white blood cells as previously described , 39. Hae® MAP 13-Plex Kits from Millipore  were used because of their higher detection accuracy and reproducibility of results compared to other vendors [Plasma concentrations of cytokines were measured using the highly sensitive xMAP technology (Luminex Corporation), which allows the simultaneous quantification of several biological analytes in a 96-well format. The MILLIPLEX vendors . These k2) test  or One-way ANOVA or Kruskal–Wallis H (K–W) test (for continuous data sets), depending on normality of data. An across-site comparison of cytokine levels was performed with the K–W test, while Dunn’s multiple comparison test was used to reveal between-site pairwise significant differences. Spearman’s correlation analyses were performed for associations of cytokines levels with age and parasite density. In addition, a Spearman’s correlation matrix was built to detect associations between cytokines. Multiple linear regression analyses were conducted to detect the variable(s) that is/are the best predictor(s) of cytokine levels. For the regression models, cytokine levels served as the outcome variables while parasitaemia, age, gender and transmission intensity served as the predictor variables. Statistical significance was generally set at P < 0.05, however, after Bonferroni’s procedure, the critical value (α) of the regression models was adjusted to 0.004.Data analyses and graphs were done using GraphPrism version 6.01  and Minitab version 17.1.0.0 (Minitab Inc.). After initial normality tests, patients’ demographics and clinical parameters were compared across the three sites either by Pearson’s Chi square (χN = 71) < Navrongo (N = 44) < Kintampo (N = 58). The proportions of both sexes were comparable across the study sites  and Kintampo (P = 0.025), however, the ages of children from Navrongo and Kintampo did not differ (P = 0.999). Parasitaemia reflected the intensity of transmission, with children from Kintampo having higher parasitaemia compared with those in Accra (P = 0.005) and Navrongo (P = 0.070), although these differences were not statistically significant for Navrongo (Table P = 0.007), with children from Accra having significantly higher haemoglobin levels compared with those from Kintampo (P = 0.008). Although haemoglobin levels in children in Accra were also higher than those in Navrongo, this difference was not statistically significant (P = 0.076). Children from Navrongo and Kintampo had comparable haemoglobin levels (P = 0.545). The median temperature at clinic also decreased with increasing transmission intensity (P < 0.001), with children from Accra having significantly higher median temperature compared to Navrongo (P < 0.001) and Kintampo (P < 0.001). Children in Navrongo also had higher median temperature than those from Kintampo (P < 0.001). Since these children were recruited at presentation to hospital with symptoms of malaria, the pattern of parasitaemia suggests that the parasitaemia threshold for clinical manisfestation of infection seems to increase with increasing transmission intensity.To investigate the role of inflammatory responses in the development of malaria parasite tolerance in endemic areas, this study examined the relationship between transmission intensity and the patterns of cytokine production in children with malaria. Using a cross-sectional approach, a total of 173 children who tested positive for malaria by RDTs and microscopy were recruited from three community hospitals in three areas with varying transmission intensities: Accra , IL-8 (P = 0.056) and IL-6 (P = 0.260) were lower in the Kintampo group compared to the Navrongo group  was the strongest predictor of all cytokine levels, except GM-CSF, for which sex was the best predictor  anchors , 49, 50,and IL-6 –54. A paand IL-6 –57.+ T-cells are exhausted [+δ2+ T cell subset, which rapidly expands and become activated during P. falciparum infection, was recently shown to be associated with repeated infections [P. falciparum exposure, and being lowest in semi-immune individuals residing in an endemic area [In high transmission areas, more frequent infections would mean an almost ‘chronic’ state of infection . Under txhausted  from perfections . This sufections . Perhapsmic area .−CD11a+CD49d+ malaria antigen-specific CD4+ T-cells inhibit the production of IL-2, which consequently may dampen IL-12 secretion, resulting in clonal depletion of Th1 cells [Alternatively, there appears to be a mechanism that dampens pro-inflammatory responses  in childh1 cells . Similarh1 cells .P. falciparum amidst decreasing transmission intensity.Altogether, findings from this study represent significant new knowledge about the mechanisms of malaria pathogenesis and parasite tolerance. The data also provide evidence and understanding of malaria parasite tolerance, an issue of utmost importance in the context of malaria control and eradication since the adverse effects of malaria resurgences are not known. While these findings need to be confirmed by additional investigations of the cellular responses underlying the patterns of cytokine production, data presented here have implications for characterizing the pathophysiology of Additional file 1. Assay Sensitivities  of analytes.Additional file 2. The distribution of age and clinical parameters of patients’ across the study sites. (A) and (C) Red line across indicates mean while error bars represent standard deviation  (B) Data is presented as a box plot with whiskers and outliers. The box represents the inter-quartile range, while the whiskers represent the 10th and 90th percentiles. The line across the box indicates the median value, closed circles represent outliers .Additional file 3. Association between cytokines and age across the sites.Additional file 4. The regression coefficients of transmission intensity as a predictor of cytokine levels.


However, this negative association happened in Africa  but not in Asia , and in the heterozygotes  but not the homo/hemizygous . There was no association between G6PD deficiency and total severe malaria . Similarly, there was no association with other malaria species. G6PD deficiency can potentially protect against uncomplicated malaria in African countries, but not severe malaria. Interestingly, this protection was mainly in heterozygous, being x-linked thus related to gender.Glucose-6-Phosphate Dehydrogenase (G6PD) deficiency overlaps with malaria endemicity although it predisposes carriers to hemolysis. This fact supports the protection hypothesis against malaria. The aim of this systematic review is to assess the presence and the extent of protective association between G6PD deficiency and malaria. Thirteen databases were searched for papers reporting any G6PD alteration in malaria patients. Twenty-eight of the included 30 studies were eligible for the meta-analysis. Results showed absence of negative association between G6PD deficiency and uncomplicated falciparum malaria (odds ratio (OR), 0.77; 95% confidence interval (CI), 0.59–1.02;  Glucose-6-phosphate dehydrogenase (G6PD) deficiency is an incomplete hereditary X-linked hemolytic disease. This enzymopathy is widespread in the tropics and subtropics, synchronizing with endemic or formerly endemic malaria2G6PD catalyzes the reaction in the pentose phosphate pathway that generates reduced form of NADPH, which is in turn responsible for glutathione (GSH) homeostasisAlthough circumstantial evidence accumulated to support the hypothesis that G6PD deficiency is protective against severe fatal malaria17871011131411Plasmodium are only Plasmodium falciparum in 17 studies35710111819202122232425262728Plasmodium vivax in one studyP. falciparum with P. vivax, P. falciparum with P. malariae, P. vivax with P. malariae, and P. falciparum with P. vivax and P. malariae) in eight studies14153132333435P. vivax which meta-analyzed with that of Leslie 2010373839The initial search on public databases yielded 1600 study reports after excluding duplicates. Titles and abstracts screening resulted in exclusion of 1364 studies based on the exclusion criteria. Only 236 studies were considered eligible for inclusion for full-text review. Further 197 studies were excluded after full-text reading. A total of 39 studies were included, which comprises 49 data sets due to multi-center study reports. However, nine studies whose data either cannot be extracted, overlapped, or combined with the rest of the data, and thus were excluded. Among the 30 included studies, the species of The characteristics of the 30 included studies are described in details in P. falciparum malaria and malaria negative individuals. The odds ratios (OR) from 19 datasets obtained from 14 studies35710141518202224293132p = 0.07) .A meta-analysis was performed to assess the frequencies of G6PD deficiency between uncomplicated  = 0.07) . There w = 0.07) . When thSensitivity analysis was performed to investigate the effect of a single study on the outcome measures. We observed significant effects of single studies on the meta-analysis result. Interestingly, the exclusion of any of four studies101415101418p = 0.001), but not for homo/hemizygous  indicating that the association is only present in heterozygous subgroup (P = 0.002) (P = 0.04) but not with children and adult subgroup (P = 0.04)  . Also, s= 0.002) . Subgrousubgroup . When th = 0.04) . This coP = 0.007) (P = 0.1)   . The stuP = 0.1) . However= 0.122)  and c.P. falciparum malaria, we assessed whether G6PD deficiency is also associated with protection from severe P. falciparum malaria. For this analysis, the comparator was uncomplicated falciparum malaria cases. Different severe falciparum malaria symptoms were considered together and then separately in relation to uncomplicated malaria. There was no association between G6PD deficiency and severe falciparum malaria, in relation to uncomplicated malaria  (P = 0.02), but not between G6PD deficiency and severe anemia or cerebral malaria (Having identified association between G6PD deficiency and mild  = 0.20) . Even wh malaria . When do malaria . The ana malaria .Plasmodium including P. vivax, P. malariae or combined co-infections. For P. vivax malaria, the OR from six datasets obtained from four papers153032P = 0.81), however, the two non-analyzable papers, Louicharoen 2009P. vivax. Similarly, there was no negative association between G6PD and malaria in patients infected with P. malariae, as reported by three datasets from two papers32P = 0.38)   and c.P. falciparum and P. vivax was considered, the OR from five datasets derived from three papers1432P = 0.87). Besides the previous co-infection, the OR from five datasets of three papers3235P. falciparum and P. malariae . Similarly, the pooled OR from four datasets reported in Jalloh 2004P. vivax and P. malariae  or combined P. falciparum, P. vivax, and P. malariae  co-infections were considered  (When combined co-infection with both nsidered . We coul = 0.85) .et al.P. vivax and P. malariae or combinations of any two or all the three species. Due to the insufficient number of reports on these species, we could not make any evident conclusion on the exact relationship especially after considering the results of the two non-analyzable papers. Therefore, more studies investigating the association between these species and G6PD deficiency are highly recommended. A negative association was found in studies performed in Africa as compared to Asia and other continents, albeit with presence of publication bias. These results may provide evidence in support of the G6PD-malaria protection hypothesis in Africa. G6PD deficiency is endemic in Africa and the African G6PD deficient patients have relatively higher enzyme activity and milder consequences than Mediterranean or Asian patients333141539et al.P. falciparum.We observed absence of negative association between G6PD deficiency not only with uncomplicated falciparum malaria but also with severe malaria . However101415Studies have demonstrated the presence of G6PD variants in geographically diverse locations, endemic or formerly endemic for malaria; suggesting the likelihood of selection by malaria17845Conversely, G6PD deficiency was not associated with protection from severe falciparum malaria. Severe malaria is characterized by severe anemia, hyperparasitemia, and cerebral malaria. The net negative odds ratios observed for severe anemia and cerebral malaria were not statistically significant. Peculiarly, the hyperparasitemia showed significant negative association with G6PD deficiency in relation to low parasitemia. There was minimal publication bias in favor of protection that did not affect the significant association. Association with hyperparasitemia may however not only indicate severity of infection but also reinfection rate. These severity factors are associated with or arguably exacerbated by hemolysis, a characteristic feature of G6PD deficiency. However, the hemolytic effect of G6PD deficiency is self-limiting, affecting mainly older erythrocytes than the newly formed ones; thereby establishing a balance between the factors. Thus, the concept of protection from severe malaria or absence thereof remains conflicting1017The inherent disadvantage of G6PD deficiency due to the associated hemolysis could be one of the factors that account for the absence of consensus on the G6PD-malaria protection hypothesis. Also, the presence of other factors that could protect from malaria, especially hemoglobinopathies like sickle cell anemia (HbS) which also overlaps with G6PD and malaria endemicity, is considered one of the major confounders of our analysis and limitations of this study.P. vivax, or P. malariae. However, the analysis was sensitive to the effect of individual studies exclusion of which resulted in negative association with uncomplicated falciparum malaria, albeit with evidence of publication bias in favor of malaria protection. The protection was found only in African countries but not in Asian. Comparing the genotypes, the negative association was found only in heterozygous females, but not in a combined group comprising homozygous females and hemizygous males. Similarly, the negative association was present in studies including only children but not with combined group comprising children and adults. With the exception of hyperparasitemia, G6PD deficiency showed no association with severe malaria. Further studies are needed to investigate the mechanism of association with heterozygous females.To recapitulate, we inferred from this analysis that G6PD deficiency may not generally protect against uncomplicated falciparum malaria, This study was performed according to the recommendations of the PRISMA statementOnly original articles that report the association of any G6PD alteration in enzymatic activity and genetic variant and malaria were included. There was no restriction made with respect to language, publication year and study design. Case reports, reviews, theses, conference proceedings were excluded. Two reviewers independently extracted the data and subsequently verified by two other reviewers. Any disagreements were resolved by discussion and consensus between the reviewers and a third party, if necessary.Thirteen electronic databases were searched including; PubMed, Scopus, Google Scholar, ISI, WHO Global Health Library (GHL), Cochrane Review Library, IBECS, POPline, Virtual Health Library (VHL), WorldCat, New York Academy of Medicine Grey Literature Report, System for Information on Grey Literature in Europe (SIGLE), and African Journals Online databases. Searches were conducted to find relevant studies published before January 2014. The initial search strings were done using PubMed and Scopus databases using the broad search term: “((Glucose-6-phosphate dehydrogenase OR G6PD) AND  and (INDEXTERMS (glucose-6-phosphate dehydrogenase OR G6PD) AND )”. Subsequently, this search string was modified to contain search terms suitable for each database. The details of search strategy and terms used in each database are presented in All original studies (published before January 2014) describing association between G6PD deficiency and malaria were included in this systematic review. All eligible studies irrespective of publication type, study design, language and publication date were considered in the qualitative systematic review. There was no restriction on certain population, age, race, ethnicity, or geographic area. Only variables reported by two or more studies and whose primary data can be extracted were included in the meta-analysis. We limited included studies to those performed on human subjects. Reviews, case studies, case series, editor correspondences, news, letters, book chapters and studies whose data could not be reliably retrieved or extracted were excluded. We omitted studies whose data is overlapped with another included study. Two reviewers independently performed initial screening and study selection. Preliminary assessment of the title and abstracts was performed to identify relevant articles. Thereafter, full texts of eligible articles were downloaded and reviewed for qualitative analysis and potential inclusion in the data synthesis. Inclusion of a study by both reviewers was conclusive while discrepancies and disagreements as regards study eligibility were resolved by discussion and/or consensus with a third reviewer.The data extracted from the included reports include: the first author, year of publication year of data, study design, data collection (prospective or retrospective), country and city of origin, characteristics of participant population , method for genetic variants detection, number of included individuals (case and control groups), outcomes and times of evaluation. When there was no data available or if there were obvious errors  in the original publication, two successive emails were sent to corresponding authors for clarification. When no clarification or genotype information was obtained after at least two emails, we will report such studies as “no data available”. Identification and elimination of potentially overlapped datasets were done under extensive care, especially for multiple reports by the same group reporting the same factors on the same subjects.p-value etc.), if available. Forest plots showing the respective OR or SMD with their corresponding 95% CI for each dataset and for the pooled data were generated. The overall effect was measured by Z-statistics with statistical significance set at p < 0.05. More clarifications and in-depth analysis were achieved through subgroup analysis.Meta-analysis to pool data from eligible studies was performed on both RevMan v5.2Chi2 test) and I2 statisticsChi2 test of heterogeneity, statistical significance was set as p < 0.10. Fixed-effects model was adopted when there was a lack of significant heterogeneity , otherwise heterogeneity among studies is assumed and random-effects model with weighting of the studies was appliedThe Cochrane Q .Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


P. vivax to prevent relapse. Recently, it is also used to reduce P. falciparum gametocyte carriage to block transmission. However, PQ metabolites oxidize hemoglobin and generate excessive reactive oxygen species which can trigger acute hemolytic anemia in malaria patients with inherited G6PD deficiency.Glucose-6-phosphate dehydrogenase  deficiency is one of the most common X-linked recessive hereditary disorders in the world. Primaquine (PQ) has been used for radical cure of A total of 252 blood samples collected from malaria patients in Myanmar were used in this study. G6PD variant was analysed by a multiplex allele specific PCR kit, DiaPlexC™ G6PD Genotyping Kit [Asian type]. The accuracy of the multiplex allele specific PCR was confirmed by sequencing analysis.Prevalence and distribution of G6PD variants in 252 malaria patients in Myanmar were analysed. Six different types of G6PD allelic variants were identified in 50  malaria patients. The predominant variant was Mahidol , of which 91.2% (31/34) and 8.8% (3/34) were males and females, respectively. Other G6PD variants including Kaiping , Viangchan , Mediterranean , Union  and Canton  were also observed.Results of this study suggest that more concern for proper and safe use of PQ as a radical cure of malaria in Myanmar is needed by combining G6PD deficiency test before PQ prescription. Establishment of a follow-up system to monitor potential PQ toxicity in malaria patients who are given PQ is also required. G6PD gene that spans 18 kb on the X chromosome (Xq28), consisting of 13 exons separated by 12 introns  . This kit can detect eight different G6PD variants: Vanua Lava , Mahidol , Mediterranean , Coimbra , Viangchan , Union , Canton , and Kaiping . Multiplex PCR was performed in a 25 μl reaction containing 3 μl of genomic DNA (approximately 20 ng), 12.5 μl of 2× multiplex PCR Smart mix, 2 μl of primer mixture and 7.5 μl of distilled water. Amplification was performed with a denaturation at 95 °C for 15 min followed by amplification steps of 35 cycles  and a final extension at 72 °C for 5 min. Each amplicon was analyzed on 3% agarose gel to check size of amplified PCR product. Each reaction was confirmed with an internal control  following the protocols provided by the manufacturer.Genomic DNA was extracted from Escherichia coli DH5α competent cells. Positive clones with appropriate insert were selected. Nucleotide sequences of cloned G6PD regions were determined by automatic DNA sequencing. At least two or three clones from each sample were sequenced to ensure sequencing accuracy. These obtained nucleotide sequences were then analyzed using EditSeq and SeqMan in DNASTAR package .To confirm the accuracy of the multiplex allele specific PCR and clarify if there were any other mutations besides the ones identified by the PCR method, samples for each variant detected by the multiplex method were randomly selected and subjected to nucleotide sequencing analysis. Variant regions corresponding to exon 6, exon 9, exon 11, and exon 12 of G6PD gene were amplified from each genomic DNA using primers described previously . To miniP. falciparum, P. vivax, and co-infections with both P. falciparum and P. vivax, respectively.A total of 252 blood samples from Myanmar malaria patients were analyzed to estimate the frequency of G6PD deficiency and its variants in these patients. The G6PD multiplex allele specific PCR analysis of 252 blood samples revealed several types of G6PD variants in 50 malaria patients Fig. . AlthougAlthough estimated malaria cases have declined remarkably in recent years, Myanmar still has the greatest malaria incidence in the Greater Mekong Region . About 6P. falciparum infected individuals with G6PD variants  and P. falciparum infected individuals with non-G6PD variants  did not differ significantly. When including P. vivax mixed infection cases, P. falciparum infection rate in individuals with G6PD variants was 66.0% (33/50) while P. falciparum infection rate in those with non-G6PD variants was 59.4% (120/202). This result is consistent with a previous report showing that G6PD deficiency may not effectively protect patients against P. falciparum infection [Fifty out of 252 malaria patients  analyzed in this study had at least one of G6PD variants. Mahidol variant (68%) was the most common G6PD variant identified followed by Kaiping (18%), Viangchan (6%), Mediterranean (4%), Union (2%), and Canton (2%) variants. This result agreed with previous studies on G6PD variants in Myanmar showing that G6PD Mahidol was the most common G6PD variant type, even though the prevalence of G6PD variants among Myanmar population varied in different regions of the country according to ethnic variation , 20, 22.nfection . Interesnfection , 27. It Class I, severe deficiency of the enzyme with chronic non-spherocytic hemolytic anemia; Class II, severe deficiency with enzyme activity < 10% of normal; Class III, moderate deficiency with enzyme activity 10–60% of normal; Class IV, very mild to none deficiency with enzyme activity 60–100% of normal; and Class V, increased enzyme activity [P. falciparum gametocytes and/or P. vivax in Myanmar, ignoring G6PD deficiency. However, considering individuals with Class II or III G6PD deficiency have potential risk associated with clinically significant AHA during treatment with PQ, results of this study suggest that more caution is needed when prescribing PQ and other 8-aminoquinoline based anti-malaria drugs for malaria patients. It is also worthy to mention that only several major Asian type G6PD variants are screened in this study. However, considering some G6PD variants are linked to other mutations including silence mutations in the G6PD gene [This study has another limitation in that G6PD enzyme activity in malaria patients could not be checked due to technical limitation in performing enzyme assay in the field using fresh blood samples. Diagnosis of G6PD deficiency based on enzyme activity is usually cumbersome. Routine laboratory assays including spectrophotometry assay  and cytoactivity . Among vactivity . Clinicaactivity , 39. Theactivity . A singl6PD gene –43, moreP. falciparum and P. vivax accounted for most of malaria cases in areas where this study was performed. However, it is likely that P. vivax is becoming predominant species in these areas with recent decrease of falciparum malaria cases [P. vivax combining with G6PD deficiency test is needed. Establishment of follow-up system to monitor possible PQ toxicity in malaria patients who are taking PQ is also required. Recently, availability of G6PD rapid diagnostic tests (RDT) has been suggested for point-of-care detection of G6PD deficiency in the field [This study is the first report on the frequency of G6PD deficiency associated with malaria in Myanmar. The overall frequency of G6PD deficiency obtained in this study was similar to or slightly higher than the average expected frequencies for the Myanmar population , 20, 22.ia cases . Therefohe field , 44–49.


The spleen plays a pivotal role in the rapid clearance of parasitized red blood cells in patients with falciparum malaria after artemisinin treatment. Prolonged parasite clearance can be found in patients who have had a splenectomy, or those with hemoglobin abnormalities and/or reduced immunity, which are all distinguishable from artemisinin resistance. This paper reports on a case of prolonged parasite clearance in a Chinese splenectomized patient with falciparum malaria imported from Nigeria.Plasmodium falciparum. The patient had not used any chemoprophylaxis against malaria in Nigeria when he worked there as a construction worker between 2009 and 2014. The patient had three episodes of malaria in Nigeria and had a splenectomy due to a traffic accident 8 years ago from the time he was admitted to hospital. The patient was orally administrated a total of 320 mg/2.56 g dihydroartemisinin-piperaquine for 2 days and intravenously administrated a total of 3 000 mg artesunate for 18 days. The axillary temperature of the patient ranged between 37.0 and 37.7 °C from Day 0 to Day 3, and blood microscopy revealed falciparum malaria parasitemia  on Day 3. The patient was afebrile on Day 4, falciparum malaria parasitemia was continuously present and then gradually decreased on the next days, and was negative on Day 21. The patient was cured and left hospital on Day 24 after no plasmodium falciparum was found in the blood on Day 21 to Day 23. No mutation was found in the K13 propeller gene when compared with the PF3D7_1343700 K13 propeller gene reference sequence.A 35-year-old Chinese male suffered 2 days of febrile illness after returning to Zhumadian city of Henan province from Nigeria on October 1, 2014. The main symptoms were febrile, including the highest axillary temperature of 40 °C, headache, and chills. A peripheral blood smear showed parasitemia  of This is the first reported case in China of prolonged parasite clearance in a splenectomized patient with imported falciparum malaria. Artemisinin resistance should be distinguished when prolonged parasite clearance is found in a malaria patient who has had splenectomy.The online version of this article (doi:10.1186/s40249-017-0259-5) contains supplementary material, which is available to authorized users. Please see Additional file Plasmodium [In humans, the spleen is the largest secondary immune organ in the body. Important functions of the spleen include removal of old and abnormal blood cells, removal of circulating pathogens, and facilitating the development of immune responses against asmodium . It has asmodium . Parasitasmodium , 4. The asmodium  may not th People’s Hospital in Zhengzhou, China on October 6, 2014, because he had a fever with the highest axillary temperature of 40 °C, headache, and chills. Prior to admission, the patient had suffered 2 days of febrile illness after returning from Nigeria on October 1, 2014. The patient had not used chemoprophylaxis against malaria in Nigeria when he worked there as a construction worker between 2009 and 2014. The patient had three episodes of malaria and had been administrated quinine, but stopped treatment as soon as he was afebrile. The patient also had a splenectomy due to a traffic accident 8 years ago from the time he was admitted to hospital.A 35-year-old Chinese male was admitted to the 6Plasmodium falciparum. Microscopy experts at the Henan Center for Disease Control and Prevention confirmed this result. The density of malarial parasites was 53 913 asexual parasites/μl on Day 0. A rapid diagnostic test  was also positive for malarial antigen. DNA of P. falciparum was identified by nested polymerase chain reaction (PCR) amplification of the small-subunit ribosomal ribonucleic acid (18ssrRNA) genes. The K13 propeller gene was amplified using nested PCR and sequenced. No mutations were found when this gene was compared to the PF3D7_1343700 K13 propeller gene reference sequence retrieved from the National Center for Biotechnology Information (NCBI) database.Upon admission, the patient’s axillary temperature was 37.2 °C, his pulse rate was 75 beats/min, his blood pressure was 93/79 mmHg, and his weight was 86 kg. Initial laboratory test results were almost normal except for a high C-reactive protein level of 35.8 mg/L and a low kalaemia level of 3.30 mmol/L  for 4 days and intravenously treated with the antibiotic ceftriaxone (2.0 g per day) for 4 days. After 72 hours of treatment, the axillary temperature of the patient was 37.4 °C and the density of falciparum parasites was 26 674 asexual parasites/μl (see Table P. falciparum malaria [Malaria had a wide geographical distribution in China but this has decreased dramatically due to the strengthening of malaria control measures in recent decades, especially in the Yunnan and Hainan provinces, and Huanghuai region –8. No lo malaria , and ora malaria . Typical malaria . The eme malaria , 18 and  malaria –21, Thai malaria , 23, and malaria .P. falciparum malaria have been reported in Nigeria [P. falciparum malaria imported from the Democratic Republic of the Congo [Over the past decade, African countries have transitioned from using chloroquine or sulfadoxine-pyrimethamine to ACTs as a first-line policy for treating uncomplicated malaria . Althoug Nigeria . There hhe Congo . RoutineProlonged parasite clearance is the basis for the current WHO working definition of artemisinin resistance  but it mPlasmodium-infected erythrocytes lose their normal deformability and become susceptible to splenic filtration, and splenic clearance of erythrocytes is enhanced by antimalarial therapy and splenomegaly [Plasmodium spp., not only because it limits the acute infection through the removal of parasites from the blood stream, but also because it modulates parasite antigen expression on the surface of infected RBCs as well as cellular and humoral immune responses [The spleen is responsible for initiating immune reactions to blood-borne antigens and for filtering the blood of foreign material, and old or damaged RBCs. It plays a crucial role in the defense against infections with viruses, bacteria, fungi, and parasites . The splnomegaly . The intesponses . Other cesponses . Falcipaesponses . HIV infesponses .P. knowlesi malaria [P. falciparum reached significantly higher levels, and mature parasite stages were more often seen in the peripheral blood of asplenic individuals [P. falciparum, fever and parasitemia are significantly more frequent in splenectomized patients than in spleen-intact patients [The delay in parasite clearance has also been reported in a splenectomized patient with  malaria . In a prividuals . In areapatients –42. Splepatients , 35, 43.patients .Recently, a molecular marker of artemisinin resistance was identified. Mutations in K13 propeller domain were shown to be associated with delayed parasite clearance in vitro and in vivo , 45. TheThis paper reports a case of prolonged parasite clearance in a splenectomized Chinese worker with falciparum malaria imported from Nigeria. Artemisinin resistance should be distinguished when prolonged parasite clearance is found in a malaria patient who has had splenectomy.


Imported malaria in France is characterized by various clinical manifestations observed in a heterogeneous population of patients such as travelers/expatriates and African migrants. In this population, host factors and parasite biomass associated with severe imported malaria are poorly known.From data collected by the Centre National de Référence du Paludisme, we identified epidemiological, demographic and biological features including parasite biomass and anti-plasmodial antibody levels  associated with different disease severity groups  in 3 epidemiological groups .P. falciparum, antibody levels, plasma PfHRP2 levels, total and circulating parasite biomass were related to severe malaria onset. Sequestered parasite biomass tended to be increased in very severe malaria, and was strongly correlated to the antibody level of the host.Age, ethnicity, absence of prior infection with P. falciparum is associated with high anti-plasmodial antibody levels which influence clinical presentation of imported malaria and its correlated circulating and sequestered parasite burden.Prior exposure to  Plasmodium falciparum infection in tropical regions also threatens non-endemic countries through imported cases  for non-normal distributions. Categorical variables were evaluated according to size and frequency. Quantitative variables with highly skewed distributions ) were log-transformed for statistical analysis and graphic representation. Chi-square test and Fisher’s exact test were used to compare categorical variables between groups. ANOVA and Kruskall-Wallis tests were used to compare quantitative variables between groups when appropriated. For all tests, a difference was considered significant when p<0.05. All reported p values are two-tailed. Bonferroni’s correction was performed for multiple comparisons between quantitative variables and groups.®, College station, Texas, USA).The association between the presence of anti-plasmodial antibody as binary variable (positive/negative serology) and adjusted potential risk factors  as disease severity sub-group , epidemiological sub-group  and age as binary variable (age<18 years-old/age>18 years-old) was investigated using logistic regression. Secondly, the association between imported malaria onset and adjusted potential risk factors  as the categorical variable epidemiological sub-groups  and antibody levels as well as age, total, circulating and sequestered parasite biomass as continuous variable was also investigated by logistic regression. Four multiple linear regressions models were finally performed to identify influential factors of PfHRP2 and parasite biomass  as the continuous variable age, disease severity and epidemiological sub-groups and antibody levels. Significant positive or negative relationships between variables were expressed by the coefficient values. For regression models, two-way interaction terms between variables were tested, but none were significant. Statistical analyses were performed using STATA, version 12  comprising 55 very severe malaria and 83 moderately severe malaria cases, and 195 were uncomplicated malaria cases (58.6%). Demographic, epidemiological, clinical and biological characteristics of the UM group and SM sub-groups are presented in Among the 333 Age of patients varied according to disease severity sub-groups p = 0.002) , with ve , with v13 [2.8 X 1012–2.5 X 1013] in very severe malaria, 4.4 X 1012 [1.5 X 1012–1.1 X 1013] in moderately severe malaria and 7.9 X 1011 [3 X 1011–2.3 X 1012] in uncomplicated malaria, p<0.001 for both comparisons; Pcirc: 2 X 1012 [1 X 1012–4.2 X 1012] in very severe malaria, 1.5 X 1012 [9.9 X 1011–2.3 X 1012] in moderately severe malaria and 2.5 X 1011 [1.0 X 1011–3.5 X 1011] in uncomplicated malaria, p<0.001 for both comparisons), but were similar in very severe malaria and moderately severe malaria groups  and 183 uncomplicated malaria cases. The plasma levels of PfHRP2 were significantly higher in the very severe malaria and moderately severe malaria group compared to uncomplicated malaria , but were similar in very severe malaria and moderately severe malaria groups . Total . Total aa groups . UnexpecP. falciparum plasma antibodies at D0 were determined for 329 patients, and classified as negative in 93 cases (28.3%), positive in 179 cases (54.4%), and strongly positive in 57 cases (17.3%). Antibody levels differed according to disease severity sub-groups (p = 0.023), negative antibody levels were more frequent in moderately severe malaria compared to uncomplicated malaria . Antibody levels also varied with regard to malaria exposure (p<0.001). Negative serology was less frequent in the first-generation migrants group (p<0.001)  .In multivariate analysis, positive antibody titers were more likely observed in uncomplicated malaria . Negative antibody levels were more likely observed in second-generation migrants and travelers/expatriates groups  .Logistic regression was performed to identify risk factors for severe imported malaria onset from age, ethnic origin, antibody levels, and parasite biomass  Tables. Linear regression was performed to identify variables influencing PfHRP2 and parasite biomass values considered as risk factors of severity, from significant variables previously identified between disease severity sub-groups  and from theoretical variables  . A negatA wide variety of clinical presentation could be seen in the context of imported malaria. Through the CNRP network and database, the study of imported severe and non-severe malaria cases, as defined by the WHO criteria established in 2000 and still in force in France, reaffirmed since 2013  despite P. falciparum in travelers/expatriates may explain this difference in clinical presentation. The high rate of prior history of malaria reported in 73.2% in the first-generation migrants groups corroborated our observations. Accordingly, it may be hypothesized that the potential protective effect of prior exposure to P. falciparum against SM, likely depends on the frequency and number of prior exposures [Plasmodium spp. but not with regard to anti-malarial immune protection, providing quantitative but not qualitative information on the antibody response. The hypothesis of probable long-term protection in first-generation migrants is consistent with our results [As reported previously, severe malaria patients, and particularly very severe malaria patients, are older than uncomplicated malaria patients , 16, 25,xposures , 27, 28. results . Persist results , 30. How results , 32. MorP. falciparum strain and the population studied, which are thus central factors in this limitation, as already discussed by Dondorp et al., [Plasma PfHRP2 concentrations, Ptot and Pcirc correlated with the severity of malaria infection as observed in univariate and multivariate statistical analysis, but did not differentiate severe forms , 33. Mat et al., . However et al., . As we u et al.,  sequeste et al., . High anOur study utilizing the CNRP network is subject to limitations. Recording of imported malaria cases is estimated to be 52% complete, and the percentage of sample transfer after notification was 75.8% in 2014, leading to a possible underestimation of particular clinical presentations as cerebral malaria . MoreoveP. falciparum antibody response, correlated to prior exposure to P. falciparum, influent the clinical presentation of imported malaria. Parasite biomass may also determine the nature of the clinical presentation and significantly interact with antibodies titers. Our data suggest the key role of prior exposure to P. falciparum in inducing protection against VSM, generating antibody levels which significantly reduce the circulating and sequestered parasite burden. A more accurate qualitative study of the protective antibody repertoire is needed to understand the host-parasite interactions in non-exposed patients in imported malaria context.Our study of imported malaria confirms that ethnic origin, and anti-S1 TablePtot, estimated total parasite biomass; Pcirc, estimated total circulating parasite biomass; Pseq, estimated sequestered parasite biomass.(DOCX)Click here for additional data file.S2 TableFGM, first-generation of migrants; SGM, Second-generation of migrants; T/E, Travellers/expatriates; VSM, very severe malaria; CI, Confidence interval; Ptot, estimated total parasite biomass; Pcirc, estimated total circulating biomass; Pseq, estimated sequestered parasite biomass. Multivariate analysis was performed on 315 patients for PfHRP2 (model 1) and 176 patients for Ptot, Pcirc and Pseq (model 2–4 respectively). Age, malaria exposure, serology status and log-transformed PfHRP2, Ptot, Pcirc and Pseq were selected, considered as influent factors. FGM and strongly positive serology were chosen as reference group for the analysis whereas age, PfHRP2, Ptot, Pcirc and Pseq were considered as continuous variable. Factors have a significant influence if p value<0.05.(DOCX)Click here for additional data file.S3 TableFGM, first-generation of migrants; SGM, Second-generation of migrants; T/E, Travellers/expatriates; VSM, very severe malaria; CI, Confidence interval; Ptot, estimated total parasite biomass; Pcirc, estimated total circulating biomass; Pseq, estimated sequestered parasite biomass. Multivariate analysis was performed on 315 patients for PfHRP2 (model 1) and 176 patients for Ptot, Pcirc and Pseq (model 2–4 respectively). Age, malaria exposure, serology status and log-transformed PfHRP2, Ptot, Pcirc and Pseq were selected, considered as influent factors. FGM and strongly positive serology were chosen as reference group for the analysis whereas age, PfHRP2, Ptot, Pcirc and Pseq were considered as continuous variable. Factors have a significant influence if p value<0.05.(DOCX)Click here for additional data file.S4 TableFGM, first-generation of migrants; SGM, Second-generation of migrants; T/E, Travellers/expatriates; VSM, very severe malaria; CI, Confidence interval; Ptot, estimated total parasite biomass; Pcirc, estimated total circulating biomass; Pseq, estimated sequestered parasite biomass. Multivariate analysis was performed on 315 patients for PfHRP2 (model 1) and 176 patients for Ptot, Pcirc and Pseq (model 2–4 respectively). Age, malaria exposure, serology status and log-transformed PfHRP2, Ptot, Pcirc and Pseq were selected, considered as influent factors. FGM and strongly positive serology were chosen as reference group for the analysis whereas age, PfHRP2, Ptot, Pcirc and Pseq were considered as continuous variable. Factors have a significant influence if p value<0.05.(DOCX)Click here for additional data file.


Malaria remains a major source of morbi-mortality among travellers. In 2007, a consensual multicenter Primary Care-Hospital shared guideline on travel-prior chemoprophylaxis, diagnosis and clinical management of imported malaria was set up in the Barcelona North Metropolitan area. The aim of the study is to assess the evolution of malaria cases in the area as well as its clinical management over the 10 years of its implementation.Plasmodium vivax cases. The number of patients who attended the pre-travel consultation was low (13.7%) as well as those with prescribed chemoprophylaxis (10%). Severe malaria was diagnosed in 34 (17.9%) patients and ICU admittance was required in 2.6% of them. Organ sequelae  were recorded in 3 patients at hospital discharge, although all three were recovered at 30 days. None of the patients died. Patients complying with severity criteria were significantly males (p = 0.04), came from Africa (p = 0.02), were mainly non-immigrant travellers (p = 0.01) and were attended in a hospital setting (p < 0.001). The most frequently identified species was Plasmodium falciparum (64.2%), P. vivax (23.2%), Plasmodium malariae (1.6%) and Plasmodium ovale (1.1%). Those patients diagnosed with P. falciparum malaria came more often from sub-Saharan Africa (p < 0.001) and those with P. vivax came largely from the Indian sub-continent (p = 0.003). Among the 126 patients in whom an immunochromatographic antigenic test was performed, the result was interpreted as falsely negative in 12.1% of them. False negative results can be related to cases with <1% parasitaemia.A total of 190 malaria cases, all them imported, have been recorded. The overall estimated malaria crude incidence was of 0.47 cases per 10,000 population/year (95% CI 0.34–0.59) with a slight significant positive slope especially at the expense of an increase in Indian sub-continent P. vivax cases imported from the Indian sub-continent. Although severe malaria cases have been frequently reported, none of the patients died and organ sequelae were rare. Conceivably, the participation of the Primary Care and the District and Third Level Hospital professionals defining surveillance, diagnostic tests, referral criteria and clinical management can be considered a useful tool to minimize malaria morbi-mortality.After 10 years of surveillance, a moderate increase in malaria incidence was observed, mostly  Nowadays malaria can be considered as a non-spreading disease—even as an infection of declining-incidence infection—in most of the countries classically regarded as highly endemic. Nevertheless, this falling number of cases has been not so evident in the European Union (UE) countries where imported malaria remains a major source of morbi-mortality among travelers , especiaAfter the first approval and implementation of the shared malaria guideline (in 2007), an observational, prospective and multicentre surveillance study has been performed in all the health facilities—located at either hospital or primary health levels—with diagnostic capability in the North Metropolitan Area of Barcelona  during the period January 2007 to December 2016. Sentinel clinicians  from the PROSICS MetroN study group voluntarily recorded all consecutive individuals with confirmed malaria at four care levels: (1) one teaching third-level hospital , (2) one International Health Unit , (3) one district hospital  and, (4) the territorial 22 Primary Care centers  also belongs to the same public provider. Individuals were considered as infected cases when any clinical suspicion of acute disease defined as the presence of antecedents of travelling to endemic areas and a clinical compatible presentation was confirmed by means of one positive thick or thin blood film. In most of the cases, in addition, an antigenic immunochromatography test  was performed. Diagnostics based on a high clinical suspicion, but with negative tests and patients residing outside the Health Area were excluded from the study.Plasmodium species , parasitaemia percentage, severity criteria (yes/no), care level , treatment, clinical evolution and outcome.The variables assessed were: age, sex, immigrant (yes/no), risk factors for severe malaria, travel destination, trip length (days), reason for travel , prior travel advise (yes/no), chemoprophylaxis (yes/no), chemoprophylactic drug  or stand-by treatment (atovaquone + proguanil), diagnostic method , Plasmodium in thin blood film; proportions of <0.5% were assumed to this value. A malaria case was considered as severe when a least one WHO criteria was identified: Glasgow (adults) <11 or Blantyre (children) coma scores) <3, Hb <5 g/dL, creatinine >3 mmol/L, pH < 7.25, hypoglycaemia <2.2 mmol/L, jaundice or bilirubin >3 mg/dL, oxygen saturation <92% or radiologically-compatible signs of APO, haemoglobinuria, spontaneous bleeding (excluding episodic epistaxis) or disseminated intravascular coagulation (DIC) criteria, seizures, shock  and parasitaemia >4%. Uncomplicated malaria cases were treated whether with atovaquone + proguanil or chloroquine depending on the species and the resistance pattern. When P. vivax/P. ovale was identified, patients received a subsequent treatment with primaquine phosphate 30 mg/kg per 14 days. Severe malaria cases were treated with quinine i.v. plus doxycycline according to the WHO-based guideline; since mid 2013 artesunate was used instead.Every person born outside the European Union (EU) was considered as immigrant. Immigrants with imported malaria epidemiologically related with a recent travel to their native country after residing for >2 years in the EU were considered as Visiting Friends and Relatives (VFRs). The major risk factors for malaria were pregnancy, age < 5 years and immunodeficiency. When assessing travel destination, a categorization according biogeographic areas was used instead of countries visited, as follows: sub-Saharan Africa, America and the Indian sub-continent (no cases have been recorded from South–East Asia or Oceania). Parasitaemia percentage refers the proportion (%) of erythrocytes with visible asexuals forms of y = mx + b; in which y = number of cases and x = time (years); m can be deduced from the Least Squares Method and represents the fact that the difference in the y coordinate between two points on a line  is proportional to the difference in the x coordinate . In short, the proportionality constant m represents the slope of the trendline and b the point where the graph crosses the y-axis. Furthermore, a tendency test (IRR: incidence relative risk) was performed by Poisson Regression.The relative frequency of the variables and their association with socio-demographic characteristics were analysed using SPSS 19.0 software . The Chi square test, with Fisher’s correction if needed, was used to compare qualitative variables. The p value was set at 0.05 for statistical significance. In order to evaluate the evolution of the disease, two time periods were defined: period 1 (2007–2011) and period 2 (2012–2016). In this line, the trend in the incidence of malaria during the study period was estimated by defining its trendline (a linear equation), which was calculated according to a model Authorization for the execution of the study was obtained after evaluation by the Ethical Review Boards of the Primary Health Area, both hospitals and the Public Health Service (PI-17-047).A total of 190 malaria cases, all them imported, have been recorded in 187 patients ; 164 (87.8%) out of those 187 were immigrants. Up to 146 (76.8%) immigrants were considered as VFRs while 18 (9.5%) malaria cases were diagnosed among newly arrived immigrants. Thus, the overall estimated malaria crude incidence was of 0.47 cases per 10,000 population/year (95% CI 0.34–0.59). Among immigrants, that ratio showed an incidence of 2.5 cases per 10,000 population/year (95% CI 2.21–2.80).On the whole, the number of patients who attended the pre-travel consultation was low  as well as those with prescribed chemoprophylaxis . All those 19 patients with malaria in spite of a proper chemoprophylaxis prescription came from sub-Saharan Africa; 17 (89.5%) of them abandoned the treatment during the travel  and 2 were considered as real chemoprophylactic failures (both involving mefloquine).P. vivax cases though none of both African or Indostani linear trend reach significance when analysed separately as shown in Fig. Socio-demographic and clinical data with a further bivariate analysis in relation to the presence of severity criteria and the malaria casuistic comparing two 5-years long periods are shown in Tables P. falciparum  and, subsequently, P. vivax , P. malariae  and P. ovale . Co-infection was recognized in 5 (2.6%) cases: 1 P. falciparum + P. malariae and 4 P. falciparum + P. ovale. Forteen cases (7.4%) were labeled as Plasmodium spp. Those patients diagnosed of P. falciparum malaria came more often from Sub-Saharan Africa (p < 0.001) and those with P. vivax came largely from the Indian sub-continent (p = 0.003). Among the 124 patients in which an immunochromatographic antigenic test was performed, the result was interpreted as falsely negative in 15 (12.1%) of them. False negative results can be related to lower <1% parasitaemia but no relation with the specific infecting specie was found. The distribution of cases according to identified species, parasitaemia degree and the performance of the antigenic test are shown in Table The most frequently identified species was During the 10 years-long study period, up to 127 malaria episodes (66.8%) in 125 patients were hospitalized and severe malaria was diagnosed in 34 (17.9%) patients. In order of frequency, the most recorded criteria of severity were hyperparasitaemia (19 patients), DIC (7), hyperbilirubinaemia (4), APO (3), Hypercreatinaemia (2), seizures (1) and low Glasgow score (1); three patients having two concurrent criteria. ICU admittance was required in 5 (2.6%) of them. Organ sequelae  were recorded in 3 patients at hospital discharge, although all three were recovered at 30 days. None patient died.P. vivax and, in a less number, of P. falciparum cases coming from Pakistan from 2012 onwards in an area where Indostani immigrants are the larger immigrant community. A similar pattern has been described in other European countries [Plasmodium identifications) [P. vivax malaria have taken place in areas ecologically similar [Different quantitative and qualitative aspects have to be taken into consideration when addressing the impact of imported malaria. With regard to quantitative relevance of the disease, surprisingly, there are very few studies estimating incidences of malaria in European countries and, moreover, they diverge considerably because of the difficulty of knowing the denominators. In the whole, the study show malaria incidences tending to be more in line with those of the two major importing-countries (France and the UK), both stating around 2000 cases/year, than those observed in Spain (with over 300–500 cases/year) . Some deountries , 7 in cocations) . This as similar   are irreplaceable, and make it necessary to endow primary care centres with specific care-points and expert personnel. During the ten years-period, two of these care-points were fully accessible, located at the International Health Unit and in the third level Emergency Area hospital. After the analysis of data, the use of immunochromatography has been abandoned.An integrated assistance model allows malaria cases to be treated at different levels of specialization without causing greater morbidity than that described in other studies. Such an efficient model was based in a net of trained general physicians able to distinguish between mild and severe malaria cases always with a close, quick and continuous support of an International Health Unit with full-time specialized personnel. Recent immigrants from sub-Saharan Africa most of them VFRs partially semi-immune to malaria and with lower risk of death  , take adA similar inter-level model could be especially adequate in areas with presence of a large proportion of African VFRs travellers, a population with little proclivity to pre-travel advice and generally reduced risk of severe malaria . In a reThe study has showed one of the lowest mortality rates (none death) ever recorded in Europe despite the fact that the percentage of severe malaria cases was 17.9%, a proportion which is close or slightly above what has been previously published . This rePlasmodium unidentified species (7.4%), particularly at primary care level. Possibly the guideline focuses more on organizational issues than on microbiological ones, an aspect to be addressed in future updates.One of the main study weaknesses concerns the relatively high number of P. vivax cases imported from the Indian sub-continent. In areas with substantial numbers of VRF immigrants, a proactive attitude and the use of updated consensual specific guidelines with the participation of primary care, district and third level hospital professionals defining surveillance, diagnostic tests, referral criteria and clinical management could be a useful tool to minimize severe cases and mortality.After 10 years of surveillance, malaria remains a relevant health problem with an even moderate increase at the expense of


Plasmodium species, Plasmodium falciparum. However, the type of RDT utilized in Sénégal does not detect other malaria-causing species such as Plasmodium ovale spp., Plasmodium malariae, or Plasmodium vivax. Consequently, there is a lack of information about the frequency and types of malaria infections occurring in Sénégal. This study set out to better determine whether species other than P. falciparum were evident among patients evaluated for possible malaria infection in Kédougou, Sénégal.Expanded malaria control efforts in Sénégal have resulted in increased use of rapid diagnostic tests (RDT) to identify the primary disease-causing P. vivax, P. ovale spp., and P. malariae were developed and validated by sequencing and DNA extracted from 475 Plasmodium falciparum-specific HRP2-based RDT collected between 2013 and 2014 from a facility-based sample of symptomatic patients from two health clinics in Kédougou, a hyper-endemic region in southeastern Sénégal, were analysed.Real-time polymerase chain reaction speciation assays for Plasmodium malariae (n = 3) and P. ovale wallikeri (n = 2) were observed as co-infections with P. falciparum among patients with positive RDT results (n = 187), including one patient positive for all three species. Among 288 negative RDT samples, samples positive for P. falciparum (n = 24), P. ovale curtisi (n = 3), P. ovale wallikeri (n = 1), and P. malariae (n = 3) were identified, corresponding to a non-falciparum positivity rate of 2.5%.P. falciparum malaria infections occur in Sénégal. Current RDT used for routine clinical diagnosis do not necessarily provide an accurate reflection of malaria transmission in Kédougou, Sénégal, and more sensitive and specific methods are required for diagnosis and patient care, as well as surveillance and elimination activities. These findings have implications for other malaria endemic settings where species besides P. falciparum may be transmitted and overlooked by control or elimination activities.These findings emphasize the limitations of the RDT used for malaria diagnosis and demonstrate that non-The online version of this article (doi:10.1186/s12936-016-1661-3) contains supplementary material, which is available to authorized users. Plasmodium species that infect humans, Plasmodium falciparum and Plasmodium vivax are responsible for most of the global mortality and morbidity. However, with intensified efforts to control the incidence of these primary species, there is rising concern over the prevalence of additional malaria species including Plasmodium ovale spp. and Plasmodium malariae into the niches previously occupied by P. falciparum and P. vivax [Among P. vivax –3. ShoulP. falciparum. For example, there are increasing reports of P. ovale spp. in various countries across the African continent, including Ethiopia, Uganda, Equatorial Guinea, and Kenya [ad hoc reports of malaria infections caused by species other than P. falciparum in Sénégal. While previous reports in Sénégal have indicated low or reduced incidence of these species [P. malariae is relatively under-reported in these regions [P. vivax infection were detected in Sénégal [Globally, there have been sporadic reports of changing trends in the prevalence of malaria species other than nd Kenya –3. Histo species , 5, ther regions . Recentl Sénégal . HoweverThe region of Kédougou in southeastern Sénégal is an area of high malaria transmission that borders the countries of Mali and Guinea. It is characterized by relatively high humidity and an ecology that supports seasonal malaria transmission, with entomological inoculation rates between 100 and 200 and incidence rates of greater than 25 per 1000 individuals –10. HumaP. ovale spp., P. malariae, and P. vivax. These assays were applied to analyse nearly 500 rapid diagnostic tests (RDT) from two health centers in Kédougou, Sénégal in order to assess the prevalence of non-falciparum species in this region.In addition to using previously-reported methods, this study also developed and validated a set of real-time PCR speciation assays to detect Kédougou  is situated in the extreme southeastern part of Sénégal, in a tropical savanna located 710 km from Dakar, the nation’s capital  as directed by the manufacturer.Plasmodium plasmepsin 4 gene based on previously-reported sequence data [plasmepsin 4 genes .First, species-specific TaqMan real-time assays were developed to target the nce data  . Amplification and detection were performed under the following conditions: 15-min incubation at 37 °C prior to amplification, initial denaturation for 15 min at 95 °C, then 40 cycles of denaturation for 10 s at 95 °C, and 1 min at 60 °C with data collection. P. malariae, P. vivax, and P. ovale spp. real-time assays were validated by Sanger sequencing  of the amplicon products, both from the synthesized plasmids and the amplification-positive samples identified in the current study.The specificities of thePlasmodium ovale curtisi and Plasmodium ovale wallikeri were identified among P. ovale–positive samples using PoTRA3 and PoTRA5 primers in a nested PCR amplification of the tryptophan rich repeat region as described previously [P. ovale wallikeri yielded a product of 245 bp, and P. ovale curtisi a 317 bp product.eviously . The PCRDNA sequences at loci of interest were determined by direct sequencing of the final PCR product using PoTRA5 primers Table . SequencP. ovale were additionally tested by high-resolution melting to differentiate sub-species using the porbp2 primers, as described previously [Samples positive for Amplicon products of these assays using control DNA templates and plasmid controls were confirmed to match sequences deposited to GenBank with 100% sequence concordance.Programme National de Lutte contre le Paludisme  were evaluated in this study . Furthermore, among the 288 samples negative for P. falciparum by RDT, 24 were positive for amplification of the P. falciparum PF3D7_0718800 gene  and P. ovale (n = 2); with one sample positive for all three species. Both P. ovale (n = 4) and P. malariae (n = 3) were identified from among the 288 RDT-negative samples  in Sénégal developed a control strategy implemented between 2006 and 2010 that involved distribution of RDT to all health facilities (2007), nationwide access to artemisinin combination therapies (2007 and 2008), and distribution of insecticide-treated bed nets (2007–2009). While the initial results of this strategy were encouraging based on the nationwide reported incidence of nce 2009 . Of concpression .P. vivax malaria in Kédougou [P. vivax was not observed in the population assessed in the current study, P. ovale wallikeri, P. ovale curtisi, and P. malariae were identified, none of which are tested for by the current RDT. It remains to be determined if this incidence is changing, particularly in comparison with reduced P. falciparum transmission in response to increased eradication efforts that target P. falciparum vectors and their characteristic behaviors, which might not be common in other Plasmodium species. Indeed, there is evidence of significant biological differences between P. ovale spp. and P. falciparum, with increased latency times reported in patients infected with the former and differences in clinical features between P. ovale curtisi and P. ovale wallikeri [falciparum species; to identify their source as importation or local transmission; their primary transmission vectors; and to differentiate between relapse, reinfection, or recrudescence events where applicable.The PNLP has also conducted nationwide cross-sectional surveys for disease surveillance. Kédougou, in southeastern Sénégal, is a region that borders Mali and Guinea with relatively high transmission, as indicated by entomological inoculation rates (EIRs) of 100–200 , 10. RecKédougou . Althougallikeri . AdditioP. falciparum infections as well as missing non-P. falciparum malaria infections in Kédougou, Sénégal. The current RDT likely detects most of the infections in this region, particularly those without co-infections; however, while the relative proportion of non-falciparum infections remains low, it may increase with improved patient access to diagnosis and treatment. Furthermore, the findings of the current study, including the presence of P. falciparum genetic material in RDT-negative samples and subsequent differences in positivity rates (39.3 vs. 44.4%), are similar to those of previous reports [The results of this study underscore the potential limitations of the routine use of standard RDT for malaria detection in Sénégal both in terms of missing  reports  that PCRP. falciparum may be due to either low parasite densities, below the level of detection of the RDT or lack of specific HRP2 loci in the parasite population that results in a negative diagnostic test. Reports of non-falciparum malaria are of concern with control and elimination efforts that increasingly rely on HRP2-based and P. falciparum-specific RDT, as these may miss infections that contribute to continued malaria transmission, and thus may undermine elimination efforts and impact morbidity and mortality due to malaria. Failure to detect malaria infections other than P. falciparum is most likely is due to the specificity of the RDT, which was not designed to detect these malaria species. Regardless of the specific reasons why the RDT failed to detect these infections, these findings point to the need for appropriate diagnosis and surveillance of malaria infection to provide both adequate clinical treatment and to direct elimination strategies to address the nature of ongoing malaria infection in a given endemic setting. Future studies are necessary to assess and track the prevalence of HRP2 deletions in endemic populations in this region.The lack of detection of This study has several limitations. First, the pre-amplification step, like nested and multi-step PCR reactions, offers increased sensitivity to detect parasites at low concentrations, but prevents us from determining the threshold cycles (Ct) values of the original samples; furthermore, RDT were administered by regular health care providers rather than staff specifically trained for malaria testing. Blood smears were not obtained from the patients, and trained microscopists were not available; therefore, parasite densities in these patients were not determined. Finally, the sampling scheme was not a cross-sectional study, so there is a potential bias in the study population. Future cross-sectional studies including larger sample sizes and asymptomatic patients are necessary to validate these findings.Despite these limitations, the findings of this study suggest the importance of assessing the true prevalence of specific malaria infections using appropriate diagnostic methods to evaluate the requirements for health care services and develop appropriate strategies for malaria elimination. Such quantification is increasingly important in order for malaria elimination programmes to implement species-appropriate detection, prevention, and treatment strategies.