Plasmodium falciparum genetic diversity, population structure and gene flows were studied.Malaria incidence worldwide has steadily declined over the past decades. Consequently, increasingly more countries will proceed from control to elimination. The malaria distribution in low incidence settings appears patchy, and local transmission hotspots are a continuous source of infection. In this study, species-specific clusters and associated risk factors were identified based on malaria prevalence data collected in the north-east of Cambodia. In addition, P. falciparum was performed. SaTScan was used to determine species-specific areas of elevated risk to infection, and univariate and multivariate risk analyses were carried out.In 2012, blood samples from 5793 randomly selected individuals living in 117 villages were collected from Ratanakiri province, Cambodia. Malariometric data of each participant were simultaneously accumulated using a standard questionnaire. A two-step PCR allowed for species-specific detection of malaria parasites, and SNP-genotyping of Plasmodium species. The clusters for Falciparum, Vivax and Ovale malaria appeared in the north of the province along the main river, while the cluster for Malariae malaria was situated elsewhere. The relative risk to be a malaria parasite carrier within clusters along the river was twice that outside the area. The main risk factor associated with three out of four malaria species was overnight stay in the plot hut, a human behaviour associated with indigenous farming. Haplotypes did not show clear geographical population structure, but pairwise Fst value comparison indicated higher parasite flow along the river.PCR diagnosis found 368 positive individuals (6.4%) for malaria parasites, of which 22% contained mixed species infections. The occurrence of these co-infections was more frequent than expected. Specific areas with elevated risk of infection were detected for all Spatial aggregation of malaria parasite carriers, and the identification of malaria species-specific risk factors provide key insights in malaria epidemiology in low transmission settings, which can guide targeted supplementary interventions. Consequently, future malaria programmes in the province should implement additional specific policies targeting households staying overnight at their farms outside the village, in addition to migrants and forest workers.The online version of this article (doi:10.1186/1475-2875-13-387) contains supplementary material, which is available to authorized users. Globally, malaria incidence has decreased by 29% between 2000 and 2012, while malaria specific mortality rates have been reduced by 45% over the same period . In a coAnopheles species (or species complexes) showing heterogeneous biting behaviour occur in the area, of which at least four are capable of transmitting the infection  [1-pi] , the Institutional Review Board of the Institute of Tropical Medicine Antwerp  and the Ethics Committee of the University of Antwerp . Gatekeepers provided informed written consent for the participation of their village. The survey participant or his/her parents or guardian provided informed written consent for individual participation.Plasmodium falciparum (139/368) and P. vivax (139/368) were the most common species, while 22.3% (82/368) were mixed infections, mainly P. falciparum/P. vivax co-infection (56/368)  was found to be significant.During the 24-day survey period, all 117 villages were visited. Blood samples from 5793 individuals were collected using a standard finger prick method. An extra list of randomly chosen individuals was provided in 43 out of 117 villages, resulting in a response rate of 70% (5793/8250). Malaria prevalence by PCR was 6.35% . 2 = 34.24, df = 1, p < 0.001). Self-reported use of bed nets was 92.4%. Among bed net users, 77.5% stated to sleep under a long-lasting insecticidal net (LLIN) provided by the National Malaria Programme, while the remaining 22.5% slept under a net bought on the market. More than half of the individuals (56.2%) reported to have stayed overnight at their plot hut the month prior to the interview, while this was only 16% for overnights in the deep forest. A gender bias was observed for individuals staying overnight in the forest , but not at the plot hut . 75% of the participants declared to wake up before 6 a.m. and 75% to go to bed before 9 p.m.More females participated in the study as compared to males  and this was demonstrated to be significant in the pooled Plasmodium analysis (p = 0.025) and for P. falciparum (p = 0.034). Note that no P. malariae cases were observed in the Khmer Ethnic population. An increased risk for a Plasmodium infection was observed in individuals with an axillary temperature ≥ 37.5 . No significant associations between Plasmodium spp. infections and sleeping times were found. Individuals that reported to stay overnight in their plot hut showed an increased risk to infection  and this was found to be significant in the pooled Plasmodium analysis and for P. falciparum (p < 0.001) and P. malariae (p < 0.001). The variables overnight stay in the forest and use of bed net were not found to be significant for any of the Plasmodium species. Odds ratios estimates for use of bed net showed a tendency, though non-significant, for a decreased risk of infection for all Plasmodium species when a bed net was used  and P. falciparum . For P. vivax an elevated risk was found in two locations, one along the river in the north  and one in a single village . In comparison to the elevated risk areas for P. falciparum, P. vivax and P. ovale, a cluster was detected for P. malariae in the south-east of the province .The plot of PCR prevalence of e Figure . In genee Figure . For alls Figure , Table 4P. falciparum infected individuals, 115 (55.6%) had an available barcoding genotype based on 7 SNP’s. For the remaining 92 individuals the parasite density was too low to proceed with the genotyping analysis. Among the 115 genotyped individuals, 19 (16.5%) had polyclonal infections, ten of which belonged to the Plasmodium falciparum cluster shown in Figure P. falciparum infection and 26 (61%) in the remaining area. Pairwise between cluster Fst values below 0.15, which is an indication of low to moderate genetic differentiation [Out of the 207 Malaria endemic countries that are in the transition progress from control to pre-elimination will benefit from supplementary interventions, targeted towards areas with an increased risk of infection. Cambodia aims to reach the pre-elimination phase in 2015 – 2020 and eliminate malaria by 2025. In 2012, already 13 out of the 24 provinces recorded less than five cases per 1,000 at risk per year, the threshold set by the WHO for entering the pre-elimination phase . HoweverPlasmodium species resulted in partly overlapping malaria clusters. In the north of the province, along the Sesan river, areas with elevated risks of infection for P. falciparum, P. vivax and P. ovale were detected, with P. ovale and P. vivax having almost identical cluster centers. An individual living inside the cluster is 2 times more likely to acquire a species-specific Plasmodium infection. At a completely different location, in the south-east of the province, an area with an elevated risk for a P. malariae infection was identified. These geographical variations could indicate the existence of various species- specific geographical niches, either due to different vector-human interactions or to environmental factors. It has for example been demonstrated that the spatial distribution of G6PD deficiency, widely accepted to be protective against malaria [Anopheles barbirostris, recently identified as a secondary vector of P. malariae parasites [Anopheles dirus[Anopheles miminus[The spatial aggregation of the  malaria , is high malaria  or that arasites , tends tes dirus and Anop miminus.Plasmodium species. Age, for example, contributed to the final model predicting P. vivax and P. ovale infections, but not in the cases of P. falciparum and P. malariae. As samples were collected during the dry season, a plausible hypothesis is that the relapsing liver stages in both P. vivax and P. ovale were responsible for this. Indeed, a recent study conducted in the Peruvian Amazon indicated the odds for relapsing liver stages to differ between age groups, but not between sexes [P. falciparum infections, while the use of a bed net contributed to the P. ovale model. The risk factor overnight stay in the plot hut seemed to be the single most important risk factor; reaching statistical significance on a 0.1 level in the univariate analysis in all but one Plasmodium species. The multivariate model selection procedure strengthened the importance of this variable as it was again selected for in 3 out of 4 Plasmodium species. This result confirmed an additional characteristic to the widely recognized risk groups in the greater Mekong subregion  [Both univariate and multivariate risk factor analyses identified age, ethnicity and overnight stay at the plot hut to be significantly associated with the overall malaria prevalence. These variables however were differentially selected for depending on en sexes . In addiigrants) , namely igrants)  and in Cigrants) . In bothigrants) , 34, 35.igrants) , 37. In P. falciparum infection but not for a P. vivax infection [Plasmodium species, and this result seem to confirm that entire families, including both males and females, spend time overnight at their plot huts where they have an increased risk obtaining a malaria infection. Forest activities such as hunting and logging, mainly involving adult males only, are frequently identified as one of the main malaria risks in Cambodia [In contrast to previous studies, the risk factors use of bed net, gender, and overnight stays in the forest were not retained for the final multivariate model. This could be due to the specific context in which the current study was performed. The blood collection took place outside the transmission season, and a widespread bed net distribution campaign ensured the majority of individuals (94%) report to sleep under a bed net. This, in combination with the fact that this study did not differentiate between net use in the village and at the plot huts, could have led to the counterintuitive result of failure to associate the risk factor bed net with malaria in 3 out of 4 species, although the risk of an overall malaria infection was a mere 3% higher for people that indicated not to sleep under a bed net  was observed, which in Asia, seemed frequently underreported [P. falciparum and P. vivax in the study area. These contrasting results could partly be explained by the differential detection methods, whereby the studies presented in the review generally used common microscopy to identify different Plasmodium species as compared to a more sensitive molecular approach used here [P. ovale and P. malariae are often missed by classic diagnostic methods such as microscopy [Plasmodium species having dormant livers stages. The relapses that can occur when no additional treatment is given for the radical cure of these malaria forms (i.e. primaquine in mild-to-moderate G6PD deficiency) can jeopardize further elimination efforts. In Cambodia, a national guideline for the use of primaquine does exist, but is not yet implemented.A high rate of mixed blood stage reported . This obreported , 41. In reported . In partsed here , 40. Addcroscopy . In eastcroscopy  and wherP. falciparum did not indicate population structure associated with the area of increased risk to infection. The diversity of the haplotypes in the province seemed to indicate random mixture over all clusters. Only Fst values below 0.15 were observed along the river, which could indicate a moderate tendency for more parasite population flow along the Sesan river. This could potentially be associated with increased movement patterns of infected individuals and/or mosquito populations along this river. However, given that these Fst values are sensitive to sample size these findings need to be confirmed.The genotyping of Plasmodium species outside the transmission season in Ratanakiri was overnight stays in plot huts at the farms located outside the village. Consequently, future malaria programmes in the province should implement supplementary strategies, aimed to target households staying overnight at their farms outside the village, in addition to migrants and forest workers.Spatial aggregation of malaria species based largely on asymptomatic infections and the identification of malaria species-specific risk factors provide key insights in malaria epidemiology in low transmission settings. The results of this study will allow targeting future research as well as future interventions aiming at elimination of malaria. The main risk factor associated with infection of three out of four human Standardized questionnaire.(DOCX 37 KB)Additional file 1:Malariometric data and prevalence per species.(DOCX 53 KB)Additional file 2:Pairwise Fst values between all clusters.(DOCX 40 KB)Additional file 3:


TLR1 and TLR6 could alter the risk of development of complicated malaria and ability of the host to control the parasite burden during acute Plasmodium falciparum infection.The factors leading to poor outcomes in malaria infection are incompletely understood. Common genetic variation exists in the human genes for Toll like receptors (TLRs) that alter host responses to pathogen-associated molecular patterns. Genetic variation in TLR1 and TLR6 in 432 patients with clinical P. falciparum monoinfection acquired on the Thai-Myanmar border were genotyped. Using logistic regression, associations with the development of complicated malaria and the percentage of infected erythrocytes (parasitaemia) on the day of presentation to clinical care (day zero) were tested.Five single nucleotide polymorphisms in TLR1 rs5743551—an allele associated with improved outcomes in sepsis—were associated with higher parasitaemia measured on day zero (p = 0.03).Genotypes carrying the T (major) allele of TLR1 rs5743551 may account for the maintenance of an allele associated with poor outcomes in Caucasians with sepsis.Since malaria exerts strong genetic pressure on the human genome, protection from parasitaemia associated with TLR1 has effects on the host response to Plasmodium falciparum malaria in Asian populations. Genotypes from TLR6 showed no evidence of association with either complicated malaria or parasite burden.These data suggest that genetic variation in  Malaria is estimated to cause approximately 850,000 deaths per year with most mortality occurring before the age of 5 years . Since dThe role of innate immunity and, specifically, innate immune responses in host defence against malaria has been reviewed previously . Common Plasmodium is a well-described cause of the sepsis syndrome, the possible contribution of TLR polymorphisms to clinical outcomes in malaria also has potential explanatory power [TLR17202A/G (rs5743551) is associated with a large increase in the severity of organ dysfunction and an increased risk of death [TLR17202A/G has been also been associated with increased Interleukin-6 (IL-6) production and decreased regulatory T cell  mediated suppression of proliferation utilizing in vitro culture systems [TLR17202A/G  is a non-coding SNP that sits within the 5′ upstream region of the TLR1 gene and, thus, cannot have a direct effect on TLR1 protein function. It has been previously demonstrated, however, that the rs5743551 SNP is associated with increased secretion of soluble mediators of inflammation in response to stimulation with Pam3CysSerLys4 (Pam3CSK4), a specific ligand for TLR1/TLR2 heterodimers [TLR1 rs5743551 is in LD with several coding polymorphisms that strongly increase cell surface expression of TLR1.Since ry power , 13. In of death . Others of death , 16. Therodimers . One potTLR1 have functional consequences in immunologic signalling and influence the host response to a broad range of microbial pathogens. Given the potential role of TLR1 in inflammatory responses to malaria, it was hypothesized that the presence of the minor TLR1A7202G allele would be associated with malaria severity and the magnitude of parasitaemia.These data suggest that polymorphisms in Plasmodium falciparum malaria. Study recruitment was from 2000–2004, a period in which malaria was endemic. Patients were excluded if they had another plausible medical explanation for fever . The age of 13 was selected as subjects were originally recruited to participate in a previously reported clinical trial [Patients (age >13 years) were enrolled from a population presenting with febrile illness to clinical sites for Hospital for Tropical Disease (Mahidol University) on the Thai-Myanmar border found to be infected with al trial . Both thal trial . PatientP. falciparum peripheral blood smear after enrolment and performed quantitation of the parasite. Species level infection was also confirmed with PCR as previously described [An expert microscopist blinded to other diagnostic testing confirmed the presence of escribed . ParasitThe institutional review board of the Faculty of Tropical Medicine, Mahidol University approved this study. Verbal and written explanations were provided in the patient’s’ native language, and informed consent was obtained from all patients or their legal guardians prior to specimen collection.TLR1  and TLR6  in the malaria cases were genotyped using Taqman-based real-time polymerase chain reaction (RT-PCR) as previously described [TLR17202A/G−) or synthesized  and run according to the recommended guidelines on an ABI Prism 7900HT . 2 % of the samples were replicated for quality control.escribed . Primer-TLR1 and TLR6 genotypes were:The two phenotypes tested for association with 10-transformed measurements of parasitaemia on day 0 for each subject. Observed genotype frequencies were compared with expected frequencies to test for deviations from Hardy–Weinberg equilibrium as previously described [TLR1 and TLR6 genotypes and the presence of complicated malaria in the Burmese, Mon, and Thai patients, logistic regression was used, assuming an additive effect as previous work demonstrated an intermediate functional phenotype in heterozygotes [TLR1 and TLR6 genotypes and day 0 log10 transformed parasitaemia, linear regression was used assuming an additive effect. For associations detected in the un-adjusted analyses, multiple linear regression analyses were performed adjusting for age and gender. Finally, to assess whether genotypic effects were consistent across ethnic groups, sensitivity analyses were performed with stratification on the basis of each of the three major ethnic groups . Statistical significance was set at p < 0.05. Analyses were performed using Stata 10.0 .(1) Complicated malaria disease versus uncomplicated disease, and (2) logescribed . Inspectozygotes . To testTLR1 and TLR6 are associated with altered innate immune responses to synthetic bacterial lipoproteins [TLR1 and TLR6 could therefore be associated with severity of malarial illness and/or the degree of parasitaemia observed in each patient on the day patients were diagnosed with clinical malaria. To test this hypothesis five different single nucleotide polymorphisms (SNPs) were genotyped, three in TLR1 and two in TLR6   followed by Mon 29.0 %), Karen (16.3 %), Burmese (12.2 %), and, to a much lesser extent Cambodian and Nepali  and susceptibility to complicated malaria [OR (CI) = 1.26 (0.93–1.71), p = 0.14, Table A trend towards an association between the “major” T allele of TLR1 and TLR6 genotypes were also tested. Table TLR1 rs5743551 and higher levels of parasitaemia. A nearly two-fold increase in mean parasitaemia in subjects carrying the “major” TT genotype relative to those carrying the “minor” GG genotype  allele of rs5743551 was associated with a parasite density roughly half of that of patients with the TT (major) allele. The G (minor) allele has been associated with increased inflammatory cytokine production in response to bacterial lipopeptides that are recognized by TLR1/2 heterodimers [Early host innate immune inflammatory responses are likely to play an important role in determining the clinical course after infection with rodimers . The dirPlasmodium berghei ANKA strain , Tlr2−/− and Myd88−/− mice have been shown by some groups to be protected from development of cerebral malaria, although the effects of TLR2 have not been consistently observed and may depend on the genetic background of the mice [Plasmodium yoelii, a model of acute uncomplicated malaria, had either a strong phenotype with impaired survival in Tlr2−/−, and Myd88−/− mice or no observable phenotypic difference [Investigations into the role of TLR signalling in malaria infection using murine models have revealed conflicting results. Both TLR1 and TLR6 signal via forming heterodimers with TLR2 , 10 All the mice –31. Infefference , 33. It P. falciparum infection, none of the genetic variants tested were significantly associated with development of complicated malaria (Table TLR1 rs5743551 demonstrated a trend towards higher odds of complicated disease (p = 0.13). Notably, while this polymorphism is in high linkage disequilibrium (LD) with TLR1 rs5743551 in Caucasian subjects, it was determined that in the Southeast Asian subjects from Northern Thailand that TLR1 rs5743618 is in very weak LD with TLR1 rs5743551 and is found at very low frequency [In an effort to clarify the role of TLRs in human malaria, several studies have used genetic approaches attempting to detect associations between common genetic variants in the genes for TLRs and clinical outcomes in patients with malaria. In a group of malaria cases from northern Thailand with confirmed ia Table , whereasrequency .TLR1 rs5743618 is associated with higher parasitaemia on day zero in an Asian population is similar to an investigation by a Brazilian group that demonstrated an association between the G allele of TLR1 rs5743618 and increased risk of developing symptomatic malaria after infection with Plasmodium [TLR1 rs5743551 in their study, so direct comparisons between their study and the studied population cannot be made. However, taken together, these studies suggest that genetic variation in TLR1 affects host susceptibility to malaria disease severity, while variation in TLR6 is less likely to play a significant role in these processes. This association is further supported by a study in a population in Cameroon that reported a different SNP in TLR1 (rs4833095) was also associated with increased parasite burden, whereas a SNP in TLR6 was not associated with any clinical phenotype [The finding that asmodium . The Brahenotype .TLR1 rs5743551 may influence host responses to malaria is provided by the finding of a significant association between the copy number of the T allele of this polymorphism and increased parasitaemia measured on day 0 of enrollment. Subjects carrying the TT genotype had a nearly two-fold higher parasitaemia than subjects carrying the CC genotype , and underlying population genomic differences. Furthermore, in the Brazilian study a substantial minority of patients were infected with P. vivax (39 %), whereas the population in Cameroon was similar to the studied population in that it was a population uniformly infected with P. falciparum. Several groups have demonstrated different host response to the various Plasmodium species. For example, higher levels of IL-10 have been documented in humans subjects infected with Plasmodium vivax when compared to individuals infected with P. falciparum [Stronger evidence that lciparum –33.P. falciparum. The current study is the first to document an association of TLR1 rs5743551 with a clinical outcome in an Asian population with malaria. TLR6 has never been strongly implicated in the host response to malaria and no association with TLR6 SNPs and clinical outcome in malaria were found.The particular strengths of the present study include detailed clinical information on a population uniformly infected with PCR confirmed TLR1 is associated with the host control of parasitaemia in Asian patients with acute febrile malaria. This assertion is consistent with the demonstrated ability of malarial GPI to activate cells through TLR2/TLR1 heterodimers [TLR1 rs5743551 may persist in higher frequencies in non-Caucasian populations because of a beneficial effect on host fitness in environments with high prevalence of malaria. This is the first plausible explanation for the relatively high frequency of an allele that is associated with worse outcomes in tuberculosis, sepsis, and leprosy [These data demonstrate that the G allele of rs5743551 in  leprosy –16. Whil leprosy . These f


Chronic malaria is usually defined as a long-term malarial infection in semi-immune subjects, usually without fever or other acute symptoms. The untreated infection may evolve to hyper-reactive malarial splenomegaly (HMS), a life-threatening complication. This paper describes the largest series of HMS ever observed outside endemic countries, and the clinical outcome after a single anti-malarial treatment. Contrarily to most authors, still reporting the traditional, long-term treatment, regardless possible further exposure, the patients in this series did not receive any further prophylaxis if they were not re-exposed to malaria infection.A retrospective, longitudinal study, describing all patients with HMS diagnosed at the Centre for Tropical Diseases of Negrar, Verona, took place over a 25-year period. HMS was defined by a longitudinal spleen diameter ≥16 cm, IgM ≥ 2.5 g/L, anti-malarial antibody titre ≥160, exclusion of other causes of splenomegaly. The short-term (≤6 months) clinical outcome after a single anti-malarial treatment was analysed and so was the long-term outcome of subjects re-exposed to malaria and submitted or not to anti-malarial prophylaxis or intermittent treatment. The association of the outcome with the main independent variables was first assessed with univariate analysis. Logistic regression was also performed.Forty-four subjects with HMS were retrieved. Of those with a short-term follow-up visit  available before returning to endemic areas, 20/22 resulted improved/cured, two were unchanged. Of 22 expatriates seen at long-term follow-up after re-exposure, 18 were improved/cured, including eight out of nine who had followed an anti-malarial prophylaxis and 10/13 who had opted for the alternative of an intermittent treatment.HMS is the most severe form of chronic malaria. A single anti-malarial treatment is probably adequate to treat HMS in the absence of re-exposure, while an adequate prophylaxis is necessary for patients exposed again to malaria transmission. Intermittent treatment would probably be the only viable approach in endemic countries. Chronic malaria is a long-term infection in semi-immune subjects. It is usually characterized by the absence of fever or any other acute symptoms, so that this condition has long been defined as asymptomatic carriage of malaria parasites . HoweverPlasmodium falciparum to this molecule.Hyper-reactive malarial splenomegaly (HMS) is probably the most severe form of chronic malaria . The maiA previous paper  describeThe main objectives of the present study were: (a) to describe the main clinical and laboratory findings of immigrants and expatriates diagnosed at CTD with HMS; (b) to analyse the short-term and long-term outcome of HMS patients after a single anti-malarial treatment, followed by long-term prophylaxis (or by intermittent treatment), for those who returned to endemic areas for malaria.The clinical records of patients were retrieved as described previously , coverinantimalarial antibody titre (IFAT-Biomérieux) ≥1/160, PLUS:massive splenomegaly   ;IgM level ≥2 SD above the local mean ;no other identifiable cause of splenomegaly or of raised IgM, such as schistosomiasis, cirrhosis, haematological conditions, and others as described previously .The following case definition for HMS was used:Final outcome was assessed on the basis of the last follow up visit.Data were exported to a pre-structured Excel file, including the main epidemiological, clinical and laboratory findings as well as the results of an ultrasound scan, as available at baseline (T1), short-term follow-up (T2) and long-term follow-up (T3) .Cured: absence of splenomegaly and normal IgM value;Improved: ≥1 cm decrease of the spleen diameter, or unchanged spleen diameter and ≥1 g/dL decrease of IgM level;Unchanged: <1 cm or no variation of the spleen diameter and <1 g/dL or no variation of IgM level;Progressed: ≥1 cm increase of the spleen diameter, or unchanged spleen diameter and ≥1 g/dL increase of IgM level.Thick and thin film microscopy, as well as the QBC Malaria Test , were performed following the routine procedures of the CTD laboratory. This is a referral laboratory for parasitic infections in Italy, where slides for quality control of laboratories of different Italian regions are prepared. Although no specific quality assessment of microscopy for the study purpose was performed (as is obvious in a retrospective study), quality control, including intra- and inter-observer reproducibility, is systematically carried out according to standardized, certified procedures.®) single dose [All patients were treated with a single anti-malarial, as for an acute malaria, once the diagnosis was established. The standard regimen followed at CTD was with oral quinine, 10 mg/kg × 3 for 3 days, followed by pyrimethamine–sulfametopyrazine  with the potential independent predictors.STATA IC 14  was used for data analysis. For categorical variables the absolute and relative frequencies were calculated. For continuous variables, the median and IQ range were considered. The association between categorical variables was evaluated using the χComitato Etico delle province di Verona e Rovigo) acknowledged the study protocol and formally authorized the study in September 2014 (protocol n. 43713).Data were entered anonymously in the database. The competent Ethics Committee  and eight were African immigrants. Seven expatriates and three immigrants were not seen again, while 29 and five, respectively, had at least one follow-up visit. Several patients had more than one follow-up visit, coinciding with periodical returns to Italy.At least one short-term follow-up visit (≤6 months) before any further exposure to malaria, was available for half of the patients of both groups . At least one long-term follow-up visit (>6 months) was available for 23/36 expatriates , and for only one immigrant (with an unclear history of re-exposure). The main symptoms, signs and laboratory findings of expatriate and immigrant patients at their first contact are summarized in Table Most of the subjects were symptomatic at presentation Table . Fever , and 6/8 immigrants (75 %), with a positive result in 17/32 (53 %) and 5/6 (71 %), respectively (p = 0.370). The parasite density was low or very low in most cases (Table Twenty-four subjects had a short-term follow-up visit (<6 months after treatment and with no re-exposure in between). The median time elapsed between initial and follow-up visit was 45.5 days (IQ range 66). One subject was impossible to classify as the spleen diameter was unchanged and the IgM value at follow-up was not available, while for another subject there was no recorded assessment of the spleen measure at follow-up. The results of the remaining 22 subjects are reported in Table Twenty-four subjects had at least one long-term follow-up visit and a reliable history concerning re-exposure or not to malaria. The median time elapsed between the initial and the last visit was 4.12 years (IQ range 2.77). Only two subjects had not been re-exposed. One was a 27 years old immigrant woman from Ghana, first observed in March 1993, who never returned to her country and was last seen after 3 years, when she was perfectly cured . The other was an expatriate missionary sister aged 63 when she was first seen in March 2001, with a huge spleen , showing nevertheless a significant improvement over time, with a 6-cm reduction of the longitudinal spleen diameter and a normalization of IgM when she was last seen in April 2004. Both had received a standard anti-malarial treatment at the first encounter, with no further prophylaxis. The results of the remaining 22 patients are summarized in Table The main findings of two particularly informative cases are briefly outlined below.6/μL, Haemoglobin (Hb) 7.7 g/L) and leucopaenia (white blood cells (WBC), 2.17 × 103/μL), a moderately low platelet count , raised lactic-dehydrogenase (LDH) , and IgM . Both the thick and thin film for malaria were negative, but the QBC showed very scanty trophozoites, compatible with P. falciparum. The anti-malarial antibody titre  was ≥160 (not further diluted). She was treated with quinine, 10 mg/kg × 3/day for 3 days, followed by a single dose of pyrimethamine–sulfametoxazole, the standard regimen for acute malaria at that time at CTD [Italian, female, non-governmental organization, volunteer in Cameroon, consulted for the first time in November 1996, when 57 years old. She had lived in the African country for 22 years. She presented with low-grade fever and a profound asthenia. The liver was moderately enlarged, while the spleen longitudinal diameter was 18 cm (she was 155 cm high). The laboratory showed marked anaemia (red blood cells (RBC), 2.67 × 106/μL, WBC 4.84 × 103/μL, PLT 145 103/μL, bilirubin 25.5 μmol/L, LDH 553 U/L, and IgM 9.2 g/L. Both the thick and thin film for malaria were negative, and so was the QBC. The anti-malarial antibody titre was 10,240. He was treated with the same regimen as Case 1. Shortly afterwards he had to go back to Uganda, therefore a short-term follow-up was not possible. An anti-malarial prophylaxis was recommended, either with doxycycline or with mefloquine . It was proposed the patient should come back to follow-up on his next return to Italy, initially planned in 1 year, considering that a long-term anti-malarial prophylaxis was not generally recommended because of potential toxicity. Nevertheless, he returned to medical observation after almost 5 years (July 2003), during which he reported having regularly taken one tablet of mefloquine once a week for the whole of the 5 years, and declared that he had “never been healthier”. The spleen diameter had decreased by 4 cm and the blood cell count, as well as the IgM value (0.33 g/L), were normal.Italian, male, Catholic missionary in Uganda for 27 years, 64 years old, consulted CTD in August 1998 for a medical check-up while he was on home leave. He reported a long history of acute malaria episodes, while in the last few years he said that he had often had malaria “without fever”. He identified the latter condition on the basis of “typical symptoms” (mainly a profound weakness), and took a short course of quinine when this occurred. The remaining clinical history was negative. He declared not having any particular complaint at presentation. The physical examination revealed a moderate hepatomegaly and a marked splenomegaly, with the lower pole well beyond the umbilical line and an echografic longitudinal diameter of 21 cm. The laboratory findings were similar to the previous case, with RBC 3.5 × 10This paper describes the largest series ever published of HMS in a non-endemic country. The spleen measure required for case definition of HMS has been variable . Most paMost of the study subjects (36) were Italians, mostly missionary people, while there were eight immigrants. The only important difference between the two populations was the median age as immigrants are on average younger people, compared to expatriates who have spent decades in a foreign country. Asthenia and hypersplenism (with a lower median RBC and platelet count) were more common in expatriates, while the two populations were similar in all other respects . Most patients were symptomatic at presentation, although symptoms were usually vague, similarly reported in literature . Fever  was strongly recommended, as it was considered that the risk of progression of the syndrome outweighed that of the drug’s side effects. The short-term outcome of most of the subjects assessed after treatment, in the absence of re-exposure, was favourable, with a comparatively quick decrease of the spleen size Fig. . The timThe term ‘chronic malaria’ has usually been used to define asymptomatic or pauci-symptomatic carriers of malaria parasites in blood –15. HowePlasmodium and tends to evolve, if untreated, similar to an auto-immune disease. According to the traditional view, long-term use of chloroquine works as an immune-modulating drug, rather than an anti-malarial [Evidence in the past few years  and frommalarial . In thismalarial , 18.Plasmodium in blood. So the Authors suggest that a negative PCR predicts a treatment failure. On the other hand, in the present and much larger series most of the patients improved at follow-up, and among those who didn’t, some had positive malaria films or QBC.This study was retrospective, and for this reason follow-up data are incomplete and very variable in time. A recall bias is likely to have occurred, and therefore the details of prophylaxis or intermittent treatment should be taken with caution. A selection bias cannot be excluded either and some patients, especially of the first years, may have been missed. The potential problems of the case definition have been discussed above. PCR for malaria is not used at CTD for routine diagnosis, therefore this information is lacking for our patients. It is possible that some of the cases with negative blood films and QBC would have been found positive by the more sensitive PCR. It has recently been suggested by McGregor et al.  that subThe hyper-reactive malarial splenomegaly (HMS) is the most severe form of chronic malaria, and not a different disease. A single anti-malarial treatment is sufficient to cure/improve most cases. However, reinfection must be prevented or immediately treated. As a lifelong prophylaxis does not appear to be feasible in malaria-endemic countries, considering the high number of chronic malaria carriers and patients with splenomegaly, research is needed in order to evaluate the efficacy of intermittent treatment as well as its best periodicity.


Plasmodium vivax and dengue co-infection as well as in individuals with malaria or dengue mono-infections in order to identify biosignatures of each clinical condition.Concurrent malaria and dengue infection is frequently diagnosed in endemic countries, but its immunopathology remains largely unknown. In the present study, a large panel of cytokines/chemokines and clinical laboratory markers were measured in patients with P. vivax malaria was diagnosed by thick blood smear and confirmed by PCR; dengue cases were detected by IgM ELISA or NS1 protein. The plasma levels of cytokines and chemokines were determined by multiplex assay.Individuals from the Brazilian Amazon were recruited between 2009 and 2013 and classified in three groups: vivax malaria (n = 52), dengue (n = 30) and vivax malaria and dengue co-infection (n = 30). Plasmodium parasitaemia levels between the groups.Individuals with malaria and dengue co-infection displayed lower levels of platelets and haemoglobin than those with malaria or dengue mono-infections . The group of individuals co-infected exhibited the highest median concentrations of IFN-γ, IL-6, CCL4 than the mono-infected groups. Network analyses of plasma cytokines/chemokines revealed that malaria and dengue co-infection exhibits a distinct immune profile with critical roles for TNF, IL-6 and IFN-γ. Further, parasitaemia levels displayed positive significant interactions with IL-6, CCL4 and IL-10 in the group of patients co-infected with malaria and dengue. No differences were observed in distribution of dengue virus serotypes and The findings described here identify unique patterns of circulating immunological markers in cases of malaria and dengue co-infection and provide insights on the immunopathology of this co-morbid condition.The online version of this article (doi:10.1186/s12936-015-0835-8) contains supplementary material, which is available to authorized users. Plasmodium infection is responsible for around one million deaths, mainly in children [Malaria and dengue fever are the most frequent arthropod-borne diseases in the world. Every year children . It is echildren , 3. Bothchildren –19.Previous studies have reported a frequent presence of malaria and dengue co-infections in different countries and implied that this fact creates challenges for reliable clinical diagnosis due to the overlap of major symptoms with malaria or dengue mono-infections , 14, 16.Plasmodium vivax malaria and dengue co-infection as well as in subjects with P. vivax or dengue mono-infections.The immunopathogeneses of dengue and malaria display common features, which include the production of multiple cytokines and the balance between pro-inflammatory and anti-inflammatory responses may regulate the clinical spectrum of these infections –25. ImpoP. vivax confirmed by PCR were recruited. Dengue subjects were diagnosed by: NS1 and RT PCR  in individuals with fewer than 6 days of fever, or by the detection of IgM ELISA as descrided by Kuno et al. [P. vivax malaria and dengue were also recruited. All patients with microscopic or molecular diagnosis of malaria caused by Plasmodium falciparum or P. vivax and P. falciparum co-infection (mixed infection), patients with serologic diagnosis of viral hepatitis , HIV, and leptospirosis were excluded. Patients with complications of dengue or malaria according to WHO criteria [Outpatients with an acute febrile syndrome who sought care in a reference hospital  in Manaus, in the Brazilian Amazon, were recruited between 2009 and 2013. Malaria individuals were diagnosed by blood thick smear and those with o et al.  in indivcriteria  were excAll clinical investigations were conducted according to the principles expressed in the Declaration of Helsinki. Written informed consent was obtained from all participants before enrolling into the study. This study was approved by the Ethics Committee of the FMT-HVD (protocol numbers: 2009/15243 and 39163/2012).Blood was obtained by venopuncture at the study enrolment and heparinized plasma was separated by centrifugation and stored at −70 °C until use in immunoassays. Circulating levels of several cytokines and chemokines, including IL-1β, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-12p70, IL-13, IL-17, IFN-γ, TNF, CCL2, CCL4, GCSF, GMCSF, were measured using a single multiplex assay according to the manufacturer’s protocol . The clinical laboratory markers haemoglobin (HB), haematocrit (HT), platelets (PTL), aspartate amino-transferase (AST) and alanine amino-transferase (ALT) were measured in fresh plasma/serum samples at the Clinical Laboratory facility from the FMT-HVD .The inferential networks were generated from Spearman correlation matrices containing values of each biomarker measured in the plasma samples. The values were input in JMP 10.0 software . Each mediator is selected as a target, and the software performs a search within the other mediators for those that are correlated, with the target calculating a correlation matrix using Spearman rank tests. As a result, the features related to the selected target are linked. The links shown in the networks represent statistically significant Spearman rank correlations (P < 0.05). In order to analyse the structure of the biomarker networks, the network density was calculated, which is, in the context of this study, the ratio of the number of edges inferred in the network over the total number of possible edges between all pairs of nodes . The denp value lower than 0.05 was considered statistically significant.In the exploratory analysis of the data, frequency tables were constructed and the Chi square test was applied to evaluate the association between categorical variables. The continuous variables were tested for Gaussian distribution within the total sample using D’Agostino and Pearson omnibus normality test. All variables were not under normal distribution, and non-parametric tests were used instead. In this context, Kruskal–Wallis with Dunn’s multiple comparison (when three groups were compared) or Mann–Whitney tests (when two groups were compared) were used to assess the differences between the clinical groups. Multinomial regression analyses adjusted for age and gender were performed to test associations between the laboratory measures  and the different clinical conditions evaluated . A hierarchical cluster analysis using the Ward’s method was performed to test if a combination of different immune-related biomarkers could cluster the study groups separately. The statistical analyses were performed using the programs GraphPad Prism 6.0 , SPSS 19.0  and JMP 11.0 . A P. vivax , dengue (n = 30) and co-infected vivax malaria/dengue (n = 30). Most participants from the malaria group were male  while the groups of co-infected and dengue patients exhibited a predominance of females . There were no differences with regard to age between the groups  when all the study groups were considered together: IFN-γ , IL-6 , IL-13 , TNF  and IL-12p70   in the group of malaria mono-infected subjects, suggesting a major role for parasitaemia in the immune profile in this clinical condition  compared to dengue fever mono-infection [Some studies have studied laboratory measurements in subjects with dual malaria and dengue infections , 6, 11. nfection –31. Othenfection , 32. On nfection .TNF polymorphisms are common and may play a role in TNF levels in the context of malaria [Heightened levels of TNF and IFN-γ have been systematically associated with increased clinical disease severity in malaria or dengue fever in many case series , 33–35.  malaria . Moreove malaria , 39. The malaria , 9, 10.In the present study, plasma levels of IL-4, IL-7, IL-12p70, and IL-13 were more elevated in subjects purely with dengue fever than in those with malaria mono-infection or malaria and dengue co-infection. Noteworthy, IL-12p70 has been previously associated with severity and protection during malaria –43, alsoAlthough the group of individuals co-infected with malaria and dengue displayed an intermediate density of the immune network compared to the other clinical groups, this group displayed the lowest values of HB and PTL, thus indicating that the relationships between the inflammatory markers may not be influenced directly by the levels of these clinical laboratory markers. However, a potential role for HB and HT in regulation of the inflammatory environment in malaria and dengue comorbidity could not be entirely discarded, and the network findings indicate that these parameters were negatively correlated with IL-7 in this group. It has been reported that IL-7 plays an important role in CD4+ T cell immune responses against the dengue virus and this cytokine is also useful for maintaining the growth and antigen-specific cytotoxic activity of CD4+ human cytotoxic T lymphocyte clones . In addiPlasmodium sp. replication. Noteworthy, PTL positively interacted with immune markers  only in the group of patients with dengue mono-infection. It has been reported that exposure of monocytes from healthy volunteers to platelets from patients with dengue induced the secretion of IL-8 [In the context of laboratory and immune markers correlations, the group of individuals presenting with malaria mono-infection displayed significant negative correlations between ALT and IL-4 and IL-7. Liver enzymes are highly inducible by the activation of immune responses and ALT was also correlated with TNF and superoxide dismutase-1 (SOD-1) levels in another case series of vivax malaria . Further of IL-8 . Thrombo of IL-8 .Parasitaemia was not different between the groups of individuals with malaria mono-infection and malaria and dengue co-infection, similarly to previous studies . HoweverAltogether, the findings of the present study indicate that concurrent malaria and dengue infections may cause a more severe disease compared to mono-infections as observed by laboratory and immune markers profiles. Overall, malaria and dengue co-infection displayed lower levels of platelets and haemoglobin and a specific immune signature with a special role for TNF compared to dengue or malaria. This detailed description of the immune response profile in subjects with malaria and dengue fever co-infection and the results depicted herein shed light into the immunopathology of this comorbid condition. Further prospective studies with larger samples and experimental models would be necessary to investigate the key mechanisms resulting in the biosignatures identified here in patients with malaria and dengue co-infection.


A deep understanding of the local epidemiology of malaria is essential for the design and implementation of setting-specific control and elimination efforts. In Côte d’Ivoire, new initiatives are underway to reduce the burden of malaria, which requires high-quality longitudinal data. The epidemiology of malaria was studied in the Taabo health and demographic surveillance system (HDSS) in south-central Côte d’Ivoire and implications for control are discussed.Plasmodium infection and parasitaemia. Haemoglobin (Hb) level was determined using a HemoCue device.Two cross-sectional surveys were carried out in the rainy season of June/July in 2010 and 2011. Inhabitants of approximately 7 % of randomly selected households in the Taabo HDSS were invited to participate. People were clinically examined, ear temperature was measured and spleen size determined. Finger-prick blood samples were collected and subjected to a rapid diagnostic test (RDT). Additionally, thick and thin blood films were prepared on microscope slides and diagnosed under a microscope for Plasmodium infection was 46.0 % in 2010 and 56.6 % in 2011, owing to a statistically significant difference (p < 0.05). Males showed a higher Plasmodium infection prevalence than females . The highest malaria prevalence was observed among infants and young children (aged ≤9 years). The risk of Plasmodium infection was significantly higher in villages compared to small hamlets and urban settings (p < 0.05). Fever, Hb level and splenomegaly were associated with parasitaemia.A total of 1187 and 1264 people in 2010 and 2011, respectively, had complete data records. The prevalence of Plasmodium infection. There is a pressing need to scale-up control interventions against malaria.Malaria is highly endemic in the Taabo HDSS in south-central Côte d’Ivoire with considerable spatial heterogeneity of  Substantial progress has been made in the understanding of the epidemiology of malaria and ways to control or eliminate the disease. In particular, integrated approaches using vector control measures, e.g. long-lasting insecticidal nets (LLINs) and artemisinin-based combination therapy (ACT) considerably reduced the burden of malaria over the last decade , 2.In Côte d’Ivoire, an over 10-year long sociopolitical crisis jeopardized malaria control. Nevertheless, different initiatives have been implemented by the national malaria control programme with financial support from the Global Fund to Fight AIDS, Tuberculosis and Malaria. However, a clear downward trend in the number of malaria cases has yet to be registered . It mustThe aim of the present study was to assess the local epidemiology of malaria in the Taabo health and demographic surveillance system (HDSS) of south-central Côte d’Ivoire . An in-dThe study protocol was approved by the institutional research commission of the Centre Suisse de Recherches Scientifiques en Côte d’Ivoire . Ethical approval was granted by the Comité National d’Ethique et de la Recherche (CNER) of Côte d’Ivoire (reference no. 1086 MSHP/CNER). Participation was voluntary and all study subjects provided written informed consent, with parents/legal guardians signing on behalf of children/adolescent below the age of 18 years.2. At the onset of operation in late 2008/early 2009, the population in the Taabo HDSS was 37,792 inhabitants. There is a small town (Taabo-Cité), 13 main villages and over 100 small hamlets. The climate is tropical, with two rainy seasons (March to July and October/November) and two dry seasons (December to February and August/September). Due to recent climate change, no clear seasonal delimitation is observed any longer and there is a tendency of just two seasons [The study was conducted in the Taabo HDSS, located in the Taabo district, some 150 km north-west of Abidjan. The Taabo HDSS is situated in a forest-savannah transitional zone and has a surface of approximately 1000 km seasons . The ave seasons .The Taabo HDSS has been launched in mid-2008 and serves as a platform for high-quality longitudinal data on all the residents, with particular emphasis on demography , health and socioeconomic status. Data are collected prospectively, usually pursuing three rounds per year to obtain key demographic data. Additionally, once every 2nd year, socioeconomic data are collected. The activities of the Taabo HDSS began by a baseline census in late 2008/early 2009, during which all the households of the district of Taabo were visited and georeferenced. Data were collected on household composition, house characteristics, household asset ownership, people’s ethnicity, nationality, religion, age, sex, education and employment.From 2009 to 2011, community-based deworming against soil-transmitted helminthiasis  and schistosomiasis (using praziquantel) was undertaken in the whole area under surveillance under the leadership of district health personnel with support of Taabo HDSS staff. A series of specific research projects provided additional treatment to study participants found positive for specific parasitic infections –15.Plasmodium diagnosis. The RDT  was used to detect Plasmodium falciparum histidine rich protein 2 (HRP 2). This RDT was shown to detect 86.9 % of test samples with wild-type P.falciparum at a concentration of 200 parasites/μl of blood and 98 % of test samples with a concentration of 2000 parasites/μl of blood, with 0 % of false positives in negative samples [Two cross-sectional surveys were conducted; in 2010 and 2011, during the main rainy season at the end of June/early July. Study participants were invited for a finger-prick blood sample that was utilized for measuring haemoglobin (Hb) level, employing a HemoCue device . Additionally, a rapid diagnostic test (RDT) was performed and thick and thin blood films were prepared on microscope slides for  samples . The res samples .Study participants were selected from the readily available Taabo HDSS database. All members of approximately 7 % of the households were selected in each of the two cross-sectional surveys. The sampling design was based on clusters stratified according to residency  and the geographical location of residency relative to the Bandama River and Lake Taabo. Four strata were distinguished: (1) households situated in hamlets that were at least 5 km away from the river or lake; (2) households located in hamlets in close proximity to the river or lake (<5 km); (3) households in the main villages far from the river or lake; and (4) households in the main villages in close proximity to the river or lake and households from urban Taabo-Cité. The total number of individuals invited to participate in the years 2010 and 2011 was 2530 and 2621, respectively. Households were randomly selected in each stratum with a probability proportional to their population size.Plasmodium asexual form or gametocyte was found after counting 500 WBC. Thin blood films were examined for Plasmodium species identification, examining 100 microscopic fields. Quality control was performed by a senior technician, who was blinded to the results obtained from the first reader. Slides with conflicting results were re-read by a third microscopist and the results discussed among the technicians until consensus was reached.Thick and thin blood films were transferred to the laboratory of the district hospital in Taabo-Cité and stained with 3 % Giemsa for 45 min. Parasitaemia was determined from thick blood films by counting the number of parasites per 200 white blood cells (WBC) or 500 WBC if less than 10 parasites had been detected, assuming a mean WBC count of 8000/μl of blood. A slide was classified as negative if no Demographic, clinical and laboratory data were double entered and validated in an Access 2007 database . Fever was defined as an ear temperature ≥37.5 °C. Anaemia was defined according to guidelines put forth by the World Health Organization (WHO) . In brie2 test. Differences in geometric mean of parasitaemia, mean age and mean spleen size between groups were analysed by Wilcoxon test (two groups) or Kruskal–Wallis test (more than two groups) in case the Shapiro normality test was significant. Odds ratios (ORs) were estimated using logistic regression to compare the risk of Plasmodium infection between groups. For all analyses, difference was regarded as significant if the p value of the test statistic was <0.05. Data analyses were performed using R statistics .Descriptive statistics were used to summarize demographic characteristics of the study sample. Comparisons between groups for categorical parameters were carried out by the Pearson’s χA total of 1187 and 1264 individuals participated in the cross-sectional surveys conducted in 2010 and 2011, respectively. The number of people sampled, stratified by age group, sex and residency, is summarized in Table Plasmodium infection, stratified by year of survey, participants’ residency and sex. The overall prevalence of Plasmodium infection in 2011 was significantly higher than in 2010 . Males showed a significantly higher prevalence of Plasmodium infection than females . The prevalence of Plasmodium infection was significantly higher among individuals living in villages compared to residents from small hamlets or urban settings (p < 0.001). Plasmodium infection occurred in all age groups. The highest prevalence of infection was observed in infants and young children (age ≤9 years). Prevalence decreased with age . Plasmodium malariae accounted for 1.1 and 0.2 % of the infections, respectively, while P.ovale was rarely observed .Three human malaria species were detected in the study area: Most infections were of low to moderate parasitaemia (16-9999 parasites/μl of blood). The proportion of infections with high parasitaemia  was 8.6 % in 2010 and 5.2 % in 2011. The geometric mean parasitaemia of positive samples was similar in both years (Table p < 0.001). With regard to sex, no difference was found in the prevalence of fever  compared to those with only light or moderate infection .Of the p < 0.01) . There was a clear decline in spleen size with age , which might, at least partially, explain the high prevalence of infection. Levels of Plasmodium infection prevalence observed in the current study were several-fold higher than those reported in a lowland village of Tanzania (13 %) [Plasmodium infection was somewhat higher in the 2011 survey compared to the preceding year. It will be important to longitudinally monitor Plasmodium infection prevalence in the general population of Côte d’Ivoire as control and elimination efforts are being escalated. The Taabo HDSS offers a unique platform in this regard and will allow for cross-country comparisons [This work presents results from one of the few repeated cross-sectional, population-based malaria prevalence surveys conducted at the district level in a highly malaria-endemic setting of West Africa. The study included all age groups, and hence, not only those who are considered at highest risk of malaria (i.e. children below the age of 5 years and women of child bearing age). Furthermore, the study provides stratified data for the three main residential settings of the Taabo HDSS; namely, hamlets, villages and a small town . High Pla (13 %) , but slia (13 %) , 22. In parisons .Plasmodium infection is dependent on the transmission intensity [Plasmodium infection rates in children, the district of Taabo can be classified as being hyperendemic for malaria [There is evidence that the age-pattern of ntensity . In areantensity , 26. Conntensity , 27. In ntensity . For all malaria .Males showed significantly higher infection rates than females. This result is supported by previous studies conducted elsewhere in Africa , Asia 3, 31 and Plasmodium infection prevalence rates in rural compared to urban areas [Plasmodium infection was similar in hamlets and Taabo-Cité, the only urban area of the Taabo HDSS. This finding indicates that, in the urban setting, ecological conditions are suitable for malaria transmission. A possible explanation is that Taabo-Cité is located in close proximity to Lake Taabo. Surprisingly though, among the rural areas studied, Plasmodium infection was more prevalent in villages than in the less developed hamlets. This observation suggests different risk factors for malaria transmission in the two types of rural settings. Interestingly, different observations have been reported for the western part of Côte d’Ivoire; among children below the age of 15 years, Plasmodium infection rates were similar in villages and hamlets [Preceding studies documented higher an areas –36. In t hamlets .P. falciparum was the predominant malaria species, while only a few individuals were found positive for P. malariae and P. ovale. These findings are consistent with epidemiological studies undertaken in 2001 in a single village of the study area [P. malariae and P. ovale can reach high prevalence rates [P. falciparum is the predominant species [In the current study, udy area  and elseudy area –41. In sce rates , but P.  species , 43.Plasmodium infection. In the current study, parasitaemia peaked in children ≤9 years of age, and thereafter showed a substantial decline, suggesting that an effective immune response is acquired in early childhood. This may explain that most infections were asymptomatic. Indeed, only a relatively small number of infected persons were found febrile in the community (15.4–21.9 %). However, it is important to note that significant proportions of febrile individuals were diagnosed as having a Plasmodium infection (55.1–67.1 %).Most infections were of low to moderate parasitaemia with no statistically significant difference between males and females. Although a marked variation in the level of parasitaemia was recorded between villages and the urban setting in 2011, a clear age-pattern was commonly observed in both cross-sectional surveys. This is a reflection of the degree of immunity to the disease, which is known to be low among children, but increases markedly over the time as a result of continuous exposure to Plasmodium infection, a considerable proportion of fever cases (32.9–44.9 %) showed no Plasmodium infection. This observation raises the question of whether febrile patients should be treated empirically for malaria. In the current study area, presumptive treatment of malaria is common, even in health facilities. As this practice results in overtreatment and might lead to the development of anti-malarial drug resistance, there is a need for RDTs and treatment of only those cases found positive [Even though fever was positively associated with positive , 44.Plasmodium infection and anaemia supports the finding that malaria plays a major role in the occurrence of anaemia in tropical areas [Schistosomahaematobium infection, inflammation, cellular iron deficiency and chronic malnutrition must be considered as important contributors to anaemia [The prevalence of anaemia was high within the surveyed population 37.3–47.6 %). Not surprisingly, children were at highest risk, which is in line with previous studies conducted in Kenya , Nigeria.6 %. Notal areas –50. Neve anaemia , 51. Thi anaemia .Plasmodium infection and splenomegaly with spleen rates showing similar trends as parasite rates. This is expected for malaria-endemic countries, where transmission is intense and perennial [The proportion of people with enlarged spleen might serve as an indicator for the intensity of malaria transmission . Evidencerennial –55. Altherennial . For exaPlasmodium infection rates, parasitaemia and anaemia in the Taabo HDSS where malaria remains the main cause of death. Indeed, the public health significance of malaria calls for concerted efforts to control this mosquito-borne disease. The prevalence of infection was particularly elevated in children below 10 years of age. Males and village residents were at higher risk than females and people living in hamlets or Taabo-Cité. The association between Plasmodium infection and fever, anaemia and splenomegaly, which is a typical feature of heavily endemic areas, demonstrates the severity of the disease. There is, therefore, a pressing need to scale-up control measures in order to reduce the burden of malaria in the Taabo HDSS and Côte d’Ivoire more generally. Further investigations on malaria risk factors will enable a comprehensive overview of the heterogeneities observed within the residence settings and the two genders to come up with setting-specific control interventions.This study provides important information on age-, sex- and residency-specific


Plasmodium falciparum are asymptomatic and constitute a reservoir of parasites for infection of newly hatched mosquitoes.In malaria-endemic countries, large proportions of individuals infected with P. falciparum, detected by rapid diagnostic test, on disease transmission and susceptibility to clinical malaria episodes. A clinical study assessed the incidence of symptomatic malaria episodes with a parasite density >5,000/μL after three screening and treatment campaigns ~1 month apart before the rainy season; and an entomological study determined the effect of these campaigns on malaria transmission as measured by entomological inoculation rate.Two studies were run in parallel in Burkina Faso to evaluate the impact of systematic identification and treatment of asymptomatic carriers of P. falciparum density was lower in the intervention arm than the control arm. This trend was not seen in those individuals aged ≥5 years. Over the year, monthly variation in mosquito density and entomological inoculation rate was comparable in both arms, with September peaks in both indices.The intervention arm had lower prevalence of asymptomatic carriers of asexual parasites and lower prevalence of gametocyte carriers during campaigns 2 and 3 as compared to the control arm. During the entire follow-up period, out of 13,767 at-risk subjects, 2,516 subjects  had symptomatic malaria. Kaplan-Meier analysis of the incidence of first symptomatic malaria episode with a parasite density >5,000/μL showed that, in the total population, the two treatment arms were similar until Week 11–12 after campaign 3, corresponding with the beginning of the malaria transmission season, after which the probability of being free of symptomatic malaria was lower in the intervention arm (logrank p < 0.0001). Similar trends were observed in infants and children <5 years and in individuals ≥5 years of age. In infants and children <5 years old who experienced symptomatic malaria episodes, the geometric mean P. falciparum had no effect on the dynamics of malaria transmission, but seemed to be associated with an increase in the treated community’s susceptibility to symptomatic malaria episodes after the screening campaigns had finished. These results highlight the importance of further exploratory studies to better understand the dynamics of disease transmission in the context of malaria elimination.Community screening and targeted treatment of asymptomatic carriers of  In 2010, as many as 1.2 million individuals are estimated to have died from malaria, revealing the considerable burden that this disease continues to place on many endemic areas in sub-Saharan Africa . FollowiMDA involves the administration of antimalarials to entire populations without knowledge of who is infected. In countries with high malaria endemicity, the majority of MDA programs have failed in their attempts to interrupt transmission or provided only a transient success ,12. GivePlasmodium falciparum infections are asymptomatic [Screening and treatment only of infected individuals is an alternative to MDA. In malaria-endemic countries, a large proportion of ptomatic -16. As aptomatic ,18. A reptomatic .P. falciparum asymptomatic carriers at a community level on the incidence of symptomatic malaria episodes with a parasite density >5,000/μL; and an entomological study to determine the effect of the screening and treatment intervention on malaria transmission as measured by EIR. The studies took place in the health district of Saponé, about 50 km south of Ouagadougou in Burkina Faso, which is an area with marked seasonal P. falciparum malaria transmission from June to November [P. falciparum asymptomatic carriers) and control arms.The description of the epidemiology of malaria is often focused on clinical parameters such as prevalence of parasitemia. However, entomological information such as vector species composition and density, proportion of infected mosquitoes, entomological inoculation rate (EIR), and insecticide resistance status are key transmission parameters essential to the planning of control measures. This article reports results from two studies run in parallel: a clinical study to evaluate the impact of systematic treatment of November . The nulet al. [The full study methodology was published by Tiono et al. . A briefP. falciparum asymptomatic carriers on the incidence of symptomatic malaria episodes in children (<5 years) and adults over a 12-month period after completion of three community screening and treatment campaigns, compared with no treatment of asymptomatic carriers. In total, 18 clusters (each comprising one village) were randomized and assigned in a 1:1 ratio to the intervention or control arm.This was a single-center, controlled, parallel, cluster-randomized study that evaluated the effect of systematic treatment of Before the study implementation phase, all inhabitants of the 18 clusters were provided with long-lasting insecticide-treated nets . Compliance with mosquito net use was checked every two months during home visits to the trial participants by Demographic Surveillance System (DSS) fieldworkers.P. falciparum asexual forms and gametocytes. In the intervention arm, the population was also screened using a rapid diagnostic test  to identify asymptomatic carriers. Those individuals with a positive RDT received treatment with artemether-lumefantrine  or artemether-lumefantrine dispersible, twice a day for three consecutive days. Subjects in the control arm were not screened by RDT – microscopy alone with delayed reading was used to ensure that study personnel and screened subjects remained unaware of subject status.During the implementation phase, inhabitants of the intervention and control clusters participated in the three screening campaigns that took place ~1 month apart between January and April 2011, before the start of the rainy season. A fourth campaign was conducted in January 2012 after the rainy season had ended to mark the end of the study at ~12 months  or history of fever within the last 24 hours. On day 1 of campaign 4, a blood sample was also collected for gametocyte assessment by quantitative reverse transcription-polymerase chain reaction (qRT-PCR), which was conducted in 1,999 randomly selected subjects drawn from the entire intervention group and a 40% subgroup of the control population. In this study, a case of symptomatic malaria was defined as fever or history of fever within the previous 24 hours and a P. falciparum was present, a count of the asexual forms against leukocytes was made using a tally counter. Counting was done based on at least 200 leukocytes according to WHO standards. If less than 10 parasites were identified from the 200 leukocyte screen, counting was extended to 1,000 leukocytes. If P. falciparum gametocytes were seen, a gametocyte count was performed against 1,000 leukocytes. All slides were read by two independent microscopists. If the ratio of densities from the first two readings was >1.5 or <0.67, or if less than 30 parasites were counted with an absolute difference of more than 10 in the number of parasites, the slide was evaluated by a third microscopist. The definitive result was the mean of the parasite density of the two most concordant reading results.The blood films obtained during visits and for symptomatic malaria assessment were air-dried and Giemsa-stained for examination by a light microscope fitted with a 100 X oil immersion lens at a single laboratory. At least 200 thick film fields were examined before a slide was declared negative. When Microscopist competency was evaluated through two equivalent quality control (EQC) programs. The first EQC was carried out by College of American Pathology proficiency testing and included a set of 5 slides provided to each microscopist for reading thrice a year. The second EQC was performed by WHO  and involved the reading of a set of 20 slides every quarter by each microscopist. Only those with a score of at least 80%, graded as ‘competent’ , were involved in the reading of trial participants’ slides.This trial is registered with ClinicalTrials.gov, number NCT01256658.The protocol and the proposed informed consent form were reviewed and approved by the Centre National de Recherche et de Formation sur le Paludisme Institutional Review Board and by the National Ethical Committee for Health Research of Burkina Faso. Prior to study initiation, a community meeting was held in each of the selected clusters to discuss the study with the community. The freedom of each individual household and household member to decide on participation was discussed to minimize the potential influence of key opinion leaders in each cluster. Individual informed consent was obtained from each participant during a visit to the household before any study procedure.From the 18 clusters selected for the clinical study, 10 were randomly selected for the entomology study. To achieve random selection, two lists containing the nine villages from each arm were generated. Using the random function in Excel , each village on the intervention list and the control list was assigned a number between 1 and 9. The villages in each arm were then listed in ascending order on the basis of the randomly assigned number. The first five villages from each list were selected for inclusion in the entomology study.Beginning at the initiation of the first screening campaign and continuing weekly until screening campaign 4 (i.e. ~12 months later), mosquitoes were collected from 10 randomly selected houses in each of the 10 selected clusters. To randomly select the houses for mosquito collection, a list was generated from the DSS database that contained the names of the heads of each household in each cluster. The random number function in Excel was then used to select 10 individual households from the list for each of the 10 clusters.Mosquitoes were collected using the indoor pyrethrum spray catch method . These cAll mosquitoes collected were transported to the laboratory where the abdomens of the females were examined under a dissecting microscope to classify them into their respective blood feeding stages: unfed, blood-fed, semi-gravid, and gravid.Anopheles gambiae s.l. was dissected from the abdomen. The random selection was done by using the random number function in Excel to select mosquitoes on the basis of the unique ID numbers given to each insect. The head-thorax portions were tested by an enzyme-linked immunosorbent assay (ELISA) that specifically detects P. falciparum circumsporozoite protein (CSP) antigen to allow estimation of the P. falciparum sporozoite index according to the method described by Burkot et al. (1984) and modified by Wirtz et al. (1987) [From each collection round, the head-thorax portion of 200 randomly selected blood-fed, semi-gravid and gravid female . (1987) ,24. To d. (1987) .An. gambiae s.l. for at least 100 insects from each collection. These were randomly selected by ID number as previously described. Mosquitoes positive for the ELISA P. falciparum CSP also underwent molecular classification. Mosquitoes belonging to An. gambiae s.l. were identified to the species level using a standard PCR technique as described by Fanello et al.[All mosquitoes collected were morphologically identified. In addition, molecular analysis was used to confirm the morphological classification of lo et al..An. gambiae s.l. mosquitoes (50 from each intervention and control cluster) were randomly selected from each collection (as described above) to undergo testing by PCR according to Martinez-Torres et al. (1998) and Ranson et al. (2000) for detection of the kdr gene mutation that confers resistance to pyrethroids [kdr allele frequency in each village of the study area, all An. gambiae s.l. collected were eligible for kdr screening regardless of whether they were alive or dead.A subsample of 500 ethroids ,28. To oThis study was analyzed at the cluster level, i.e. the cluster was considered as an experimental unit and observations were based on individual subjects in each cluster. All hypothesis testing, except human biting rate and EIR, was conducted one-sided at a 0.05 level of significance. No adjustments for multiplicity were performed.5000), survival analysis was carried out using PROC LIFETEST in SAS®, assuming hazard ratio remains constant over time, and considering only the number of subjects at risk at that time point of consideration as denominator. Time was measured in days from the day of diagnosis of asymptomatic carriage to first SMRC5000 in the study period. The survival probability was estimated based on a Kaplan-Meier estimate. Missing time was censored at time of withdrawal or completion of the study period including immigrant and emigrant subjects.To account for both the occurrence and timing of the first RDT-confirmed symptomatic malaria episode with a parasite density >5,000/μL  and the proportion of sporozoite-positive mosquitoes. The daily EIR was multiplied by the number of days in the corresponding month to obtain an estimate of the monthly EIR. Then, the monthly EIRs in each arm were summed up to obtain the cumulative annual EIR. To examine the influence of the intervention on human biting rates and EIR, a two-sided Wilcoxon rank sum test at 95% level of significance was adopted.et al. (2013) [et al. (2013) [These results were reported previously by Tiono . 2013) . A total013 . A t. (2013) .The prevalence of asymptomatic carriage of asexual parasites over time is presented in Table 5000 following screening campaign 3 showed that in the total population the two treatment arms were similar until Week 11–12, i.e. the beginning of the malaria transmission season. After this time, the probability of being free of malaria was lower in the intervention arm (logrank p <0.0001). Similar trends were observed in infants and children <5 years (logrank p = 0.0356) and in individuals ≥5 years of age  in the intervention vs the control arm was 82,203 vs 104,120 during the first episode, 81,583 vs 95,248 during the second episode, 88,599 vs 91,514 during the third episode, and 83,532 vs 97,966 at any episode  and 3.8% (95% CI 3.2% – 4.6%) respectively in the intervention and the control arms. Similar patterns were observed between arms when the participants were stratified by age category.Gametocyte density as measured by microscopy at the end of the trial was at least 2-fold higher in the control arm than in the intervention arm. When gametocyte density was measured by qRT-PCR, the difference between the two arms was even greater Figure .kdr gene mutation was slightly higher in the control arm  than the intervention arm . The intervention arm mean (SD) biting rate was 1.12 (1.20) bites per person per night, vs 1.12 (1.19) bites per person per night in the control arm. Over the year, EIR and mosquito density results were also comparable in both arms, with peaks for both observed in September . The species composition collected in the two arms of the study was similar  [Community screening and targeted treatment of asymptomatic carriers of l. 2013) .P. falciparum infection that protects against the development of clinical episodes [The results may suggest that clearance of asymptomatic infections increases an individual’s risk of contracting a symptomatic malaria episode. A similar ‘rebound’ effect has been observed following the use of chemoprophylaxis in malaria-endemic areas -31. It hepisodes . Consequepisodes ,34. As sThe development of clinical and parasitological immunity to malaria is marked by the ability of individuals to control disease and parasite density . In thisP. falciparum sexual stages is an essential factor for reducing human-to-mosquito transmission of malaria parasites by preventing the fertilization and development of the parasite in the mosquito midgut. In addition, it has been shown in previous work that naturally acquired immunity against Pfs48/45 and Pfs230  is a function of recent exposure rather than of cumulative exposure to gametocytes [Among other factors, malaria transmission depends on the presence of infectious sexual parasites (gametocytes) in human peripheral blood. While individuals with subpatent gametocytemia are able to infect mosquitoes, previous research carried out in Burkina Faso has shown that individuals with microscopically detected gametocytes are two-fold more likely to be infectious compared to those with submicroscopic gametocytes  . Althougetocytes . TherefoA potential confounding factor is the invasion of intervention clusters by infected mosquitoes from surrounding villages that did not receive LLINs and thereby contributed to the spread of infection; however, as such invasion is also likely to occur in the control clusters the effect of this factor on the overall results is likely to be marginal. Additionally, RDT sensitivity (vs microscopy) during the three screening campaigns was 92.4%, 84.0% and 77.8%. These findings suggest that by using RDT and subsequent microscopic confirmation a notable proportion of asymptomatic carriers were undetected. The use of more sensitive molecular techniques, possibly in combination with RDT, may merit exploration in future mass screening and treatment interventions.kdr mutation observed was comparable to that observed in 2010 by Badolo et al. in a village located 30 km from Ouagadougou with the same agricultural practices as the study area [Overall, the malaria transmission dynamic in the study arms is comparable to previous reports from the area . The humudy area .P. falciparum seemed to be associated with an increase in the treated community’s susceptibility to malaria episodes once the screening campaigns had finished. In the context of malaria elimination, these results clearly show the importance of further exploratory studies to better understand the dynamics of disease transmission and possible immunological factors involved. They also underline the fact that clearing the reservoir of malaria parasites with effective antimalarial drugs might not be enough to interrupt malaria transmission in high-transmission settings; association with malaria transmission-blocking tools might be required. In the absence of an effective malaria transmission-blocking vaccine, drugs with known transmission-blocking potential such as primaquine [Community screening and targeted treatment of asymptomatic carriers of imaquine  and iverimaquine  might haAT has received honoraria from Novartis Pharma AG, Basel, Switzerland to attend Advisory Board meetings to discuss this study and manuscript. AM is an employee of Novartis Healthcare Private Limited, and KH is an employee of Novartis Pharmaceuticals Corporation. MG, NS, IN and SS declared no competing interests.All authors were involved in the design of the study, data interpretation, and defining the content for and critically reviewing the manuscript. AT, MG, NS, IN and SS were involved in data collection, while AT, MG, AM and KH conducted the data analysis. AT, MG, NS, AM and KH were involved in writing the manuscript. All of the authors had full access to data in the study, discussed the results, reviewed the draft manuscript and agreed on the final version. KH, the corresponding author, had final responsibility for the decision to submit the manuscript for publication. Editorial assistance was provided by Louisa Reed from PreScript Communications, with funding from Novartis Pharma AG.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2334/13/535/prepubIncidence of first symptomatic malaria episode with a parasite density >5,000/μL.Click here for file


In China, the prevalence of malaria has reduced dramatically due to the elimination programme. The continued success of the programme will depend upon the accurate diagnosis of the disease in the laboratory. The basic requirements for this are a reliable malaria diagnosis laboratory network and quality management system to support case verification and source tracking.The baseline information of provincial malaria laboratories in the China malaria diagnosis reference laboratory network was collected and analysed, and a quality-assurance activity was carried out to assess their accuracies in malaria diagnosis by microscopy using WHO standards and PCR.Plasmodium species were identified with great accuracy by microscopy and PCR. However, Plasmodium ovale was likely to be misdiagnosed as Plasmodium vivax by microscopy.By the end of 2013, nineteen of 24 provincial laboratories have been included in the network. In the study, a total of 168 staff were registered and there was no bias in their age, gender, education level, and position. Generally Plasmodium species can be identified fairly accurately by microscopy and PCR. However, laboratory staff need additional trainings on accurate identification of P. ovale microscopically and good performance of PCR operations.China has established a laboratory network for primary malaria diagnosis which will cover a larger area. Currently,  The epidemiology of malaria in China has changed dramatically ,2, and tThe provincial and national laboratories are not only required to directly participate in the patients diagnosis and review samples from hospitals, but should also review samples diagnosed by lower level laboratories. For microscopic diagnosis each year all positive slides must be reviewed by all-level laboratories, and no less than 10% of negative slides generated in clinics, then no less than 3% of the former, and no less than 1% of the former again must be reviewed by the county prefecture and provincial levels, respectively. Finally, at least once a year the national laboratory performs reviews and QA activities to cover all the provinces.Plasmodium 18s rRNA gene as mandatory methods in the provincial and national laboratories, while rapid diagnostic test (RDT) is optional. This is because using microscopy, Plasmodium parasites can be rapidly characterized and quantified using standard procedures . St. St13]. RNA gene ,11,15. TP value <0.05 was considered statistically significant.Baseline information from each laboratory was verified by an expert group  through field investigation. All collected data were entered into Microsoft Excel and analysed using SAS 9.2. A The programme was reviewed and approved by the Ethical Committee of National Institute of Parasitic Diseases, China CDC. All participants provided written informed consent for the study.In total, 19 provincial reference laboratories that met all requirement for inclusion in the network such as laboratory room, devices, organization, and technical demands were established in 2011, 2012 and 2013, respectively  ranging in ages from 25 - 59 years (40.26 ± 10.62) with 61.9% being males (104/168) and 38.1% females (64/168). Among them 45.8% (77/168) had bachelors’ degree and 35.7% (60/168) had a graduate degree and the remaining had a college degree. In addition, 96 individuals (5.05/laboratory) were full-time staff of the malaria laboratory while 72 worked part-time. The position of the individuals was responsible for laboratory management, microscopy, molecular biological detection, and management of QA and biosafety, respectively.2 = 0.1539, P = 0.9259. Gender: Fisher’s exact probability, P = 0.7677. Education level: Fisher’s exact probability, P = 0.3238. Work style (full-time or part-time): Fisher’s exact probability, P = 0.6412.The 19 laboratories were classified into three groups (G1 ~ 3) Figure  based onPlasmodium vivax-positive slide marked ‘13-1-MM10’ as Plasmodium ovale. The P. ovale positive slide of ‘13-2-MM05’ was the most difficult for participants with nine failing to identify it and most of them (6/9) incorrectly identifying it as P. vivax (Table P = 0.2338).In total, only two participants correctly identified all 20 slides. Seven failed in one slide, six incorrectly identified two slides, two individuals failed in three slides, and one each incorrectly identified four and six slides, respectively. Particularly, 12 participants identified the P = 0.3569).Among the results reported, 84.21% (48/57) of samples were detected correctly; 63.16% (12/19) of staff detected all samples correctly and 26.32% (5/19) failed in one sample. Interestingly, two participants detected only one sample correctly . Besides, microscopists , who achieved the top level 1, 2 or 3 efficiency in diagnosing malaria through a microscope and were assessed in China by a WHO-Certified Level-1/expert microscopist in 2012 [Plasmodium species by microscopists in national laboratory was 91.67 (55/60), 86.67 (39/45) and 95.56% (43/45). Overall, these demonstrated the high competency of Plasmodium species identification by Chinese malaria microscopists.Currently, participants in the provincial laboratories can efficiently identify most  in 2012 , in the Plasmodium detection in China ranges from 62 to 66%, which is based on three national technique competitions for diagnosis of parasitic diseases conducted in 2011 [Plasmodium falciparum and P. vivax species identification was about 65% [However, malaria diagnosis in China still faces the challenges. First, the microscopy competency at lower levels needs improvement. The average accuracy of  in 2011 , 2012 [1 in 2011  and 2013bout 65% ,18. In ebout 65% ,19,20, tPlasmodium species in China. Although total malaria cases reported in recent years decreased markedly, malaria importation poses a constant threat to mainland China with the proportion increasing every year. The major species imported to China are the dominant P. falciparum from African continent and P. vivax from Asian countries, but other species, such as Plasmodium malariae and P. ovale have also been reported [Second, it is difficult to identify rare reported ,21,22.Plasmodium ovale has been reported throughout the world, and its natural distribution [P. ovale received little attention primarily due to sporadic malaria cases associated with this species [P. ovale malaria has increased due to importation by travellers to sub-Saharan Africa [P. ovale malaria include common cold-like mild symptoms and are accompanied with low parasitaemia, and individuals infected this species are given the same treatment as P. vivax [P. ovale from P. vivax by examining peripheral blood films stained with Giemsa [P. ovale malaria cases in China were misdiagnosed as P. vivax malaria [P. ovale, and strengthen the training at different levels.ribution  is limitribution -27 and tribution -30. In C species . The incn Africa -36. The P. vivax . It has h Giemsa . Consist malaria ,39 as waThe diagnosis of malaria parasites by nested PCR in the present assessment is at medium level with an accuracy of 84.21% (48/57). The ease and speed of PCR makes it an important tool in malaria elimination programmes and should be improved to detect asymptomatic infections. Since various PCR methods have been developed for malaria diagnosis -44, it iIn addition, more comprehensive assessments including slides and dry blood spots with different parasite densities should be considered in future.Plasmodium species using microscopy and PCR is at medium to high level. The biggest problem is the misdiagnosis of P. ovale as P. vivax microscopically. It is essential for more laboratories at different levels apply to join the network to obtain the required training and sustain malaria elimination in China. Special emphasis should be on the identification of locally rare Plasmodium species, such as P. ovale.A malaria diagnosis reference laboratory network has been established in China and is in its initial stages. The identification of