Plasmodium vivax and Plasmodium ovale are often considered the malaria parasites best adapted to long-term survival in the human host because of their latent exo-erythrocytic forms. The prevailing opinion until the middle of the last century was that the maximum duration of Plasmodium falciparum infections was less than two years. Case reports and series investigating blood donors following accidental malaria infection of blood transfusion recipients and other sporadic malaria cases in non-endemic countries have shown clearly that asymptomatic P. falciparum infections may persist for up to a decade or longer (maximum confirmed 13 years). Current policies in malaria-free countries of excluding blood donors who have lived in malarious areas are justified. Vigilance for longer than three years after declaring elimination in an area may be needed. Plasmodium vivax and Plasmodium ovale are often considered the malaria parasites best adapted to long-term survival in the human host because of their latent exo-erythrocytic forms. Yet Plasmodium malariae, which has no latent forms, can persist asymptomatically in the blood for decades, and perhaps throughout the entire life of the host [P. malariae infections have been revealed by blood donation [the host . Many ofdonation , with ilPlasmodium falciparum malaria comes first from the early descriptions of neurosyphilis patients who were given malaria. Malariatherapy, the brainchild of the Viennese neuropsychiatrist Julius Wagner-Jauregg, was introduced as a treatment for neurosyphilis in the 1920s, and was widely used until supplanted by penicillin in the 1950s. The malariatherapy parasite of choice was generally P. vivax rather than the more dangerous P. falciparum, but all species were used. Detailed studies of syphilis patients infected with strains of P. falciparum reported durations of infection between 50 and 500 days depending on the infecting strain [Characterization of the natural history of untreated or partially treated g strain , 4.P. falciparum infections was debated by malariologists during the first era of malaria eradication (in the 1950s) as it was a key consideration in the investigation of any sporadic cases in the post-elimination period. A review by Covell in 1960 presented the prevailing opinion that P. falciparum infections generally did not continue beyond one year although he noted nine cases where infection had recurred between one and two years after the primary infection, and he cited Ciuca’s review of 12,842 cases of experimental malaria from Romania showing a maximum persistence of P. falciparum infections of 27 months [P. falciparum could persist in humans for at least three years. One example was probable transfusion malaria from a donor who must have been parasitaemic for more than two years. Another was a presumed recrudescence of falciparum malaria in a young woman who had a documented episode of malaria three years previously, after which she had resided in an area considered free of malaria transmission [P. falciparum infections continued for up to 121 weeks [The duration of in the 190s as it P. falciparum malaria at the end of the malaria season remained chronically infected throughout the dry season and the following rainy season with the same genotype [A mathematical modelling approach has been used to estimate the duration of falciparum infections using repeated cross-sectional survey data from other cohorts, such as the subjects in the Garki project in the 1970s. Estimates for the total duration of infection across all age groups were long at 602 days  581–625) for Pare-Taveta (in Kenya/Tanzania), 734 days (95% CI 645–849) for West Papua, and 1,329 days  for Garki in Nigeria . Until mgenotype .In high-transmission areas asymptomatic parasitaemia is very common. The ability of individuals to control their parasitaemia at low levels without symptoms (premunition) results from the gradual acquisition of immunity following repeated or protracted exposure. In these areas uncomplicated and severe malaria are predominantly diseases of young children. Superinfection is usual with individuals harbouring multiple, genetically unrelated, parasite clones in a constantly changing equilibrium, often generating transmissible densities of gametocytes but remaining below the combined asexual parasite density that provokes illness –14.Plasmodium falciparum is usually responsible. Anti-malarial antibody titres are high but microscopy examination of blood smears is usually negative. Persistent or recurrent active malaria infection is confirmed by response (shrinkage of the spleen) to effective anti-malarial treatment (often in a prolonged course to prevent re-infection). Typical histopathological findings in the spleen are non-specific cellular hyperplasia and lymphocytic infiltration without malaria pigment. Two cases of symptomatic falciparum malaria occurring post splenectomy (performed for suspected lymphoma) were reported from France. Both were in immigrants who had not visited a malaria-endemic area for at least two years. Their diagnoses were changed to HMS retrospectively and indicate persistent active infection throughout this more than two years interval [Sometimes the host immune response to repeated malaria infection is excessive, manifest by the syndrome of hyper-reactive malarial splenomegaly (HMS); marked splenomegaly with hypersplenism and hypergammaglobulinaemia (in particular raised IgM). interval . Similarinterval . ExampleP. falciparum[P. falciparum cases. Assuming a multiplication factor of approximately ten-fold per cycle and a pyrogenic threshold of 108 parasites in total in an adult, this suggests an inoculum of ~1,000 parasites in a blood bag or bottle of approximately 500 mL. Thus the likely donor density approximated an average of 2 parasites/mL, well below the pyrogenic (and microscopy detection threshold) density of ≥50,000/mL.The first report of malaria as an accidental consequence of an artery to vein blood transfusion was described in 1910 by Woolsey . The effA search of Pubmed and Embase was performed for case reports of transfusion or transplant transmitted falciparum malaria or for cases of falciparum malaria presenting more than six months after leaving an endemic area. Cited references were used to identify other cases. Cases of transfusion-transmitted malaria from the USA were searched for in the annual MMWR malaria surveillance reports. Searches were also performed in websites of blood banks and the international haemovigilance network . ReportsTwenty-nine cases of transfusion and transplant-transmitted falciparum malaria are summarized in Table Plasmodium falciparum gametocytaemia was reported in three of the cases. In one case, gametocytes were the only parasite stage seen on the blood film a month after a fever episode, suggesting the patient had self-cured the infection. Five cases reported having taken antimalarial drugs previously. In 13 cases the symptomatic episode coincided with pregnancy (seven women) or was associated with a co-morbidity, e.g., diagnosis of malignancy (one), diabetes mellitus (one), post head-injury (one), sickle-cell anaemia (one), HIV (two cases), which suggests a reduction in host immunity may have unmasked the latent infection.Table The ability to detect asymptomatic carriers of malaria parasites depends on the sensitivity of the detection method used. Blood smear microscopy by experienced microscopists will detect parasite densities of ≥50/μL reliably. More recently, molecular methods have been employed to detect sub-patent parasitaemias. PCR is considerably more sensitive than microscopy or rapid diagnostic tests based on antigen detection but typically blood samples have been taken from finger-prick blood spots on filter paper, thus a volume of only ~5 μL has been amplified. This obviously misses densities which are below one parasite in 5 μL or 200 parasites/mL. In general, most PCR methods have limits of detection of ≥1,000 parasite/mL. Using larger blood volumes parasite densities down to ~20/mL can be detected, several orders of magnitude below the microscopy level of detection .Malaria infection may also be assessed using serology, although interpretation is difficult. A variety of crude and more refined test antigens have been evaluated. Typically seroprevalence increases with age while the correlation with patent parasitaemia goes down , 62. TheLongitudinal serological studies have been conducted in immigrants who have moved to countries that are malaria free. In a Spanish study IgG antibody levels against the erythrocytic antigens AMA-1 and MSP-142 (3D7 and FVO strains), EBA-175 and DBL-α, did not correlate with the time since migration . SerologAnopheles gambiae in West Africa [et al. studied the infectivity of long-term asymptomatic P. falciparum malaria to laboratory-bred Anopheles atroparvus following experimentally induced infections in ten individuals [th day after infection.There is good evidence that chronic sub-patent infections are a source of onward mosquito transmission of malaria. In semi-immune individuals with persistent infections, gametocytaemia at densities below the limit of microscopy detection is often infectious . In 1931t Africa . Ciuca eividuals . They obP. falciparum can survive for extended periods in the human host. The previously quoted maximum duration of two years should be revised; 13 years is the maximum confirmed duration reported to date. The transfusion malaria cases suggest the parasite continues its erythrocytic life cycle at very low densities, kept in check by the host’s immune response. This is a common pattern for malaria parasites in all animals, and is the most likely explanation for persistence of both P. falciparum and P. malariae in man. There is no evidence for protracted dormancy or an exo-erythrocytic parasite stage of these parasites, although it is possible that the entire life cycle could take place in the vascular spaces in the placenta or spleen. Incomplete antimalarial drug treatment may have a modulatory effect on the infection early on. As well as the poorly understood host’s response to the parasite, P. falciparum possesses sophisticated immune evasion mechanisms, notably the differential serial expression of var genes encoding for PfEMP1, the major surface antigen of the blood stage [P. falciparum parasites survive for prolonged periods in the human host without exhausting their repertoire of antigenic variation is unclear. The appearance of symptoms years after infection may reflect waning of host immunity. The late recrudescing infections presented in Table These reports show unequivocally that od stage . How P. P. falciparum might be. The World Malaria Report estimated that in 2012 there were between 135 and 287 million malaria cases of which 91% were caused by P. falciparum[It is not possible to estimate how common chronic infection with lciparum. Even ifApproximately 107 million units of blood donations are collected globally every year . Very feP. falciparum[These observations on the longevity of falciparum infections and the sub-microscopic parasite burden are relevant to immigrants and refugees who have left malarious areas. In a parasite prevalence study of 195 African refugees in Italy in 2007, 62 patients tested positive for malaria using molecular detection methods, of whom 13 were gametocytaemic. Microscopy confirmed the positive results in 14 of the 62 cases, of which 13 were lciparum. The repIn countries targeting malaria elimination, achieving this aim is likely to take longer if the sub-microscopic parasites are not eliminated. Improved high-throughput and low-cost methods are needed to detect these individuals harbouring low parasite densities. Conventional PCR methods still miss infections with densities below 1/uL. Serology has been proposed as a useful tool to detect changes in malaria transmission in settings where transmission is in decline and appears to be a sensitive method for the detection of malaria infection but its specificity is unclear, as is the usual duration of detectable antibodies once an infection has cleared. There is a need to investigate the role of serology in defining asymptomatic carriage, certainly in lower transmission and pre-elimination countries.Plasmodium falciparum can persist in the human host for longer than thought previously. Current policies in malaria-free countries of excluding blood donors who have lived in malarious areas are justified. Vigilance for longer than three years after declaring elimination in an area may be needed.


Regular formation of an erythrocyte monolayer in each microchamber was observed following dispersion of an erythrocyte suspension in a nuclear staining dye, SYTO 21, onto the chip surface and washing. About 500,000 erythrocytes were analyzed in a total of 4675 microchambers, and malaria parasite-infected erythrocytes could be detected in 5 min by using the fluorescence detector. The percentage of infected erythrocytes in each of 41 patients was determined. Accurate and quantitative detection of the parasites could be performed. A good correlation between examinations via optical microscopy and by our chip system was demonstrated over the parasitemia range of 0.0039–2.3438% by linear regression analysis (R2 = 0.9945). Thus, we showed the potential of this chip system for the diagnosis of malaria.Accurate, sensitive, rapid, and easy operative diagnosis is necessary to prevent the spread of malaria. A cell microarray chip system including a push column for the recovery of erythrocytes and a fluorescence detector was employed for malaria diagnosis in Uganda. The chip with 20,944 microchambers (105 μm width and 50 μm depth) was made of polystyrene. For the analysis, 6 μl of whole blood was employed, and leukocytes were practically removed by filtration through SiO Malaria, a mosquito-borne infectious disease, is one of the major human infectious diseases, there having been approximately 214 million clinical cases and 438,000 fatalities in 2015 alone55791011122) nano-fibers to isolate erythrocytes from whole blood and a fluorescence detector with a CCD camera instead of centrifugation and DNA microarray scanner, respectively  by filtration through this push column was confirmed .To achieve the confinement of erythrocytes in each microchamber, we optimized the hydrophilicity of the cell microarray chip surface by subjecting it to reactive ion-etching2 = 0.9945). By the RDT, all 41 samples were decided as positive for malaria infection. On the other hand, 40 samples were positive, and 1 sample was negative (sample no. 32), by the PCR analysis.Blood samples from 41 patients were examined under a light microscope with Giemsa staining, and malaria parasites were found in 37 of them, with parasitemia ranging from 0.0039% to 2.3438% . MalariaFluorescence-positive erythrocytes, which were equivalent to malaria parasites, could not be detected in any of 50 healthy donors by use of the cell microarray chip system (data not shown).Bright-field images of malaria parasite-infected erythrocytes , leukocyTM, Ambion, Inc., TX) to obtain purified erythrocytes from whole blood. Furthermore, we employed a DNA microarray scanner with confocal laser scanning for the detection of fluorescence-positive, malaria-infected erythrocytes. However, this methodology is not suitable in the field setting because of high cost, complicated technical handling, and its time-consuming aspect. In our present study, we employed a push column without any power supply and a leukocyte isolation filter instead of centrifugation, as well as a fluorescence detector with a CCD camera instead of an expensive DNA microarray scanner. Although almost all of the leukocytes (over 99.9%) in the whole blood remained in the outer tube by use of the push column, a nominal number of leukocytes were found among the isolated erythrocyte fraction on the cell microarray chip. The condensability of leukocyte nuclei is apparently greater than that of nuclei of malaria parasites, as reported previouslyEarlier we reported a cell microarray chip system for highly sensitive, accurate, and rapid detection of malarial parasites in malaria cultures4 to 57.6 × 104 cells were examined in 25 clusters on the cell microarray chip. These numbers of erythrocytes were still sufficient to obtain high-sensitive detection of malaria parasites.It required over 22 min to scan a whole cell microarray chip  for the detection of malaria parasite-infected erythrocytes by using the fluorescent detector with CCD camera (data not shown). A reduction in diagnosis time is important in field use, and so we only scanned 25 clusters (4675 microchambers), requiring only 5 min. In our previous study, each microchamber accommodated about 130 erythrocytes from a healthy donor, and so 600,000 erythrocytes in total would be expected to be analyzed on a cell microarray chip when only 25 clusters are examinedP. falciparum diagnosis is recommended for malaria parasite-infected patients16In the present study, the detection limit in the cell microarray chip system was 0.0033%, and this sensitivity was greater than that of most routine diagnostic laboratories using Giemsa staining (0.01%). Although there were no malaria parasite-infected erythrocytes in 4 samples  by Giemsa staining or by use of the cell microarray chip, we did obtain positive results for these 4 samples by the RDT and/or PCR method. False positivity is known as one of the disadvantages of RDTsPlasmodium falciparum infection (n = 41), as determined by use of SD BIOLINE Malaria Ag P.f  as a rapid diagnosis test (RDT), were examined for the presence or absence of P. falciparum-infected erythrocytes with conventional Giemsa-staining microscopy and with PCR. This RDT is a lateral flow chromatographic immunoassay for the quantitative detection of P. falciparum-specific protein, i.e., histidine-rich protein II (HRP2).Peripheral venous blood was obtained from 41 patients who were suspected of having malaria or who were receiving malarial treatment at the outpatient care of the Lacor Hospital at Gulu, Uganda. Blood samples positive for 2S2O5 solution was performedFour microliters of whole blood from malaria patients was smeared so as to produce a thin film on a slide. Each slide was stained with 5% Giemsa  stain in phosphate-buffered saline (pH7.2), and then examined under a light microscope  at a magnification of x 1,000 for the counting of malaria parasitesPlasmodium small subunit ribosomal RNA genesP. falciparum, rFAL 1,5′-TTAAACTGGTTTGGGAAAACCAAATATATT-3′ (664–693) and rFAL 2,5′-ACACAATGAACTCAATCATGACTACCCGTC-3′ (869–840) were employed as primers for the 2nd PCR. For both PCRs, 1.0 μl of DNA sample was mixed with Ex Taq buffer  containing 0.2 μl of each primer, 2 mM MgCl2, a 0.2 mM concentration of each deoxynucleoside triphosphate, and 0.5 U Ex Taq (Takara Bio Inc.) in a final volume of 20 μl. Conditions for the first PCR were as follow: step 1, 95 °C for 4 min; step 2, denaturation at 94 °C for 1 min; step 3, annealing at 58 °C for 2 min; step 4, extension at 72 °C for 2 min; step 5, 25 repeats of steps 2–4, and 72 °C for 3 min. Those for the second PCR were as follow: step 1, 95 °C for 5 min; steps 2, denaturation at 94 °C for 1 min; step 3, annealing at 58 °C for 2 min; step 4, extension at 72 °C for 2 min; step 5, 30 repeats of step 2–4, and 72 °C for 5 min.Malaria DNA was extracted from blood blotted onto a filter paper  by using a DNA extraction kit  according to the manufacturer’s instructions. Nested PCR was performed to confirm the presence of malaria in the blood samples. Sequences of the oligonucleotides used as primers for nested PCR were based on the 2-nano-fibers, and the leukocytes remained in the outer tube. To confirm the efficiency of erythrocyte recovery and removal of leukocytes from whole blood, we counted the numbers of erythrocytes and leukocytes with a hemocytometer before and after the filtration of whole blood with this push column. The efficiency of erythrocyte isolation and removal of leukocytes was calculated as follows: number of erythrocytes in the eluted portion/number of erythrocytes in whole blood × 100(%) and number of leukocytes in whole blood − number of leukocytes in the eluted portion/ number of leukocytes of whole blood × 100(%), respectively.The push column  was employed for the isolation of erythrocytes from whole blood . BrieflyAs previously reported, a cell microarray chip with 20,944 microchambers  was made from polystyrene by the Lithographie Galvanoformung Abformung process by Starlight Co. Ltd. , which is a nucleus-specific fluorescence dye  was added to 250 μl of isolated erythrocytes, and the mixture was dispersed manually onto the cell microarray chip by using a pipette, followed by 10 min of standing, to allow the erythrocytes to settle down into the microchambers under gravitational force. Then the excess erythrocytes on the cell microarray chip surface were removed by gentle washing with RPMI 1640 medium . Twenty-The percentage of parasitemia in each patient was determined as follows: [the number of erythrocytes detected as malaria parasite-infected ones by the fluorescence detector/the average number of erythrocytes confined in microchamber × 187 (microchambers) × 25 (clusters)] × 100.We also obtained blood samples from 50 Japanese volunteers who had never been to a malaria-endemic area and were negative by RDT. These blood samples were also analyzed by using the cell microarray chip to examine the presence or absence of fluorescence-positive erythrocytes according to the above-mentioned procedure.This study was approved by the Institutional Review Board of the National Institute of Advanced Industrial Science and Technology for the use of human derivatives for biomedical research, and by the Ethics Committee of Uganda National Council for Science and Technology. All subjects provided written informed consent for the collection of samples and subsequent analysis. All experiments were carried out in accordance with the approved guidelines.How to cite this article: Yatsushiro, S. et al. Application of a cell microarray chip system for accurate, highly sensitive, and rapid diagnosis for malaria in Uganda. Sci. Rep.6, 30136; doi: 10.1038/srep30136 (2016).


Plasmodium falciparum is needed. A blood-stage vaccine is desirable to deal with morbidity and mortality secondary to P. falciparum infection, and thus would be a valuable strategy in the fight against malaria.Complex and widespread with about 90% of the global burden in sub-Saharan Africa, malaria remains a significant health problem. Malaria vaccines have the chance to make significant inroads, with RTS,S vaccine candidate as the most advanced candidate. However, the need for a highly efficacious, long-lasting vaccine is still unmet. Continued support for a repertoire of tools for P. falciparum serine repeat antigen-5 formulated with aluminum hydroxyl gel (BK-SE36) is a blood-stage malaria vaccine candidate. The processing of SERA5 is believed to mediate parasite egress from the infected red blood cell. Field studies show good correlations of anti-SERA antibodies to malaria protection against blood parasitemia, malaria symptoms and severe disease. A Phase 1a trial demonstrated its safety and immunogenicity in malaria naïve Japanese adults. A Phase 1b trial was conducted in the malaria endemic area of Northern Uganda to assess the vaccine’s safety and immunogenicity. BK-SE36 had an acceptable safety and immunogenicity profile. Post-trial, Stage 2 subjects were age-matched to 50 control individuals to compare malaria episodes 130–365 days post-second vaccination. Log-rank test reveal significant differences in BK-SE36 vaccinees vs control group in time-to-first episodes with parasitemias ≥5,000/μL and those with ≥5,000/μL + fever . BK-SE36 vaccinees have fewer multiple malaria episodes with high parasitemia. We also observed that the antibody titers against SE36 were boosted after episodes of natural infection. Above results show that BK-SE36 could provide some protection against natural P. falciparum infection until 1 year post-second vaccination.The


As the geographical distribution of malaria transmission becomes progressively clustered, identifying residual pockets of transmission is important for research and for targeting interventions. Malarial antibody-based surveillance is increasingly recognised as a valuable complement to classic methods for the detection of infection foci especially at low transmission levels. The study presents serological evidence for transmission heterogeneity among school children in The Gambia measured during the dry, non-transmission season.19). Antibody distribution was modelled using 2-component finite mixture model with cut-off for positivity from pooled sera set at 2-standard deviation from the mean of the first component. Factors associated with a positive serological status were identified in a univariate model and then combined in a multilevel mixed-effects logistic regression model, simultaneously adjusting for variations between individuals and school.Healthy primary school children were randomly selected from 30 schools across the country and screened for malaria infection (microscopy) and antimalarial antibodies  asymptomatic. The overall seroprevalence was 12.7% (527/4140) with values for the schools ranging from 0.6% to 43.8%. Age  and parasite carriage  were strongly associated with seropositivity.A total of 4140 children, 1897 (46%) boys, were enrolled with mean age of 10.2 years . Microscopy results available for 3640 (87.9%) children showed that 1.9% (69) were positive for Serological responses to malaria parasites could identify individuals who were or had been infected, and clusters of residual transmission. Field-adapted antibody tests able to guide mass screening and treatment campaigns would be extremely useful. In the past decade, there has been a significant but uneven reduction in malaria indicators Classically, malaria endemicity and transmission are described by the parasite prevalence and the entomological inoculation rate (EIR) respectively, and these methods remain useful in many settings. Nevertheless, in low transmission settings, the paucity of infected mosquitoes, the need to collect and analyse large number of samples coupled with declining sensitivities of microscopy, RDTs and EIRs reduce the efficiency of these methods Where malaria transmission is stable, children less than 5 years old bear the greatest burden of malaria so the intensity of malaria transmission and the impact of interventions are often established in this age group. However, with decreasing transmission, older children may become increasingly at risk of malaria As part of a number of ongoing studies on the dynamics of transmission in low endemic settings, the methods previously tested in the pilot study were applied in a nationwide schools survey to describe and document the variation in malaria transmission across the whole country. This paper reports on the results and discusses the use of seroprevalence data to describe trends in transmission.In May 2012, at the end of the dry season and before the onset of the rains, a cross-sectional malaria seroprevalence survey was carried out among primary school pupils across The Gambia. The country has a population of about 1.8 M people with 39% less than 15 years old Summary data on school attendance by district and region was obtained from the Ministry of Education. There are 411 primary schools distributed across 37 educational districts in six administrative regions A copy of the information sheet describing the study together with an invitation to meet the study team on a pre-defined date at the school premises were distributed to the caregivers of the selected children who were asked to take them home. On the scheduled day, members of the research team met with the caregivers, explained the study, answered questions and asked for a written informed consent for their children to participate to the survey.Paracheck-Pf rapid test kit  and an antimalarial give if positive, where negative; the study nurse gave treatment based on the most likely alternative diagnosis.Consenting caregivers were asked about bed net availability and use at home, and indoor residual spraying (IRS) of their homes in the preceding year. Enrolled children had their body temperature checked and a blood sample collected by fingerpick for slide microscopy, haemoglobin (Hb) estimation with a portable device  and on to a filter paper for subsequent antimalarial antibody measurement. Febrile children (temperature >37.5°C) were screened for malaria using a P. falciparum malaria parasites was determined by reading 100 high power fields under oil immersion. Where a log10 difference in parasite density between the two reads was noted, a third read by a senior microscopist was used to resolve the result.Blood slides were stained with 10% Giemsa for 10 minutes and read independently by two microscopists. The presence of 19 (MSP119) were determined by indirect ELISA with samples eluted from collected filter papers using previously published methods Antibodies against merozoite surface protein-119 antibodies to be about 20% A previous dry season survey in the eastern part of the country showed the prevalence of MSP1Collected field data were double entered into a database on OpenClinica . After cleaning, the data was analysed using STATA version 12.1 . Summary characteristics were presented for continuous (means and standard deviations) and categorical (proportions) variables for each school.Antimalarial antibody distribution was modelled by fitting pooled positive sera using a two-component finite mixture model and the cut off for positivity set at two standard deviations away from the mean of the first component. This derived cut-off titre was applied across all children to determine proportion of seropositive children in the study population and in each school. The prevalence of parasitaemia (microscopy), seropositivity and anaemia (Hb <11 g/dl) across schools and the correlation between malaria infection and seroprevalence were determined.Factors associated with a positive serological status were identified in a univariate model and then combined in a multilevel mixed-effects logistic regression model, which simultaneously adjusts for variations between individual children and across schools. The goodness of fit was measured by calculating the Pearson residual and by visually assessing the agreement between these parameters using a Bland-Altman plot.The Gambia Government/MRC Joint Ethics Committee approved the study (SCC 1318) and the Directorate of Basic and Secondary Education at the Ministry of Basic and Secondary Education granted access to the schools. A written informed consent to participate was obtained from caregivers on behalf of all children enrolled in the study.A total of 4140 children were enrolled, 1897 (46%) were boys. The ages of the children ranged between 4 and 20  years and were similar between schools. The mean bednet ownership was 87% (range 42%–99%) and the large majority  of homes had reportedly been sprayed with an insecticide within the previous year .The mean Hb was 12.5 (SD 1.3) g/dl and 8.4% (348/4140) children had an Hb <11 g/dl; the odds of anaemia were not significantly lower in boys compared to girls . A history of fever in the preceding 24 hours was reported in 4.9% (203/4140) children of whom 4.4% (9/203) had an axillary temperature ≥37.5°C at the time of the survey. These were all negative for malaria using the rapid malaria test kit.Plasmodium falciparum monoinfections. Most  of those infected were asymptomatic, i.e. without any history of fever. The odds of infection did not vary with age .Microscopy results were available for 87.9% (3640/4140) children, 1.9% (69/3640) were positive and all were The prevalence of malaria infection, measured by microscopy, was spatially variable with the highest prevalence seen in schools in the eastern part of the country. In 27 of the 30 schools, malaria prevalence was less than 5%  while in the three remaining schools, the prevalence ranged between 9% and 15% . Infecte19 antibodies and seroprevalence at the schools ranged between 0.6% and 43.8%  component  was set nd 43.8% . There wParasite carriage (a positive slide result) was associated with an 8-fold increase in the odds of being seropositive , anaemia, bednet ownership, IRS and parasite carriage (positive blood slide), only age  and parasite carriage  remained strongly associated with seropositivity .In settings with seasonal malaria transmission, parasite carriage is extremely low during the dry season and confined to particular locations and individuals Therefore dry season malaria surveys could provide insights on the duration of parasite carriage as most  these infections were probably acquired during the previous transmission season, and on where transmission is likely start after the onset of the rains.Some studies suggest that the proportions of infected and clinically ill persons are higher in these foci than in the surrounding population This is probably the most extensive description of the burden of malaria infection in The Gambia at the end of the dry season when malaria transmission has practically ceased and supports the evidence for heterogeneity and declining transmission across the country Such low numbers of infected individuals at the end of the dry season could be targeted to reduce the parasite reservoirs and thus transmission. Nevertheless, there are probably sub-patent infections in these areas, undetected by microscopy, which could still restart transmission Parasite density in asymptomatic individuals may be influenced by acquired immunity and seasonal variations in transmission, and these could affect the sensitivity of screening tools. Both parasitological (microscopy) and serology data show countrywide variation in transmission but with weak correlation between the two measures. Seropositivity estimates have also been seen to be weakly correlated with estimates by PCR although detection rates are higher in the latter compared to microscopy Malaria risk can vary geographically and by both individual and community variables, sometimes with conflicting effects It is likely that transmission will become more heterogeneous as it declines so interventions need to be matched to persons and locations where they are likely to have the maximum effect, This data suggests that serological tests could identify areas of persistent malaria transmission even where parasite-infected individuals are undetected by routine tests. Potentially, they could be used either for surveillance in a (pre-) elimination status or as a prelude to interventions, e.g. mass treatment, aiming at to the elimination of the parasite reservoir Dry season parasite and serological surveys are useful tools for identifying malaria foci as well as for describing short and medium term trends in malaria transmission. Both tools are able to detect heterogeneity in transmission across the country though their results are weakly correlated. Field-adapted serological tests would be extremely useful as a complement for mass screening and treatment programs.


Malaria transmission in Mali is seasonal and peaks at the end of the rainy season in October. This study assessed the seasonal variations in the epidemiology of malaria among children under 10 years of age living in two villages in Selingué: Carrière, located along the Sankarani River but distant from the hydroelectric dam, and Binko, near irrigated rice fields, close to the dam. The aim of this study was to provide baseline data, seasonal pattern and age distribution of malaria incidence in two sites situated close to a lake in Selingué.Geographically, Selingué area is located in the basin of Sakanrani and belongs to the district of Yanfolila in the third administrative region of Mali, Sikasso. Two cross-sectional surveys were conducted in October 2010 (end of transmission season) and in July 2011 (beginning of transmission season) to determine the point prevalence of asymptomatic parasitaemia, and anaemia among the children. Cumulative incidence of malaria per month was determined in a cohort of 549 children through active and passive case detection from November 2010 through October 2011. The number of clinical episodes per year was determined among the children in the cohort. Logistic regression was used to determine risk factors for malaria.The prevalence of malaria parasitaemia varied significantly between villages with a strong seasonality in Carrière  compared with Binko . Children 6–9 years old were at least twice more likely to carry parasites than children up to 5 years old. For malaria incidence, 64.8–71.9 % of all children experienced at least one episode of clinical malaria in Binko and Carrière, respectively. The peak incidence was observed between August and October (end of the rainy season), but the incidence remained high until December. Surprisingly, the risk of clinical malaria was two- to nine-fold higher among children 5–9 years old compared to younger children.A shift in the peak of clinical episodes from children under 5–9 years of age calls for expanding control interventions, such as seasonal malaria chemoprophylaxis targeting the peak transmission months. Dams and irrigation schemes resulting from water resource development projects (WDP) transform ecosystems and can exacerbate a number of potential breeding sites for mosquito vectors due to a higher availability of water bodies and microclimate modifications. However, there is conflicting evidence on the potential role of WDP in the malaria burden in Africa based on variations in endemicity, seasonality and the type of water management and drainage systems –4. In stIn Mali, the agro-hydropower dam of Sélingué was built in 1982 and represents the most important centre of energy production in Mali, of approximately 200 million kW-h a year. The presence of an artificial lake introduces a change in the environmental setting of Sélingué with villages located in a dry area such as Carrière, and others in more humid areas with permanent presence of water pools due to irrigation, such as Binko , while older children remained mostly asymptomatic with parasitaemia . Publish3 of water and has an area of 409 km2. From June (at the beginning of the rainy season), the water fills the dam, which remains high until January. Then, under the combined effects of evaporation and water output for power production, the water level decreases slowly. Lake Sélingué rises and falls in the rainy and dry seasons allowing agriculture on the irrigated perimetre, primarily rice cultivation and vegetable production, managed by the Office of Rural Development of Selingué. Fishing and aquaculture is sustained on the lake when water levels allow.Sélingué is located in the Guinean Sudan Savanna areas of Mali, 130 km from the capital Bamako. The climate features a dry season from November to May and a wet season from June to October. The Sélingué Dam is a hydro-electric and irrigation dam brought into service in 1982 and located in the Sikasso Region, on the Sankarani River, one of the affluents of the Niger River. The retaining basin of the dam forms the artificial Lake Sélingué. The average rainfall over the basin is 1500 mm per year, although the gradient is significant between the more humid south and the drier northern parts of the basin. When full, the lake stores 2.2 kmThe Sélingué Health District is one of the sentinel sites for malaria surveillance maintained by the National Malaria Control Programme (NMPC). It is the smallest health district in Mali covering seven health zones composed of seven to ten villages each. Each health zone has a community health centre run by a nurse providing basic primary care and referring severe cases to the district health centre located at Selingué. This study was conducted in the villages of Binko and Carrière, each hosting a community health centre. The distance from each village to the referral district hospital is 3 km. The two villages have different ecology features. Binko is located 5 km northwest of Lake Sélingué and 2–3 km from the irrigated perimeters where rice is cultivated perennially. The population size estimated from household census is approximately 4608 inhabitants of which 19 % are under 5 years of age and 17 % between 5 and 9 years old. The inhabitants are mostly sedentary farmers involved in rice cultivation and vegetable production. The ethnic distribution is dominated by the Malinke (80 %) followed by Bambara and Bozo at 5 and 4 %, respectively. The remaining 11 % is comprised of Fulani, Songhoi, Sarakolé and Senoufo.Carrière is located at the northeast edge of the lake in a relatively dry area where ground water pools depend on rainfall. The population size is 2916 inhabitants of which 20 % are under 5 years and 16 % between 5 and 9 years old. This population is primarily formed of Bozo fishermen who migrated from the central Niger Delta (Mopti and Ségou regions). The Bozo represent around 67 % of the population while Bambara and Soninkés represent 4 % each. The remaining 24 % of ethnic groups are described as follows: 3.8 % Sarakolé, 3.5 % Songhoi. Other groups, such as Senoufo, Minianka and Fulani, represented about 8 %. The village is characterized by transient migration as the Bozo fishermen move north for 3–4 months (February–May) each year, when the water level in the lake is low and unfavourable for fishing.Initially a house-to-house census was carried out in April 2010 to characterize the study population. A total of 1665 and 678 children under 10 years of age were enumerated in Binko and Carrière, respectively. Based on a conservative prevalence of malaria parasitaemia of 40 %, a precision of 5 %, and 95 % confidence interval, the minimum sample size required for a cross-sectional survey was 223 for Binko and 196 for Carrière. Using random selection, a total of 80 households were selected from a list of 323 in Binko (25.0 %) and 61 households out of 165 in Carrière (38 %). Households selected were also participating in the first cross-sectional study. All children aged 6 months to 9 years of age from the selected households were enrolled in the cohort study after informed consent. Individual interviews were carried out with the mother or guardian of enrolled children to obtain information on bed net ownership and usage as well as demographic and socio-economic status according to the Malian national poverty line .In each village two cross-sectional malariometric surveys were carried out among children between 6 months to 9 years of age. The first survey was conducted in October 2010 (end of the rainy season) corresponding to the peak of malaria transmission season and the second survey in July 2011, 1 month after the beginning of the rainy season. Each survey was carried out at the health center where parents or guardians were invited to bring their children irrespective of health condition. The participation was voluntary and withdrawal was possible throughout the study. The consent form was written in French, but was explained in the local language to participants by the investigators and a literate village representative.®Pf by Orchid biomedical Systems, Goa, India). Haemoglobin levels were determined using a Hemocue® 301 reader. RDT-positive children with mild malaria were treated with artemether–lumefantrine (AL) according to the national malaria policy, while children with severe malaria received AL upon transferral to the Sélingué district health centre. Microscopy reading of thick blood smears was performed with ocular 10, objective 100 and the species and numbers of malaria parasites were counted per 300 leukocytes. Quality control of microscopy readings was complete on 10 % of randomly selected slides per site.All children enrolled were clinically examined and axillary temperature measured with an electronic thermometer. Finger prick was performed on all children for on-site diagnosis of malaria by rapid diagnostic test (RDT), detecting histidine-rich protein 2-HRP2: ParacheckChildren enrolled in the cohort study were followed from 1 November, 2010 to 31 October, 2011 by a team composed of one medical candidate and two local guides at each community health centre. Passive detection of cases was defined as parents of enrolled children that seek treatment at the health centre for any symptoms of illness. Active case detection was done on a weekly basis by performing a daily visit to random households to ask for any sick child in the cohort. By the end of a week all households with enrolled participants were visited by the team. Parents of children seeking treatment or seen at home with children with fever were interviewed on the history of illness and the children were examined clinically. Blood smear slides were read retrospectively at the Malaria Research and Training Centre in Bamako, so that the treatment decision was based on the result from RDT on field.Malaria parasitaemia was defined as malaria infection or parasite carriage. Symptomatic cases in this study were all patients presenting with fever plus malaria infection, while asymptomatic cases were participants with malaria infections without any symptoms at the time of diagnosis.Plasmodium falciparum carriage across age groups. X2-test was used to test for differences in age-specific prevalence and between villages. The odds ratio was used to measure the differences in risk among age groups, across villages and between seasons.Data were analysed using SPSS version 14.0 for Windows. Descriptive statistics were used to summarize demographic and malaria indicator data. The seasonal variation of fever, asymptomatic and symptomatic malaria  by village and by survey was estimated. Parasite density was stratified for a malaria-positive patient to highlight the level of The monthly cumulative incidences of malaria by health centre were calculated by dividing the number of malaria cases (defined as fever plus positive blood smear) recorded during the period by the total population per month to minimize the effect of short-term migration in the cohort. Because of the difficulty to perform slide reading in the field, treatment of cases was based on RDT results by national policies for malaria treatment at community level. Two or more consecutive malaria episodes occurring within 3 weeks of the first episode were considered recrudescent infections and treated as a single episode.A logistic regression was performed to identify malaria risk factors. The reference group for comparison was the one for children between 5–9 years of age. The risk of households presenting at least one child with malaria during the follow-up period was compared according to household socio-economic status. The socio-economic score was calculated based on the following variables: monthly self-estimated income (above US$150), type of house, type of bedding, presence of in-house electricity, running water, toilet, and/or shower, ownership of a car, motorcycle and/or bicycle.The study was approved by the institutional review board of the Faculty of Medicine and Pharmacy and Dentistry, in Bamako, Mali.In Table Plasmodium falciparum represented more than 89 % of the Plasmodium-positive infections in both surveys. When stratifying the density of P. falciparum infections among children with positive parasitaemia, the majority of children were infected with <1000 parasites/µl in both villages. The difference between villages was significant only for the range 1–1000 parasites/µl, (p < 0.001 at the end of the transmission season and p = 0.01 at the beginning of the transmission season). The geometric mean of parasites/µl (95 % CI) was statistically different between the two villages at the beginning of the transmission season with 271 (81–362) for Carrière and 108 (72–117) for Binko (p = 0.01).The prevalence of symptomatic malaria was also higher in October for both villages, showing a statistiaclly significant difference between Binko (9.2 %) and Carrière (15.9 %), p < 0.01. Gametocytes carriage rate shows a significant difference between the two sites in July with, respectively, 1.9 % in Binko and 6.7 % in Carrière (p < 0.01). The overall prevalence of anaemia among children was 28.2 % in Binko and 34.4 % in Carrière in October (p = 0.14) compared to 20.9 % in Binko and 20.2 % in Carrière in July (p = 0.99). The differences between surveys for the same village were not statistically significant (p > 0.05). When stratifying by mild and severe anaemia, Carrière presented a higher prevalence of mild anaemia (27.2 %) compared to Binko (18.4 %), at the end of the transmission season (p = 0.02).Table P. falciparum parasites/µl) was observed among children aged 1–4 years in Binko and children 5 years old and over in Carrière with respectively 8.3 and 9.5 %. Whereas during the October survey, respectively 43.6 and 69.2 % of infected children aged from 1–4 years in Binko and Carrière ranged from 1001–5000 P. falciparum parasites/µl while among infected children 5 and more years old, 62 % in Binko and 71.4 % in Carrière had a parasitaemia equal or greater than 5000 P. falciparum parasites/µl  from 6 months to 9 years of age were enrolled. The study participants lived within 80 households in Binko  and 61 households in Carrière . In Binko, 87.1 % (296/340) of the participants were successfully followed during the 1-year period compared to 84.3 % (253/300) in Carrière. The majority of the subjects lost-to-follow-up had their households moved to another village/town for more than 3 months. The baseline characteristics of the study population are presented in Table In Binko, 151 children (52.3 %) experienced a total of 195 episodes of acute clinical malaria while the remaining 145 children (47.7 %) did not experience any malaria-related symptoms. In Carrière, 134 children (52.9 %) experienced 141 episodes of clinical malaria and 119 children (47.1 %) did not. Table ersus 68.4 % in Carrière, presented a P. falciparum density between 1 and 1000 parasites/µl. in both villages. Only 10.1 % in Binko and 19.1 % in Carrière had infections of >5000 parasites/µl. No significant difference was observed when comparing the distribution of parasite density among infected children between the two villages. The prevalence of anaemia was greater than 60 % in both villages of which, mild anaemia represented 45.3 % in Binko and 47.8 % in Carrière, respectively (p = 0.83).The majority of children, 70.8 % in Binko vMalaria incidence showed a clear seasonal pattern with 65–70 % of all new malaria cases occurring between August and January, with a peak in October corresponding with the end of the rainy season. The incidence was very low from February to June (five to 40 per 1000) but never reached zero. Malaria incidence was below 50 % during the dry season whereas an increase was observed approximately 1 month after the rainy season start (June) and peaked in October Fig. .Fig. 3MoThe risk of acute malaria increased with age group in both villages Table . In BinkA comparison of malaria disease risk between different age groups across the study population of the cohort study was performed. Several risk factors were examined, including malaria infection at enrolment, age, socio-economic status, and bed net ownership. Only parameters shown to be significant are presented in Table Children free of malaria infection at the time of enrolment had a significantly lower risk of experiencing clinical malaria during the one-year follow-up period . Age was also a significant risk factor as the odds ratio of clinical malarial was lower for all ages below five. The lower risk was statistically significant for children under 1 year of age  and 1–2 years of age  as compared to children over 5 years (p < 0.01). The risk of being positive for malaria was nine-fold higher in children aged 5–9 years compared to children aged up to 2 years in these villages. The socio-ecomic status of the household was highly associated with malaria as households below the national poverty line displayed a 3.2 higher risk of having at least one child with malaria as compared to households above the poverty line .Anopheles gambiae, and therefore contribute to sustain malaria transmission during the dry season. In contrast, Binko is located at 3–4 km from the rice fields of the irrigated perimeter. The malaria transmission pattern for this village may be associated with the practices of irrigated rice cultivation, as irrigation schemes have been found to increase the densities of An. gambiae s.s. during the early crop stages in the dry season [An. gambiae s.l. during the dry season in the fishing hamlet Fourda, located in the immediate vicinity of the River Niger compared with the Kéniéroba, a more distant village from the river [Malaria epidemiology is known to be dependent on several factors including socio-economic factors, access to health care and the local environment . Prior ty season , 17. Thehe river , 15. Theversus 70 % utilization among children under 5 years of age) [The high prevalence of asymptomatic parasitaemia reaching up to 58 % during malaria transmission contrasts with a high coverage of LLINs. Since 2007, the NMCP has organized regularly free distribution of LLINs to households with children under 5 years old (one net for one to two children). In this study, about 93 % of households reported that they owned at least one LLIN and 82 % of children below 10 years old reportedly slept under an LLIN the night prior to the survey. Both ownership and utilization rates are higher in this study compared with the national DHS 2010 (85 % household ownership  of age) . The higThe risk of clinical malaria was twice as high among children aged 5–9 years compared to children between one and 4 years of age. This constitutes a shift in age groups at high risk for clinical episodes of malaria from children below five to the 5–9 years old living with malaria as generally reported , 20. TheSevere malaria, also described in this study, was mainly dominated by severe anaemia with a cumulative incidence of 15 % in both villages. Anaemia remains a major public health issue and requires further studies to identify the causes of anaemia among children in the region. Several competing causes of anaemia, including schistosomiasis and worm infections, are heavily present . FurtherAlthough malaria is present throughout the year in the area, the prevalence of parasitaemia as well as the clinical disease incidence showed a seasonal pattern with a clear peak at the end of the rainy season in October. Other studies conducted in Mali have reported a similar transmission peak at the end of the rainy season  –23. UnliSchistosoma haematobium in Sélingué is up to 85.9 % among schoolchildren with 43.8 % representing heavy infection. The transmission of Schistosoma mansoni is confined to the Sélingué Dam area with a prevalence of 12.5 % [Anaemia is prevalent among children in the study area, specifically among the under 5 years old. This study does not find a significant correlation between anaemia and malaria infection. The finding calls for probably the presence of other causes of anaemia. Other parasitic diseases, mainly schistosomiasis, are highly endemic in the area. The prevalence of f 12.5 % . This stMalaria incidence remains highly seasonal in Sélingué but prolonged over 2–3 months after the rainy season, presumably associated with irrigation related to the agro-hydropower. No secondary peak during the dry season in relation to the irrigation scheme was observed. Since malaria prevention strategies and case management target specific vulnerable groups (children under 5 years of age and pregnant women), this study shows that the proportion of children over five carrying malaria parasites in these communities is much higher at any season compared to the younger ones. If Mali has adopted universal LLINs coverage since 2011, control interventions such as free treatment strategy remains focused on children under five while SMC covers only children under 2 years old. The findings call for expanding both SMC and free ACT to school-age children to cover the peak transmission months in order to meet an important change in the disease burden.


Plasmodium falciparum infections (n = 316) sampled from a single location in Malawi before (2006 and 2007) and after enhanced intervention (2008 and 2012). The SNP data generated were used to examine temporal changes in the proportion of multiple-genotype infections (MIs), mean number of heterozygous SNPs within MIs, parasite genetic diversity (expected heterozygosity and genotypic richness), multilocus linkage disequilibrium and effective population size (Ne). While the proportion of MIs, expected heterozygosity, genotypic richness, multilocus linkage disequilibrium and Ne were unchanged over time, the mean number (±standard deviation) of heterozygous SNPs within MIs decreased significantly (p = 0.01) from 9(±1) in 2006 to 7(±1) in 2012. These findings indicate that the genetic diversity of P. falciparum malaria parasites in this area remains high, suggesting that only subtle gains, if any, have been made in reducing malaria transmission. Continued surveillance is required to evaluate the impact of malaria control interventions in this area and the rest of Malawi, and to better target control interventions.With support from the Global Fund, the United States President's Malaria Initiative (PMI) and other cooperating partners, Malawi is implementing a comprehensive malaria control programme involving indoor residual spraying in targeted districts, universal coverage with insecticide-treated bed nets, use of rapid diagnostic tests to confirm the clinical diagnosis of malaria and use of the highly effective artemisinin-based combination therapy, artemether-lumefantrine (AL), as the first-line treatment for malaria. We genotyped 24 genome-wide single nucleotide polymorphisms (SNPs) in  This has led to significant reductions in the numbers of malaria cases and malaria-related deaths in several countries . Since 2Plasmodium falciparum exhibits a highly outbred population structure characterized by extensive outcrossing, high incidence of multiple-genotype infections (MIs), low levels of linkage disequilibrium, and a low prevalence of genetically identical infections  population structure characterized by high levels of self-fertilization, limited recombination, few MIs, high levels of linkage disequilibrium and a high incidence of genetically identical infections  and after (2008 and 2012) the intensification of control efforts. Ndirande Health Centre is the only public health facility in Ndirande Township, and serves a catchment area with a population of ∼300,000 people. In recent years, this area and the rest of Malawi have seen increased uptake of malaria control interventions including increased use of ITNs, introduction of rapid diagnostic tests to confirm the clinical diagnosis of malaria and switch to a more efficacious drug, AL, as the first-line treatment for malaria. To our knowledge, the population in this area does not have easy access to other drugs, and AL is available through prescription only. We employed a passive case detection system to identify children with uncomplicated P. falciparum malaria amongst children seeking care at the health centre. No special effort was made to find unsuspected or asymptomatic disease in the community where the enrolled children lived. At least 46 malaria-infected blood samples were sampled per year. This sample size allows the detection of a 30% difference in the proportion of MIs before and after enhanced intervention with 80% power and 95% confidence assuming a pre-intervention prevalence of 78%. During the sampling of 2006–2008 infections, we avoided sampling members of the same household and serial sampling of the same person by carefully matching records of patients already enrolled into the study with those of prospective study participants. We did this to preclude parasite relatedness structure driven by similar host genetic background. However, when collecting malaria-infected blood samples in 2012, no special effort was made to avoid sampling members of the same household or serial sampling of the same individual. All samples were collected with the approval of the child's parent or guardian under the auspices of antimalarial drug resistance studies and health facility-based cross-sectional malaria surveys approved by the University of Malawi College of Medicine Research and Ethics Committee.Malaria-infected blood samples were collected from 316 children less than 5 years old presenting to Ndirande Health Centre in Blantyre with uncomplicated 2.2Samples from 2006 to 2008 were in the form of whole blood, and were extracted for parasite DNA using Qiagen DNA Mini Kits . Samples from 2012 were in the form of filter paper blood spots and were extracted using a two-step protocol to maximize parasite DNA yield. Blood was first eluted from filter paper blood spots using Gensolve Kits , followed by parasite DNA extraction with 96-well QIAamp DNA Blood Kits  . The con2.3P. falciparum laboratory strains 3D7, V1S, W2-8E, DD2, HP710 and an artificial mixture of W2-8E and 3D7 were included in each genotyping run as positive controls. W2-8E is a parasite clone isolated from W2 by dilution cloning while HP710 is a clone from the Thai-Myanmar border. To perform the assay, a master mix consisting of 2.95 μl of commercial grade nuclease-free water, 0.05 μl of the 40x Taqman SNP assay and 5 μl of the TaqMan Universal PCR Master Mix  per reaction was added to each well of a 96-well real-time PCR plate  pre-loaded with 2 μl (10 ng) of each parasite DNA sample. The concentration of parasite DNA in the template was standardized across all samples to avoid the potential technical bias of scoring more heterozygous loci in samples with high parasite densities. Parasite DNA samples were amplified on the StepOne real-time PCR instrument (Applied Biosystems), followed by analysis of the results with Applied Biosystem's proprietary Allelic Discrimination software. Where the software did not give genotype calls directly, we made allele calls manually by examining both the amplification plot and the multi-component plot. To examine if the 24-SNP molecular barcode assay has enough resolution power, we also genotyped six parasite clones isolated from a multiple-genotype infection sampled at Ndirande Health Centre in 2008. These clones were previously genotyped using a custom-made 384-SNP GoldenGate assay, and shown to be either closely related (differing only at a few of the 316 successfully genotyped SNPs) or genetically identical   using the molecular barcode assay as described previously . The named SNPs) .2.4P. falciparum genetic diversity indices (expected heterozygosity and genotypic richness), multilocus linkage disequilibrium, effective population size, proportion of identical parasite infections, and to examine if there was significant genetic differentiation between parasite samples collected before and after the intensification of malaria control efforts. SNP data for multiple-genotype infections (MIs) were used to examine temporal changes in the proportion of MIs and the mean number of heterozygous SNPs within MIs. We conducted analyses both by the year of sampling and by pooling samples from early (2006 and 2007) and late (2008 and 2012) sampling periods.We identified multiple-genotype infections by examining the number of heterozygous SNPs in each infection genotyped. Because human blood-stage malaria parasites are haploid, a single-genotype infection is expected to have only one allele at each SNP locus while a multiple-genotype infection is expected to carry multiple alleles. However, in this study, we used a minimum of two heterozygous SNPs as a threshold for classifying MIs because one random SNP out of the 24 SNPs genotyped was occasionally wrongly scored as heterozygous even in well-characterised monoclonal infections. SNP data for single-genotype infections were used to estimate SNP allele frequencies, and to measure temporal changes in 2.5ps) between all pairwise comparisons of single-genotype infections and clustered infections on the UPGMA tree based on the genetic distance metric, 1-ps, using PHYLIP  index (R measures the proportion of unique genotypes present in the samples and is estimated as: R = (G − 1)/(N − 1) where G is the number of unique genotypes and N is the sample size (ST) index  index  to examiple size . In addiT) index , estimatT) index .2.7AS (standardized index of association), which compares the observed variance in the numbers of alleles shared between parasites with that expected when parasites share no alleles at different loci . NeV was estimated using the pseudo-maximum likelihood method implemented in the programme, MLNE v. 1547 . This is because in such a case, negative binomial regression provides a better fit than the Poisson model. All tests were implemented in STATA version 10.1 .Proportions were compared using the Chi-square test while means were compared using the Two sample 33.1P. falciparum malaria before and after enhanced intervention. Summary statistics for parasite sampling and basic genetic data are presented in P. falciparum genome (PlasmoDB version 5.0) was monomorphic in all the four temporal samples . The rest were polymorphic with minor allele frequencies ranging from 0.03 to 0.50 . Of the 295 parasite infections that we successfully genotyped, 217 were classified as multiple-genotype infections (MIs) while the remaining 78 were single-genotype infections.We successfully genotyped 295 out of 316 parasite infections sampled from children less than 5 years old presenting to Ndirande Health Centre with uncomplicated http://dx.doi.org/10.1016/j.actatropica.2014.11.008.Supplementary Table S1 related to this article can be found, in the online version, at P. falciparum genome, PlasmoDB version 5.0, was monomorphic in all parasite isolates analysed  just like in a previous study, which examined parasite genetic diversity in malaria parasite infections from Blantyre . Patients infected with identical parasite genotypes were from different households. There was no evidence of serial sampling of identical parasite genotypes from the same individual.Relationships amongst single-genotype infections sampled and laboratory controls are shown on the UPGMA tree . As expe3.3p = 0.823). In contrast, the average number of heterozygous SNPs within MIs decreased significantly from 9 ± 1 in 2006 to 7 ± 1 in 2012 (p = 0.01).We observed subtle changes in the composition of multiple-genotype infections over the time frame observed . The inc3.4He and the genotypic richness index, R (He for parasite samples collected in 2006 (He = 0.37 ± 0.16) did not differ significantly (p = 0.34) from that of 2012 samples (He = 0.32 ± 0.17). Similarly, the mean He for samples from both 2006 and 2007 was 0.38 ± 0.15 compared with 0.37 ± 0.16 in samples from both 2008 and 2012 (p = 0.58). Similarly, R was 0.95 [0.74–1.00] in 2006 compared with 0.93 [0.68–1.00] in 2012 (p = 0.81).We observed no significant temporal change in parasite genetic diversity as expressed by the expected heterozygosity index, index, R . The mea3.5FST = 0.030 ± 0.036), 2007 and 2012 (FST = 0.038 ± 0.043), 2007 and 2008 (FST = −0.013 ± 0.018), and between pooled samples from 2006 to 2007 compared with samples collected in 2008 and 2012 combined . Similarly, there was no significant multilocus linkage disequilibrium (p > 0.05) in all temporal samples.There was no significant genetic differentiation between parasite samples collected in 2006 and 2012 (3.6p > 0.05) over time . Poisson regression analysis revealed an average of 2% decline in parasite density per year . However, univariate negative binomial regression analysis revealed that the number of heterozygous SNPs within MIs was not associated with parasite density (p = 0.062), season (p = 0.056), patient age (p = 0.903) or sex (p = 0.979). The only variable associated with a reduction in the number of heterozygous SNPs within MIs is the year of sampling. The lack of association between the number of heterozygous SNPs within infections and parasite density suggests that the observed temporal decrease in the mean number of heterozygous SNPs within MIs was not simply a result of sampling low density infections. The mean number of heterozygous SNPs within MIs can provide an indirect measure of the number or relatedness of parasite genotypes within MIs  or very prolonged (moderate Ne for many generations). Therefore, we may have failed to detect changes in Ne and He because the drop in transmission is not large enough or has not been sustained for long enough to significantly alter Ne and He. In addition, declining populations of small organisms like P. falciparum tend to have relatively large population sizes, which are less sensitive to the loss of genetic diversity. It is possible that not enough of the parasite population was sampled to allow accurate estimation of these parameters since our power calculations were based on detecting change in the proportion of MIs. With respect to Ne, He and Ne is detectable. Given the exponential relationship between Ne and genetic drift, it is perhaps overly optimistic to expect Ne to change over the time frame observed (1–5 years). The Ne metric is very sensitive to Ne decreasing from 10 to 1 or 100 to 10 but detecting the change from 10,000 to 1000 or even 1000 down to 100, is very difficult  and a Wellcome Trust Intermediate Fellowship in Tropical Medicine and Public Health (grant # 099992/Z/12/Z) to Dr Standwell Nkhoma. Collection of 2012 samples was supported by the National Institutes of Health, USA through grant # 1U19AIO89683-01 to Professor Terrie Taylor and Dr Miriam Laufer.This study was supported by a Research Initiative Award (grant # IA10) from the


DDX39B (BAT1) encodes an RNA helicase known to regulate expression of TNF and IL-6. Elevated levels of these two cytokines are associated with increased severity of clinical malaria. The aim of this study was to investigate the relationship between single nucleotide polymorphisms (SNPs) in the DDX39B, TNF and IL6 genes and the clinical outcomes of patients with Plasmodium vivax malaria.NlaIII or NcoI. The plasma levels of cytokines were determined using ELISA.Cross-sectional investigations were carried out in two regions of the Brazilian Amazon where several studies on the pathogenesis of vivax malaria had been performed. Individuals were categorized according to infection status as well as clinical presentation into the following groups: uninfected, asymptomatic infection, mild infection, or complicated infection. Polymorphisms were identified using PCR restriction fragment-length polymorphism analysis and the restriction enzymes DDX39B-22C > G was associated with absent or decreased manifestations of malaria and the C allele was a risk factor for disease complications. Study participants heterozygous for TNF-308 (GA) and DDX39B-348 (CT) had higher TNF levels than wild-type participants. Haplotypes that included DDX39B (-22C > G and -348C > T) and TNF polymorphisms were not directly associated with mild or complicated malaria infections; however, haplotypes AGC, ACC, GGT, AGT and ACT were associated with increased TNF levels. Participants with genotype combinations GC/CC/GG/GG and GG/CT/GG/GG (DDX39B-22/DDX39B-348/TNF-308/IL6-176) had decreased and increased risk of mild malaria, respectively, compared with asymptomatic and uninfected participants. GC/CC/GG/GG was linked to decreased TNF and IL-6 levels.The G allele of DDX39B and IL6 SNPs who had vivax malaria. These findings support the postulation that a set of mutations in immune-related genes is associated with inflammatory mediators and the clinical outcomes of patients with malaria.This is the first study to describe patients with  Plasmodium vivax malaria is a major worldwide threat, with two to three billion people remaining at risk of infection  box polypeptide 39B; BAT1: HLA-B associated transcript 1; RNA: Ribonucleic acid; TNF: Tumor necrosis factor; IL-6: Interleukin 6; SNP: Single nucleotide polymorphism; CXCL10: C-X-C motif chemokine 10; CRP: C-reactive protein; FUNASA: Brazilian National Foundation of Health; PCR: Polymerase chain reaction; FIOCRUZ: Oswaldo Cruz Foundation; HAV: Hepatitis A virus; HBV: Hepatitis B virus; HCV: Hepatitis C virus; HDV: Hepatitis D virus; HIV: Human immunodeficiency virus; FMT-HVD: Fundação de Medicina Tropical Dr Heitor Vieira Dourado; DNA: Deoxyribonucleic acid; OR: Odds ratio; CI: Confidence interval; HWE: Hardy-Weinberg equilibrium; IQR: Interquartile range; MHC: Major histocompatibility complex; IκBL: Inhibitor of κB-like protein.The authors declare that they have no competing interests.VRRM performed the experiments, analyzed the data, and wrote the manuscript together with BBA and MBN. LCLS and GCG helped perform the experiments. BBA performed the field study and sampling in Rondonia and performed the plasma measurements and wrote the manuscript. BMLM was responsible for the field study and sampling in Manaus. MVGL supervised the clinical study and sampling in Manaus. MBN conceptualized the study, supervised the clinical study in Rondonia, and helped with data interpretation and the writing of the manuscript. All authors have read and approved the final version of the manuscript.


Plasmodium falciparum infection and a clinical indicator (i.e. fever) were measured among children in three villages of central Côte d’Ivoire  and associations with net coverage explored. In Bozi and Yoho, LLINs were provided by the national malaria control programme, prior to the study and an additional catch-up coverage was carried out in Bozi. In N’Dakonankro, no net intervention was conducted.Sleeping under a net, particularly a long-lasting insecticidal net (LLIN), is associated with reduced malaria morbidity and mortality, but requires high coverage and adherence. In this study, parasitologically confirmed P. falciparum infection was determined by microscopy and a rapid diagnostic test (RDT). Fever was defined as an axillary temperature ≥37.5°C. A questionnaire was administered to obtain demographic data and net usage.Three cross-sectional surveys were carried out; two in the dry season (February 2010 and November 2011) and one in the rainy season (May 2012). Among 897 children aged <15 years, P. falciparum according to microscopy in the third survey was 74%, 81% and 82% in Yoho, N’Dakonankro and Bozi, respectively. Meanwhile, 46% of the children in N’Dakonankro, 44% in Bozi and 33% in Yoho slept under a net. The risk of P. falciparum infection did not differ between net-sleepers and non-net-sleepers. Fewer children had parasitaemia ≥1,000 parasites/μl of blood in Bozi in the third compared to the first survey. Fever was poorly correlated with P. falciparum infection. The risk of P. falciparum infection did not depend on the village of residence, presence of fever or sleeping under LLIN the night before the survey. Conversely, it was higher in the rainy season and among older children.The proportion of children infected with P. falciparum is highly prevalent, the use of nets was associated with significantly lower levels of parasitaemia. The apparent lack of effect on P. falciparum infection and fever might be explained by the relatively low net coverage in Bozi and Yoho and the relatively short period (<2 years) during which the impact of nets was measured.In an area where  Controlling malaria remains a formidable public health challenge, particularly in sub-Saharan Africa, where 80% of all cases and 90% of all malaria-attributable deaths occur, mainly among children below the age of 5 years . MalariaIn countries like Côte d’Ivoire, the challenge now is to roll out interventions at a level sufficient to gain and maintain effective malaria control. With regard to LLINs, they were distributed from 2006 to 2007 to children aged <11 months, along with routine immunization, and to pregnant women at antenatal clinics in 37 health districts. In 2008, LLINs distributions took place in 19 health districts with financial support of the Global Fund to Fight AIDS, Tuberculosis and Malaria to children aged <11 months at health centres, and to all pregnant women attending antenatal clinics. In the same year, an additional 18 health districts benefitted from LLINs during measles vaccination campaigns of 5-year-old children and vitamin A supplementation. In 2009, the national coverage of LLINs was estimated at 24.4% and the usage rate at 16.3%. The coverage and usage rate of LLINs of pregnant women was 19.8% and 13.9%, respectively, whilst the respective percentages for children under the age of 5 years were 30.2% and 28.1%. In Bouaflé health district in central Côte d’Ivoire, LLINs coverage was 47.4% and usage rate 20.2% .Plasmodium infection. The key findings pertaining to malaria transmission have been presented elsewhere [P. falciparum infection and fever) and net coverage, during the dry and rainy seasons in rural settings of central Côte d’Ivoire with different levels of malaria transmission and vector control interventions. Two villages (Bozi and Yoho) are located in the health district of Bouaflé and received LLINs by the PNLP. Additionally, in Bozi, LLINs were distributed in a catch-up intervention to all households inhabited by children aged <15 years and pregnant women. In the third village (N’Dakonankro), no specific net interventions were carried out. As RDTs had been introduced in Côte d’Ivoire and were brought to scale, we also wanted to compare the performance of RDT versus microscopy for the diagnosis of P. falciparum infection.This study was part of a broader project aimed at measuring the impact of nets on malaria transmission, as determined by entomological inoculation rate (EIR), and different parasitological and clinical indicators, such as prevalence, parasitaemia and morbidity due to lsewhere . In brieThe study protocol was reviewed by the institutional review board of the Centre Suisse de Recherches Scientifiques en Côte d’Ivoire . Ethical approval was granted by the national ethics committee of Côte d’Ivoire (reference no. 02-2011/MSLS/CNER-P). Oral informed consent was obtained from parents/guardians of participating children and heads of households, whilst children provided oral assent. Oral rather than written informed consent was obtained because of high illiteracy rates in rural Côte d’Ivoire. The purpose and procedures of the study were explained to all participating households in the local language using lay terms. It was emphasised that participation is voluntary and people could withdraw anytime without further obligation. Children with a positive RDT and axillary temperature ≥37.5°C were given an ACT (i.e. artemether-lumefantrine) free of charge.The study was carried out between July 2009 and May 2012 in three villages of central Côte d’Ivoire; Bozi ; N’Dakonankro  and Yoho  [Plasmodium parasite prevalence ranges between 40% and 86% [In central Côte d’Ivoire  and 86% . There i and 86% .A LLIN campaign was implemented in November 2008 by the PNLP, and both Bozi and Yoho benefited from this intervention , an additional 150 LLINs were distributed to households in Bozi inhabited by children aged <15 years and pregnant women. Households were coded and clearly marked after receipt of a LLIN. Household heads were instructed how to use and wash LLINs.Figure In February 2010, a baseline cross-sectional survey was carried out in the three villages. All children aged between 7 and 15 years who attended grades 1–4 in the primary schools were invited for a finger-prick blood sample. Furthermore, mothers and caregivers of under 7-year-old children were invited to bring their children to a designated community location where finger-prick blood samples were taken. Net use was reported by schoolchildren themselves and the team considered these nets as LLIN because they had been offered by the PNLP. A total of 897 children participated, corresponding to 98.9% of those invited (n = 907). In November 2011, 6 months after free LLIN catch-up in Bozi, a second survey was carried out, including 169 children. One year later, in May 2012, a third survey enrolled 416 children. Many children were absent during the second and third survey Figure .Pf test; ICT Diagnostics, Cape Town, South Africa) were performed at the same time. The slides were air-dried and then transferred to a nearby laboratory where they were stained with Giemsa for 45 min. Slides were examined under a microscope at high magnification by the same experienced technician throughout the study. Plasmodium density was estimated by counting the number of parasites per 200 leucocytes. Whenever fewer than 10 parasites were recorded, reading was continued for a total of 500 leucocytes. Parasite count was converted to parasites/μl of blood, assuming a standard count of 8,000 leucocytes/μl of blood. For quality control, 10% of the slides were randomly selected and re-examined by a senior technician.Thick and thin blood films were prepared on microscope slides and an HRP-2 based RDT . Clinical malaria was defined as children with either a positive RDT or a positive Giemsa-stained microscope slide (or both) plus axillary temperature ≥37.5°C. Prevalence was the number of positive children divided by the total number of children examined. We used the results from the thick and thin blood film examinations for estimating the prevalence and the relationship with net coverage.Pf test were calculated, considering microscopy as the diagnostic reference standard. The kappa statistic was used to assess the concordance between ICT ML01 malaria Pf test and microscopy. Univariate logistic regression was employed to assess the effect of one variable on risk of P. falciparum infection. A multivariable model was used to assess the overall effect of the aforementioned variables. Chi-square (χ2) or Fisher’s exact test, as appropriate, was used to compare groups.Sensitivity, specificity and predictive values of the ICT ML01 malaria Overall, there were 1,482 contacts, equally distributed across the three villages on the three occasions ; 61%  58-63%) occurred on the first survey, 11% (95% CI 10-13%) on the second (dry season) and 28% (95% CI 26-30%) on the third (rainy season). The mean age of surveyed children ranged from 6.7 (95% CI 6.4-6.9) to 8.7 (95% CI 8.1-9.4) years at the first survey, and from 10.7 (95% CI 10.5-10.8) to 12.2 (95% CI 11.7-12.6) years at the third survey , 144 matched , for a between-reader agreement of 98.8% (95% CI 94.0-100%), 94.1% (95% CI 71.3-99.9%) and 95.1% (95% CI 83.5-99.4%) for the three surveys, respectively. Discrepancies occurred for slides with low parasitaemia.P. falciparum differed across the three villages at the end of the dry season (first survey). A higher prevalence was found in Yoho  compared to N’Dakonankro  and Bozi  (both p < 0.001)  than in the other two villages (p = 0.002) Table ; it dropThe percentage of children who reportedly had slept under a net the night before the first survey was 24.9% (95% CI 20.1-30.2%) in N’Dakonankro (mainly ITNs), 15.2% (95% CI 11.2-19.8%) in Yoho (mainly LLINs) and 21.6% (95% CI 17.1-26.6%) in Bozi (mainly LLINs)  , 24.2% (95% CI 17.6-31.8%) and 26.9% (95% CI 19.5-35.6%) reported to have slept under a net the night before the interview in N’Dakonankro, Yoho and Bozi, respectively. A significant difference was observed between surveys only in Yoho (p = 0.031).P. falciparum infection (determined by microscopy) during the first and the third survey in the three study villages. At the third survey in N’Dakonankro, 81% of children were infected and 46% slept under a net (mainly ITNs), while in Yoho, the respective figures were 74% and 33% (mainly LLINs) and in Bozi, 82% and 44% (mainly LLINs) =1.17, 95% CI 0.92-1.48).Figure Children who had fever on the day of survey and had slept under a net were 20%, 14% and 19% at the first survey in N’Dakonankro, Yoho and Bozi, respectively. Only 12 subjects were found to have fever at the third survey overall. The univariate logistic regression showed that the risk to be febrile was almost halved for children who reported having slept under a net .Overall the proportion of symptomatic malaria infection was 45%, 67% and 64% in N’Dakonankro, Yoho and Bozi, owing to a statistically significant difference between villages  but did not differ over time.P. falciparum infection . Of the total 1,482 subjects who had a recorded fever and were tested for malaria parasites by either microscopy or RDT, concordance was 0.41 and 0.43, respectively  and lowest in Bozi , but there was no statistically significant difference between sites. No association was found for fever and net usage. The risk of malaria infection increased significantly with age within this cohort of children under the age of 15 years and was significantly higher during the rainy than the dry season.Table P. falciparum-positive slides , 314 (31.6%) had a parasite count >1,000 parasites/μl of blood. The number and proportion of children who had a positive slide with >1,000 parasites/μl of blood at the first survey was 60 (36.4%) in N’Dakonankro; 73 (40.3%) in Yoho; and 50 (36.0%) in Bozi. At the third survey, the respective numbers and percentages were 34 (31.2%) in N’Dakonankro; 20 (17.7%) in Yoho; and 23 (22.5%) in Bozi. The difference between the first and the third survey in the study villages was statistically significant in Bozi and Yoho , but not N’Dakonankro.Parasite counts were mostly low, particularly at the third survey. Of the 994 Among the 1,482 malaria tests subjected to both microscopy and RDT, 63.9% were RDT-positive and 64.4% were microscopy-positive, with moderate agreement between the two diagnostic approaches . Considering microscopy as the diagnostic reference standard, RDT sensitivity at the first survey was 79.6% and specificity was 61.0%. At the third survey, sensitivity and specificity for RDT to diagnose malaria were 76.1% and 60.7%  and malaria infection and all-cause fevers. Civil unrest, with important population movements , renderePlasmodium infection did not depend on the village of residency, presence of fever or recent net use. Conversely, as expected, the risk of malaria was related to rains and increased with age within the age-range of the population investigated (children aged <15 years). This observation is consistent with previous studies conducted in western Côte d’Ivoire [vs. 171 asexual forms per μl of blood). Some malaria cases were observed at the end of the dry season, possibly because malaria transmission occurred year-round with probable low intensity during this season.Multivariable analysis showed that the risk of d’Ivoire  and in Zd’Ivoire , where mPlasmodium falciparum infection was found to be highly prevalent among children in the three study villages in central Côte d’Ivoire, corroborating previous findings [findings ,23. WhilCan fever be used as an indicator to assess the impact of preventive measures like net use on malaria risk? In the current study the answer is no. The relationship between malaria parasitaemia and fever has been reviewed recently . In our Plasmodium infection or fever in relatively high exposure endemic areas. There was no obvious difference between N’Dakonankro where people used mainly ITNs and Yoho and Bozi where LLINs were the predominant net type. ITNs prove generally protective in children by reducing uncomplicated malaria incidence, malaria parasite infection prevalence and malaria-attributable mortality [The targeted catch-up distribution of LLINs in Bozi had no apparent effect on malaria infection or all-cause fevers, as compared to the other villages, but Bozi was the only village where parasitaemia decreased between the first and the third survey in the children seen on both occasions. Similar results were published previously in a comparable epidemiological setting in Côte d’Ivoire , which cortality ,32. For ortality , whilst ortality . A possiortality  or 3 yeaortality  of follovs. 74% for counts <1,000 parasites/μl of blood. The overall prevalence ranged between 50% and 80%. These results could be explained by the fact that RDT based on PfHRP2 detection on the one hand do not reliably detect lower-density parasitaemia [PfHRP2 persists several days after parasites disappear [There was only moderate agreement between RDT and microscopy; both sensitivity and specificity of RDT were lower than expected ,36. Senssitaemia , and on isappear .Our study has several limitations. First, seasonal differences between surveys might have been a confounding factor for the lack of association between net coverage and the risk of malaria. It should be noted, however, that the three villages experienced average and comparable rainfall over the course of the study. Second, we did not collect information on factors that might have impounded on the association between malaria risk and net usage, such as socioeconomic status, parents’ education attainment, household size, exact number and type of net per household and per person and number of sleeping spaces protected by nets. Third, there was substantial patient attrition between surveys . This was largely due to the sociopolitical political crisis of 2010, which pushed people to move into secured areas; when the war was over, the return of displaced populations was progressive while those who did not move were not primarily concerned with the study. However, the relatively large sample size (~300 children were seen both at the first and third survey) should at least partly overcome this limitation.Lessons learned from this study are that surveys are important to target interventions, and should cover a period sufficiently long to detect trends and quantitate the effects of interventions. The suitability of RDT in these conditions should be studied in more detail. Although our study did not show any appreciable associations between net usage and malaria infections or fevers, the use of nets (mainly LLINs) may have been associated with lower parasitaemia.ACT: Artemisinin-based combination therapy; CHW: Community health worker; CI: Confidence interval; CSRS: Centre Suisse de Recherches Scientifiques en Côte d’Ivoire; EIR: Entomological inoculation rate; HRP2: Histidine rich protein 2; IRS: Indoor residual spraying; ITN: Insecticide-treated net; LLIN: Long-lasting insecticidal net; OR: Odds ratio; PNLP: Programme National de Lutte contre le Paludisme; RDT: Rapid diagnostic test.The authors declare that there are no competing interests.AFO implemented the study, analysed the data and drafted the manuscript. PLO assisted with the data analysis and revision of the manuscript. MD, GR and MT contributed to the design of the study and the revisions of the manuscript. JU contributed to the design of the study and assisted in the drafting and revision of the manuscript. BGK designed the study, coordinated field activities and assisted with the data analysis and revision of the manuscript. All authors read and approved the final manuscript.