Detection of hotspots depends on the sensitivity of commonly used diagnostic tools. In the elimination stage, malaria control programs should consider polymerase chain reaction testing to guide detection of asymptomatic malaria hotspots. Malaria control strategies need to respond to geographical hotspots of transmission. Detection of hotspots depends on the sensitivity of the diagnostic tool used.We conducted cross-sectional surveys in 3 sites within Kilifi County, Kenya, that had variable transmission intensities. Rapid diagnostic test (RDT), microscopy, and polymerase chain reaction (PCR) were used to detect asymptomatic parasitemia, and hotspots were detected using the spatial scan statistic.Eight thousand five hundred eighty-one study participants were surveyed in 3 sites. There were statistically significant malaria hotspots by RDT, microscopy, and PCR for all sites except by microscopy in 1 low transmission site. Pooled data analysis of hotspots by PCR overlapped with hotspots by microscopy at a moderate setting but not at 2 lower transmission settings. However, variations in degree of overlap were noted when data were analyzed by year. Hotspots by RDT were predictive of PCR/microscopy at the moderate setting, but not at the 2 low transmission settings. We observed long-term stability of hotspots by PCR and microscopy but not RDT.Malaria control programs may consider PCR testing to guide asymptomatic malaria hotspot detection once the prevalence of infection falls. See the editorial commentary by White on pages 1051–52.) or lactate dehydrogenase (pLDH). This tool has greatly improved the ability to provide diagnostic services in rural areas of sub–Saharan Africa because RDTs require minimal training and rely on immune-chromatography, which avoids the need for electricity  and and18] aExamination for malaria parasites using RDTs, microscopy, and PCR was performed by trained laboratory technicians and was standardized across the sites. Blood samples were obtained from all children aged <15 years whose consent to participate in the study had been obtained , 17. ChiP. falciparum in the blood. Rapid diagnostic test stocks were stored in air-conditioned rooms with monitored temperature and humidity. Quality assurance for the stored test kits was conducted regularly before use.Rapid diagnostic tests  were used to detect the presence of HRP2 specific to P. falciparum, Plasmodium malariae, and Plasmodium ovale).Thick and thin blood smears were Giemsa stained and examined using light microscopy at 1000× magnification for malaria parasites and malaria species, respectively. Malaria infection and parasite counts by microscopy were determined independently by 2 readers, and discordant readings were resolved by a 3rd reader. The number of parasites per 200 white blood cells (WBCs) was counted, and parasite density per microliter of blood was calculated using an average count of 8000 WBCs/µL of blood, as described elsewhere [P. falciparum multicopy 18S ribosomal RNA genes, as described elsewhere [For PCR analysis, DNA was first extracted from 30 µL of whole blood using QIAxtractor machine . The DNA was eluted in 100 µL, from which 5 µL of DNA were amplified by quantitative PCR. This was done using a TaqMan assay for the lsewhere , except lsewhere . We usedlsewhere . Plates P- alues) were compared. The risk ratio herein is defined as the risk of malaria within a hotspot divided by the risk outside the hotspot.Individuals who had complete data on RDT, PCR, and microscopy were included in the analysis. Hotspots are defined as geographical areas experiencing significantly higher prevalence of asymptomatic parasitemia than would be expected by chance. In our study, we assess chance using the spatial scan statistic  through Parasite prevalence was computed by imposing spatial grids on the data and collapsing to the mean prevalence within each cell of the grid. This was done with grids of variable sizes—0.5 × 0.5 km, 1 × 1 km, and 2 × 2 km—selected a priori to allow for a sensitivity analysis that would examine the potential bias resulting from the modifiable areal unit problem and repeated by year. The association between parasite prevalence by PCR and by microscopy or RDT was assessed for the various grid sizes. Furthermore, we compared the stability of spatial heterogeneity of PCR and microscopy datasets by examining Spearman’s rank correlation coefficient between parasite prevalences within grids separated in time.The degree to which hotspots overlap was defined as the fraction of homesteads within the intersection of hotspots detected by PCR and microscopy or RDT divided by the total number of homesteads within the hotspots. Only homesteads within primary hotspots (most likely cluster regardless of significance) and any other significant secondary clusters were included in the computations.Hotspots of malaria transmission were mapped on Google Map extracts in R version 3.3.1 . Graphs,P. falciparum parasite density measured by PCR and by microscopy among those testing positive  and strong association between detection by PCR and detection by microscopy (Supplementary Table 1) . Parasite densities by PCR and microscopy were log-normally distributed  and not in either of the low transmission sites. No P. vivax case was reported in any of the sites.Malaria species were only examined by microscopy. Overall, the prevalences of malaria by species in the 3 sites were 9.67% (n = 830/8581 films), 0.16%(n = 13/8004 films), 0.60% (n = 48/8014 films), and 0% (n = 0/8014 films) for P. falciparum  and . We identified 1 significant primary hotspot of P. malariae , a borderline significant secondary hotspot , and a nonsignificant hotspot of P. ovale . The hotspots of P. falciparum, P. malariae, and P. ovale overlapped geographically (supplementary figure 1). Further analysis was restricted to P. falciparum.In pooled data analysis from Junju, we identified 2 statistically significant hotspots of Plasmodium falciparum was detected by PCR, RDT, and microscopy. Significant hotspots of malaria transmission by the 3 diagnostic tools were observed in the Junju and Ganze sites. However, hotspots of malaria transmission in Ngerenya were statistically significant only when measured by PCR and RDT and not statistically significant when measured by microscopy . However, the associations were weaker in low transmission settings (In the Junju site, the prevalences of parasitemia within grids were predictive of the prevalences in the following year. The stability appeared to be greater for PCR and microscopy, which remained significant for intervals <5 years, and less stable for RDT prevalences, which were significantly predictive of the prevalence in the following year for intervals only up to 2 years.In contrast, the prevalences of parasitemia within grids in Ganze were not predictive for the following year by any measure (Plasmodium falciparum parasite prevalence has frequently been used as a marker of transmission intensity and is widely used in detection of hotspots of asymptomatic parasitemia. However, the estimated prevalence of parasitemia has been shown to vary substantially with the diagnostic tool used. Polymerase chain reaction and other molecular techniques are significantly more sensitive than microscopy and RDT for detection of malaria parasites, especially at lower transmission intensities where parasite densities are lower [re lower , 8. ThisWe observed substantial heterogeneity of malaria transmission in the 3 sites, as has been previously described . HotspotThere were significant correlations between PCR and microscopy and between PCR and RDT parasite prevalences within grid cells imposed on the data at 3 different spatial scales. The correlations were stronger in Junju than in Ngerenya and Ganze . The preAs would be expected, PCR densities were lower than microscopy densities , and theP. falciparum parasites do not express the HRP2 protein, leading to false negatives [We observed stable hotspots of asymptomatic parasitemia in the Junju cohort but not in Ganze, and we were not powered to assess stability of hotspots in Ngerenya. Hotspots were similarly stable when detected by PCR or microscopy but not RDT . The advegatives . These fegatives , 29, theThe main limitation of our study is that data were collected from geographical areas of close proximity on the Kenyan coast. However, these geographical areas captured a range of transmission intensities during a period when transmission was falling . AlthougClinical malaria case monitoring has also been used to identify hotspots of malaria transmission . HoweverMalaria control programs increasingly need to adopt targeted malaria control at low transmission intensities. Our findings suggest that PCR, RDT, and microscopy can potentially determine hotspots at moderate transmission intensities, but PCR testing has a diagnostic advantage as transmission intensity falls. Therefore, malaria control programs should consider PCR testing when the prevalence of infection is low.The Journal of Infectious Diseases online. Consisting of data provided by the authors to benefit the reader, the posted materials are not copyedited and are the sole responsibility of the authors, so questions or comments should be addressed to the corresponding author.Supplementary materials are available at supplementary figure 1Click here for additional data file.supplementary figure legendsClick here for additional data file.Supplementary TablesClick here for additional data file.


Sick neonates in malaria endemic areas are frequently transfused with donor blood unscreened for malaria parasite. Consequently, they are at risk of transfusional malaria which can lead to increased neonatal mortality. The study aimed to determine the burden of transfusional malaria in neonates to help in policy formulation on prevention of transfusional malaria.One hundred and sixty four neonates admitted into the neonatal unit of a tertiary hospital over a 10 month period who were scheduled for blood transfusion were screened for malaria parasites pre-transfusion, at three and 14 days post transfusion using Giemsa stained thick and thin films. Donor blood was screened for malaria parasites at the point of transfusion. Neonates who developed malaria parasitaemia post transfusion were followed up for signs of malaria.All recruited neonates tested negative to malaria parasite pre- transfusion. One hundred and twenty (73.2%) were term neonates with 94(57.3%) aged 1-7days. Four (2.4%) neonates developed malaria parasitaemia three days post transfusion and all four developed fever that resolved on treatment for malaria. Three (1.8%) of 164 donor blood samples had malaria parasitaemia and all three (100%) neonates who were transfused with the infected donor blood developed malaria parasitaemia post transfusion. However, one neonate who developed malaria parasitaemia post transfusion was transfused with non-infected donor blood.The prevalence of transfusional malaria in this study is low (2.4%). However, 100% of neonates who received malaria infected donor blood developed transfusional malaria. We therefore recommend routine screening of donor pre-transfusion, testing of neonates who develop fever post transfusion and treatment of those who test positive to malaria. Blood transfusion is a lifesaving procedure that involves introduction of blood and blood products into the circulatory system of an individual. It is a common procedure in hospitalized children under the age of five years in malaria endemic countries because of their vulnerability to severe anaemia. Other grEven though blood transfusion can be lifesaving, it is associated with complications such as transmission of infections, immunological reactions and volume overload. Infective agents that can be transmitted to recipients of blood transfusion includes; viruses, bacteria, spirochetes and protozoa. Transfusional malaria (TM) is the most common protozoal transfusion transmissible infection in malaria endemic countries and it iSick neonates who undergo blood transfusion in sub-Saharan Africa especially in Nigeria are at risk of TM. This is because several studies have shown that the prevalence rate of malaria parasitaemia in donor blood across the SSA countries and in Nigeria is high with a range of 0.7%-55% and 4.7%Furthermore, the aforementioned studies were conducted in a mixed population of adult and children and so the findings may be different in the neonatal population especially with the recent report of increasing prevalence of neonatal malaria.,17 Also,The aim of this study was to determine the incidence and risk factors of transfusional malaria in neonates in South-West Nigeria.This cross sectional descriptive study was carried out at the neonatal intensive care unit of a tertiary hospital in South-West Nigeria between February and November 2015.Lagos University Teaching Hospital has two NICU; in-born and out-born units for admitting neonates delivered within the hospital and those referred from other facilities respectively. The hospital has a blood bank that runs 24-hours services providing screened and cross-matched blood products when required. Blood is obtained from voluntary donors, families and friends who are routinely screened using questions and temperature check to exclude high risk donors who may transmit infections and potential donors with a recent history of fever; temperature greater than 37.5°C; symptoms of ill health, current or recent use of malaria medications in addition to those who have risky behavior are excluded from being donors. Donors are also routinely screened for HIV, hepatitis B, hepatitis C and syphilis prior to donation. Malaria parasite is not routinely screened for in the donor blood prior to transfusion.Admitted neonates who were scheduled for whole blood or packed red cell transfusion and who satisfied the following criteria were consecutively recruited into the study: mothers/care givers gave written informed consent, were negative when tested for malaria parasites prior to transfusion, were not on malaria medication and not on breast milk from mothers on malaria medication within three days pre or post-transfusion. Neonates who received more than a blood component and had multiple transfusions where excluded. A pretested questionnaire  was usedApproval for the study was obtained from the Lagos University Teaching Hospital Health Research and Ethics Committee (HREC). A written informed consent was obtained from the mothers/care givers of the neonates and information obtained was handled confidentially. Feedback on the test result was communicated to the mothers/care givers. The neonates were followed up post transfusion and the result of those with malaria parasitaemia was promptly made available to the managing physicians for appropriate treatment.Thick and thin smears were prepared from blood samples obtained from the blood bag and from neonates pre transfusion, three days and 14 days post transfusion. The smears were prepared and subsequently stained with freshly prepared Giemsa solution according to standard procedure. The staiTransfused neonates were followed up post transfusion for signs and symptoms attributable to malaria such as fever, vomiting, diarrhea, jaundice and refusal of feed. Axillary temperature of neonates was measured prior to transfusion and for 14 days (8hourly and any time the parent/care giver felt the neonate’s body was warm) post transfusion. Those who developed fever or symptoms were tested for malaria parasitaemia and those who tested positive were promptly treated and a repeat malaria microscopy carried out 14 days post-transfusion to determine parasite clearance.The data obtained was inputted and analyzed using the Statistical Package for Social Sciences (SPSS) version 20.0. Frequency distribution tables and bar chart was generated for categorical data. Test of hypothesis for categorical data was done using the Chi-square and Fishers exact test. The Kolmogorov-Smirnov test was used to test for normality of continuous variables. Continuous variables were expressed as means and standard deviations when normally distributed or median and inter-quartile range when skewed. The Student’s t test was used to test for statistical difference between normally distributed continuous data. Spearman’s rank order correlation was used to determine the relationship between parasite density in neonates and parasite density in transfused blood. Binary logistic regression was used to determine the odd ratios and statistical significance of some predictors of TM. Level of significance was set at p value of <0.05.One hundred and sixty four neonates concluded the study schedule; one hundred and thirty-three (81.1%) and thirty one (18.9%) were recruited from the out-born and in-born units of the hospital respectively. One hundred and four (63.4%) of the study participants were males with a male to female ratio of 1.7:1. The overall mean age was 9.1 ± 6.5 days, with 57.3% of the study population aged 1–7 days and 42.7% aged 8-28days. One hundred and twenty (73.2%) neonates were delivered at term and the mean gestational age was 37.0 ± 3.2weeks. Of the forty-four neonates delivered preterm, 21 (47.7%) were late preterm. The mean birth weight was 2656.9 ± 794.3 grams with 66.5% of the participants having normal birth weight. A total of one hundred and three (62.8%) of the participants were transfused on account of severe hyperbilirubinaemia; forty-five of these participants had both severe hyperbilirubinaemia and anaemia. Of the sixty-one participants who were transfused on account of anaemia from various causes, majority (47.5%) of them had sepsis as the cause of anaemia. One hundred and forty-six (89.0%) of the study population were transfused with whole blood while eighteen (11.0%) were transfused with packed cells. One hundred and thirty (79.3%) received stored blood while thirty-four (20.7%) received fresh blood. The median age of stored blood was 2.0 days with a range of 0 to 19 days. Most (62.7%) of the participants had double volume exchange transfusion. P. falciparum were identified in all three transfused donor blood samples. The median parasite density was 525 parasites/μl of blood (range 30–6029).Three (1.8%) out of one hundred and sixty-four transfused donor blood samples used in the study were positive for malaria parasites. Trophozoites of P. falciparumdetected in their peripheral blood with median parasite density of 908 parasites/μl of blood (range 71–4079 parasites/μl of blood). All four implicated donor samples in post transfusion malaria were whole blood; three of which were fresh and one stored for five days. Two of the fresh donor samples (each 15mls and 20mls) were used for top-up transfusion in preterm neonates whiles the remaining two; one fresh (420mls) and one stored (500mls) were used for double volume EBT. One of the implicated donor sample (20mls) which was fresh and used for top-up transfusion was negative for malaria parasites.It was observed that four (2.4%) out of one hundred and sixty-four participants developed malaria parasitaemia three days post transfusion and all had trophozoites of p<0.001).  of the remaining 161 participants who were transfused with malaria negative donor blood developed post transfusional malaria. The association between receiving malaria positive blood and development of post transfusional malaria in the neonates was found to be statistically significant (<0.001). There was a positive and strong correlation between malaria parasite densities in neonates (recipients) three days post transfusion with malaria parasite density in transfused donor blood. p<0.001) and birth weight (p = 0.022).  and aged 1–7 days (75%). However, a statistically significant relationship was observed across gestational age ( 0.022). p = 0.028). All the participants with post transfusion malaria were of blood group O and received whole blood but there was no statistically significant relationship between blood components received (p = 1.00), blood group (p = 0.319), type of transfusion (p = 0.724) and the development of malaria parasitaemia post transfusion.  and this relationship was statistically significant  of malaria parasitaemia in the donor blood. A similar incidence rate of 2% was reported by Owusi-Oforiet al in a mix al[et al in a sim al[et al and alsoet al[Prevalence rate of 1.8% for malaria parasitaemia in donor blood observed in the current study is lower than the previous reports in Nigeria where a range of 4.1% in the South-Western to 74.1%et al who founThe decline in the prevalence of malaria parasitaemia in transfused donor blood recorded in the study may be a reflection of effective malaria control with the recent scale up in vector control through the use of insecticide treated mosquito nets (ITNs) and indoor residual spraying (IRS), community awareness programs, better diagnostics and use of artemisinin-based-combination therapy (ACT). The WHO reported a significant improvement in the use of ITNs, IRS, diagnostic testing for malaria with corresponding increase in the use of ACTs in Africa. The repoet al[et al[et al.[A significant association was observed between malaria parasitaemia in transfused donor blood and parasitaemia in the neonates post transfusion. Similarly, the parasite density in the transfused donor blood correlated positively and strongly with parasite density in the neonates post transfusion. These findings support transmissibility of malaria parasites from asymptomatic blood donors to recipients of blood. All three participants who received malaria infected donor blood developed malaria parasitaemia post transfusion representing a 100% risk to the recipients. On the contrary, Ali et al and Owus al[et al reportedal[et al. ThereforOne participant (0.6%) who was transfused with freshly donated non-infected donor blood developed parasitaemia post transfusion. The likely reasons for this finding may be that malaria parasite infection was acquired through the bite of an infected mosquito and parasites were in the liver stage of development (pre-erythrocytic schizogony) during the initial microscopy pre-transfusion. The age of the participant (20 days) further supports acquired malaria as the mode of transmission excluding congenital malaria which is expected to occur in the first week of life. It is however possible that parasite in donor blood might have been missed by microscopy which has been reported to have low sensitivity at low parasite density. The non-use of polymerase chain reaction (PCR) as the diagnostic tool in the present study is a major limitation as it use may have increased the diagnostic yield.The proportion of the participants who developed malaria parasitaemia post transfusion was significantly influenced by gestational age, birth weight and duration of storage of blood. On further analysis using logistic regression, it was observed that low birth weight (LBW) and fresh blood transfusion were predictors of TM. The higher risk of developing TM with decreasing birth weight is of public health concern considering the rapid increase in this population resulting from increased survival due to better neonatal intensive care. These neonates are particularly at greater risk because they are born earlier before maternal transfer of immunoglobulin. Therefore, they may be prone to severe TM. Participants who received fresh blood were observed to be 12 times more likely to develop TM when compared to those who were transfused stored blood. This finding portends a serious health implication for neonatal care where small volume transfusion with fresh blood is often practiced. Routine screening of freshly donated blood prior to transfusion is further emphasized.et al[It was observed in the study that even though the subjects who developed TM had normal temperatures pre-transfusion, all (100%) of the participants developed a fever post transfusion with a range of 37.7°C- 39.1°C recorded between days two and five post transfusion. Ali et al reportedThe study has shown100% concordance rate of malaria infection in neonates transfused with malaria infected donor blood. Also, neonates with low birth weight and transfused with fresh donor blood are more likely to develop TM. This high burden of TM in neonates necessitates policy formulation. The authors recommend routine screening of freshly donated blood prior to transfusion to neonates and children under 5 years and follow up of all recipients of blood transfusion. Also, future studies using PCR is recommended.We were unable to use polymerase chain reaction which is a more sensitive and specific method for malaria parasite testing.All blood donors over the study period could not be tested for malaria parasite at the same time point.S1 Appendix(DOCX)Click here for additional data file.S2 Appendix(DOC)Click here for additional data file.


Urinary Schistosomiasis and malaria are endemic in Sub-Saharan Africa. There are public health concerns and implications of these parasites. This study sought to assess the prevalence of malaria, urinary schistosomiasis, and anaemia in children of school going age in two municipalities in Ghana.S. haematobium, P. falciparum infection and the haemoglobin concentration of respondents. A total of 404  school children between ages 9 - 14 years (mean age 11.8 ± 1.4 years) were recruited for the survey. Urine and blood samples were collected using standard operating procedures for urinary schistosomiasis and malaria diagnosis. Haemoglobin concentration was measured using a Hemocue® Hb 201 m.A cross-sectional study design was used to investigate the prevalence of S. haematobium and P. falciparum respectively with a small proportion (0.9%) of the respondents infected with both parasites. The prevalence of anaemia in the study population was 59.9%. The risk of developing anaemia was not associated with being infected with any of the parasites. All co-infected children had anaemia.The prevalence of mono-infection was 4.7 and 12.9% for High prevalence of anaemia was observed within the study population. Prevalence of malaria was higher compared to schistosomiasis. Interventions to address the high levels of anaemia is required within the community.The online version of this article (10.1186/s40794-018-0063-7) contains supplementary material, which is available to authorized users. Malaria and urinary schistosomiasis are parasitic infections with grave public health implications. Both parasites have been associated with poverty and several related factors which may result in their distribution . Some prS. haematobium is the most common form of schistosomiasis in Ghana. Schistosomes are often referred to as blood flukes because they live in blood vessels. Its transmission requires an intermediate and suitable snail host. Contamination of water bodies with human excreta and urine releases the primary larvae (miracidiae) which infects the snails and develops into free-living secondary larvae in the snail host. Water-related activities such as swimming, fishing, irrigation or farming, fetching of water and washing clothes or dishes expose humans to the cercariae. The cercariae penetrate the skin thereby infecting humans in the process   municipality than in Ga South (6.6%) [, P. falciparum and S. haematobium infection  school children had concomitant on Table .p < 0.05) in the haemoglobin level between S. haematobium positive and negative individuals. There was a significant difference in the mean haemoglobin concentration of respondents with light and heavy infection for S. haematobium. Children with heavy infection had a lower mean haemoglobin level than children with light infection. P. falciparum infected children had lower haemoglobin concentration compared with children not infected with P. falciparum , reported in this study was lower than the prevalence in a similar population [P. falciparum infection could be attributed to the use of malaria prevention methods such as insecticide-treated nets and insecticides. Previous studies in same study site revealed a prevalence between 54 and 60% for urinary schistosomiasis [The overall prevalence of 4.7% reported for urinary schistosomiasis in this study was less than the prevalence reported in similar studies in Nigeria and Ethiopia , 11, 15.parum 12.%, reportsomiasis . The yeasomiasis .The total prevalence of anaemia in the study was 59.9% which is indicative of a severe public health problem . In EthiThe mean haemoglobin concentration of the study population was 11.6 ± 1.5 g/dl. This is because the prevalence of mild anaemia was higher than the prevalence of moderate and severe anaemia. This finding is similar to other studies among children of school going age , 20.P. falciparum infected children was lower than S. haematobium infected respondents, malaria was not a discriminatory factor to anaemia. This could be attributed to the high endemicity of P. falciparum among the study respondents resulting in misclassification. Children who were lightly infected with S. haematobium had a higher haemoglobin concentration because they are less likely to have haematuria. The Plasmodium parasites break down red blood cells and increase the clearance of both infected and uninfected red blood cells and a reduction in erythropoiesis [P. falciparum infection was reported in Nigeria [p-value < 0.05) thus contributing to the higher haemoglobin concentration in S. haematobium infected respondents. The intensity of S. haematobium infection is positively correlated with hemoglobin concentration [Although haemoglobin concentration of opoiesis . Similar Nigeria . Respondntration .S. haematobium and P. falciparum infection had a lower hemoglobin concentration than children with mono-infection [All respondents with coinfection had anaemia. In Ethiopia children with concurrent nfection . The conThe reported prevalence of hookworm infection in Ghana ranges from 3.2 to 10% , 23. TheFurther studies, taking into consideration, these confounders should be carried to obtain the true relationship between these risk factors and haemoglobin concentration in school children. This is necessary to understand the causes of the high levels of anaemia for the formulation of appropriate public health intervention.High prevalence of anaemia within the study communities is of concern. Investigating common causes of low hemoglobin concentration such as helminth infection and nutritional intake among others is required to address the challenge. Furthermore, public health interventions to reduce the prevalence of malaria and schistosomiasis particularly among school going children is warranted.Additional file 1:Socioeconomic Status Tool. (DOCX 48 kb)


The pathophysiology of the placental malaria is not fully understood. If there is a fetal sex–specific susceptibility to malaria infection, this might add to the previous knowledge on the immunology, endocrinology and pathophysiology of placental malaria infections.This study was conducted to assess whether the sex of the fetus was associated with placental malaria infections.A cross-sectional study was performed including a secondary analysis of a cohort of women who were investigated for prevalence and risk factors  for placental malaria in eastern Sudan. Placental histology was used to diagnose placental malaria infections.P = 0.007) and women who had female new born were at higher risk for placental malaria infections .Among 339 women enrolled, the mean (SD) age was 25.8 (6.7) years and parity was 2.7 (2.2). Among the new born babies, 157 (46.3%) were male and 182 (53.7%) were female. Five (1.5%), 9 (2.7%) and 103 (30.4%) of the 339 placentas had active, active-chronic, past-chronic malaria infection on histopathology examination respectively, while 222 (65.5%) of them showed no malaria infection. Logistic regression analyses showed no associations between maternal age or parity and placental malaria infections. Women who have blood group O (OR = 1.95, 95% CI = 1.19–3.10; Fetal gender may be a novel risk factor for placental malaria. In this work the female placentas were at higher risk for malaria infections than the male placentas. There is a great in interest in recent years in the female/male distribution during pregnancy and its interaction with maternal and perinatal health . Many maPlasmodium falciparum malaria infection during pregnancy is a major public health problem especially in sub-Saharan Africa, where it is a major cause of maternal and perinatal morbidity and mortality [ortality ,10. The ortality –13. The ortality . This st2 that were taken and recoded immediately. Following delivery, new born sex and weight were recorded.This is a secondary analysis of a cohort of women who were investigated for prevalence and risk factors for placental malaria in eastern Sudan using placental histology as tool for diagnosis for placental malaria infections ,16. The P. falciparum parasites per 200 white blood cells was recorded. Maternal blood groups were investigated by the agglutination method.Thick blood films for malaria were prepared from the maternal, placental  excised from the centre) and cord blood and were Giemsa-stained and the number of asexual 3 full thickness placental blocks were taken from the placenta and kept in neutral buffered formalin for histopathology examinations. The biopsy samples were processed by embedding them in paraffin wax using standard techniques. In every case, the thick paraffin sections were stained with hematoxylin-eosin and Giemsa stains. Histology was evaluated using the criteria of Bulmer et al [Around three cmer et al . PlacentThe histopathology and hematology studies were performed by two independent readers namely HAH for hematology and AAM for histopathology.2 tests, respectively. Binary regressions were performed with placental malaria infections as a dependent variable and the independent variables age, parity, residence, education, antenatal care, maternal blood group (O versus non O) and new-born gender were entered in the model if their univariate P was <0.20. Odds ratios and 95% confidence interval were calculated and P < 0.05 was considered significant.Data were entered using SPSS for windows (version 20.0). Means and proportions were compared between two groups  using Student’s t-test and XThe study received ethical clearance from the Research Board at the Faculty of Medicine, University of Khartoum.Thirty –two women were excluded from the study because they had incomplete data (18), had antepartum hemorrhage (11) or had thyroid diseases (3). Three hundred and thirty nine women had complete data include the placental malaria infections and the sex of the new born and their data were analysed. Of these women, 139 (41.0%), 265 (78.2%), 113 (33.3%) and 66 (19.55%) women were primiparae, had rural residence, were illiterate and had no antenatal care, respectively. While 32.2% of these women used bed nets, only (5.3%) used intermittent preventive treatment in the index pregnancy. The mean (SD) of the age of the enrolled women was 25.8 (6.7) years and mean (SD) parity was 2.7 (2.2), respectively. Of the participants, 79 (23.3%), 74 (21.8%), 22 (6.5%) and 164 (48.4%) had blood group A, B, AB and O, respectively.One hundred and fifty seven (46.3%) of the new born babies were males and 182 (53.7%) were females. The mean (SD) of the birth weight was 3081.4 (530) g and it was not different between males and females [3122.7 (526.0) vs. 3045.8 (534) g, P = 0.184. There was no significant difference in the maternal age, parity, education, residence, antenatal care level, or proportion of women using bed nets in the index pregnancy between women with male and female new born babies, Maternal and placental blood films for malaria were positive in three cases. Two maternal, placental and cord settings had positive blood films for malaria. All the positive blood films for malaria were found to have a positive placental malaria infection on histology.One hundred and seventeen placentas had malaria infection. Five (1.5%), 9 (2.7%) and 103 (30.4%) of the 339 placentas had active, active-chronic, past-chronic malaria infection on histopathology examination respectively, while 222 (65.5%) of them showed no malaria infection.While there was no significant difference in age, parity, education, residence and antenatal care level between women with placental malaria infection and women without placental malaria infection, women with placental malaria infection had a significantly higher number of O blood group and had a female new born (78 (66.7%) vs104 (46.8%), P < 0.001), P = 0.007) and women who had female new born were at higher risk for placental malaria infections , Logistic regression analyses showed no associations between the age, parity, education, antenatal care and placental malaria infections. Women who have blood group O , immune system modulation and increase in the receptors mediating parasite cytoadhesion need to investigated as possible mechanisms of association between female sex and placental malaria infection ,21. One Although the effects of fetal sex on the long-term health of the mother are still not completely understood, these are now obvious in two maternal diseases namely asthma and preeclampsia ,24. It iThe placenta not only transports nutrients and oxygen from the maternal circulation and returns waste products to the maternal circulation, but also it has a major endocrine and immunological function being responsible for synthesizing many hormones and cytokines that might have effects on the physiology of both the mother and the fetus ,27. ThesThe pathophysiology of placental malaria is not fully understood and the finding of the sex–specific susceptibility will add to the previous knowledge on the immunology, endocrinology and pathophysiology of placental malaria infections.P. vivax in the area [The submicroscopic malaria was not investigated (through polymerase chain reaction) and this point should be considered as one of the limitation of the current study. Previous study has shown that 32.0% of pregnant women in eastern Sudan had submicroscopic malaria . The datthe area .The current study supported the recent findings that fetal sex might influence the maternal milieu. In this work the female placentas were at higher risk for malaria infections than the male placentas.The study received ethical clearance from the Research Board at the Faculty of Medicine, University of Khartoum, Sudan.S1 Table(XLSX)Click here for additional data file.


Malaria transmission is highly heterogeneous, especially in low endemic countries, such as Cambodia. This results in geographical clusters of residual transmission in the dry, low transmission season, which can fuel the transmission to wider areas or populations during the wet season. A better understanding of spatial clustering of malaria can lead to a more efficient, targeted strategy to reduce malaria transmission. This study aims to evaluate the potential of the use of serological markers to define spatial patterns in malaria exposure.Plasmodium falciparum and two Plasmodium vivax). The antibody half-lives range from approximately six months until more than two years. Geographical heterogeneity in malaria transmission was examined using a spatial scan statistic on serology, PCR prevalence and malaria incidence rate data. Furthermore, to identify behavioural patterns or intrinsic factors associated with malaria exposure (antibody levels), risk factor analyses were performed by using multivariable random effect logistic regression models. The serological outcomes were then compared to PCR prevalence and malaria incidence data.Blood samples collected in a community-based randomized trial performed in 98 high endemic communities in Ratanakiri province, north-eastern Cambodia, were screened with a multiplex serological assay for five serological markers . The main risk factors for all It is possible to identify similar malaria pockets of higher malaria transmission together with the potential risk factors by using serology instead of PCR prevalence or malaria incidence data. In north-eastern Cambodia, the serological markers show that malaria transmission occurs mainly in adults staying overnight in plot huts in the field. Pf.GLURP.R2 showed a shrinking pocket of malaria transmission over time, and Pf.MSP1.19, CSP, PvAMA1 were also informative for current infection to a lesser extent. Therefore, serology could contribute in future research. However, further in-depth research in selecting the best combination of antigens is required.The online version of this article (doi:10.1186/s12936-016-1558-1) contains supplementary material, which is available to authorized users. The rison 3.1.0 ) with lnon 3.1.0 . Incidennd ‘car’ , 37). La . Furthermore, gender appeared to be significantly different for CSP, Pf.MSP1.19 and Pv.MSP1.19, and sleeping in the forest for CSP and Pv.MSP1.19. However, the IRR of these last two risk factors are so close to one, that this is probably negligible.Methods that can identify stable areas of transmission over time are suggested to be most effective for assessing geographical variations in malaria exposure. Therefore, Ab-responses acquired with cumulative malaria exposure, measured over several seasons, were recommended for implementation in geographical clustering analyses . During Where most studies only focused on PfAMA1 and PfMSP1.19 , 16–19, P. falciparum Ags Pf.GLURP.R2, Pf.MSP1.19 and CSP. These serological markers could map the transmission risks with more precision and accuracy, as they provide the ability of distinguishing recent from past exposure [The previous study performed by Kerkhof et al.  has led exposure , 41. TheP. falciparum and P. vivax Ags. The largest pockets were located around the most northerly site of the ‘Tonle San River’ for all Ags. In comparison with the PCR prevalence data, P. falciparum exhibited similar pockets, whereas for P. vivax differences were seen. The similar pockets found between the PCR prevalence rates and sero-reactivity are in line with a study performed by Bousema et al. [In Ratanakiri, significant malaria pockets were observed for both a et al. , that obP. falciparum situated around the capital ‘Ban Lung’ of the Ratanakiri province. Different studies [When comparing the serologically based pockets with the incidence based pockets, the pockets neighbouring Vietnam were comparable, while the most northerly pockets at the ‘Tonle San River’ were slightly shifted to the West. There are malaria incidence based pockets found for  studies , 42, 43 That most pockets were perceived around the river confirms findings from other studies that also found more malaria pockets along open water bodies , 44, 45.P. falciparum and P. vivax was acceptable (between 72 and 95 %) to predict PCR and malaria incidence pockets. However, sensitivity was in general much lower, except in predicting P. falciparum PCR pockets (between 74 and 95 %). In comparison, sensitivity of P. falciparum and P. vivax incidence in predicting PCR pockets lies between 10 and 50 %.The specificity of serological markers for When looking at the different serological markers, variable patterns were observed, going from malaria pockets that move between the east and west in November 2012 and 2013  to lasting pockets that became smaller (Pf.GLURP.R2) or remained similar in size (PvAMA1). These varying patterns require further investigation related to the differences in immunogenicity and persistence of the Ab-responses , 46. TheP. falciparum malaria were seen for age, ethnicity and overnight stay at the plot hut. There were also differences observed in gender and sleeping in the forest, however, this is most probably negligible, as the IRR was very close to 1. Significant elevated risks for vivax markers were seen for age, whereas staying in plot huts showed to be a risk factor for Pv.MSP1.19 only. The P. falciparum outcomes are in line with a previous study performed in the same area by Sluydts et al. [P. falciparum and P. vivax could be explained by that fact that P. vivax shows relapse patterns that influence the serological outcomes, and that longer half-lives were observed for the P. vivax Ags in a previous study [Risk factors related to sero-reactivity were identified by univariate and multivariable analyses. Significant elevated risks for s et al. . In thiss et al. , 17, 26.s et al. . Therefos et al. . Differeus study .Overall, these outcomes confirm the findings of Sluydts et al. , and arePlasmodium life cycle and having a longevity ranging from very short (~1 months) to long (year round), might lead to other promising results. However, methods to acquire the exact Ab-persistence are still in its infancy [P. falciparum Ag AMA1 in the high endemic setting of Tanzania. Further development in quantifying exposure over different timescales, as well as the measurement of very recent exposure, serological approaches will provide a major contribution in estimating spatio-temporal patterns of risk [This study contributes in the validation of serological markers to distinguish very recent from past exposure, as suggested by Sturrock et al. . This is infancy . While P infancy  on P. fa of risk . The use of risk , as wellP. vivax.Identification of pockets with higher malaria transmission would be essential when adopting a malaria elimination strategy. Present study shows that PCR parasite prevalence, malaria incidence rates or serology, show equivalent results in identifying malaria pockets. The attempt to validate serological markers that are most likely to reflect current and past exposure led to Pf.GLURP.R2 showing a shrinking malaria pocket over time. Moreover, Pf.MSP1.19, CSP and PvAMA1 are also reflective for recent malaria transmission to a lesser extent. This means that serology can provide promising information for future research, especially in evaluating short-term interventions for malaria elimination. However, there is still more research required in selecting a promising combination of Ags, in particular for


Plasmodium falciparum infection are not optimal for non-invasive, population-based screening for malaria. It was hypothesized that a mass spectrometry-based metabolomics approach could identify urinary biomarkers of falciparum malaria.Currently available diagnostic techniques of P. falciparum infection confirmed with microscopy, and 25 controls of adults with negative blood smears for malaria matched on age and sex. Urinary samples were collected from these individuals during presentation at the clinic, and a second sample was collected from both cases and controls 4 weeks later, after the cases had received anti-malarial medication. The urine samples were screened for small molecule urinary biomarkers, using mass spectrometry-based metabolomics analyses followed by multivariate analysis using principal component analysis and orthogonal partial least square-discriminant analysis. The chemical identity of statistically significant malaria biomarkers was confirmed using tandem mass spectrometry.The study used a case–control design, with cases consisting of 21 adults in central Ethiopia with a diagnosis of P. falciparum infection were distinct from healthy controls. After treatment with anti-malarial medication, the metabolomic profile of cases resembled that of healthy controls. Significantly altered levels of 29 urinary metabolites were found. Elevated levels of urinary pipecolic acid, taurine, N-acetylspermidine, N-acetylputrescine and 1,3-diacetylpropane were identified as potential biomarkers of falciparum malaria.The urinary metabolic profiles of cases with P. falciparum infection.The urinary biomarkers of malaria identified have potential for the development of non-invasive and rapid diagnostic test of The online version of this article (doi:10.1186/s12936-017-1875-z) contains supplementary material, which is available to authorized users. Plasmodium falciparum  and 17 (81%) were male. The mean age for the controls was 32.1 years  and 20 (80%) were male. The validation of the LC–MS performance for metabolomics analysis is presented in Additional file 21 individuals with Typical LC–MS base peak chromatograms obtained from urine samples of malaria patients and healthy controls are shown in Fig. 2Y and Q2 values were R2Y = 0.993 and Q2 = 0.583  and healthy controls (C1) datasets only, total separation between the two groups were observed Fig. . The OPL and healA list of urinary metabolites which make a major contribution to the predictive OPLS-DA model between malaria patients and healthy controls is shown in Table P. falciparum infection in humans. The analysis clearly identified a number of candidate biomarkers that are elevated in individuals with active infection confirmed by blood-smear microscopy. Levels of these molecules decrease after treatment with anti-malarial medication, suggesting that these molecules are biomarkers of active infection.This is the first study to use a metabolomics approach to identify urinary biomarkers for P. falciparum infection in humans. A case–control study design was used, with prospective data collection in cases after they were treated and had recovered from the original infection, and also in controls. This allowed the candidate molecules identified in the cross-sectional study to be tested for their response to treatment, and hence reduced the possibility of false-positive outcomes as a consequence of multiple hypotheses testing that is a concern with this type of statistical analysis. However, these observations are preliminary and require confirmation in other datasets, before we can be confident that these associations are causal and these molecules are clinically useful biomarkers for infection with P. falciparum infection.The strengths of these data include the prospective testing of the hypothesis that a metabolomics approach can identify biomarkers for P. falciparum infection, there is a constant dynamic metabolic interplay between the host and the parasite during the course of infection that may perturb the biochemical profiles of both the parasite and the host. The parasite invasion induces a constellation of responses by the host which are collectively known as “active-phase responses” [The increased level of succinic acid, taurine, alanine and pipecolic acid in malaria patients was consistent with previously reported studies –14, but sponses” . This phsponses” . Hence, sponses” .Plasmodium infection was consistent with previous in vitro studies [P. vivax infected patients [An increased level of alanine was observed in malaria patients compared to healthy controls, suggesting that lactic acid was converted to alanine, suggesting evidence of enhanced glycolysis pathway activity during the course of infection, consistent with recent observations . However studies , 19. Recpatients , 11. Thil-lysine (methylated derivative of lysine), alanine, l-threonine, N-acetylglutamine (metabolite of glutamine) and N-acetylasparagine (metabolite of asparagine) were observed in the urine of malaria patients but not healthy controls. This finding was consistent with previously reported studies, in which abnormal levels of amino acids and amino acid metabolites were found in the urine and plasma of patients infected with P. falciparum [Abnormally high levels of pipecolic acid, trimethyl-lciparum . A high lciparum , suggestP. falciparum infection is consistent with the observation that acute kidney injury occurs during malaria infection [Plasmodium-infected erythrocytes compared to non-infected ones.The increased excretion of urea in cases with nfection  and has nfection . Signifinfection  reportedP. falciparum infection. This approach identified a number of candidate molecules that are associated with the presence of and recovery from P. falciparum infection in human. This approach has the potential to lead to a non-invasive urinary diagnostic test for P. falciparum infection.A metabolomics analysis of urine samples from a case–control study was used to identify possible diagnostic urinary biomarkers of Additional file 1. Further methods.Additional file 2: Figure S1. PCA score plots overview obtained from all malaria and control urine samples. Figure S2. MS/MS spectra comparison of tentitively identified taurine in the urine sample of malaria patients against its authentic standard. Figure S3. MS/MS spectra confirmation of tentitively identified succinic acid, creatinine, uridine and L-threonine in the urine sample of malaria patients against their authentic standards.


Plasmodium falciparum in culture, in the mouse model and in rhesus monkeys. MB was also shown to have a potent ex vivo activity against drug-resistant isolates of P. falciparum and P. vivax. In preclinical studies, MB acted synergistically with artemisinin derivates and demonstrated a strong effect on gametocyte reduction in P. falciparum. MB has, thus, been considered a potentially useful partner drug for artemisinin-based combination therapy (ACT), particularly when elimination is the final goal. The aim of this study was to review the scientific literature published until early 2017 to summarise existing knowledge on the efficacy and safety of MB in the treatment of malaria.Methylene blue (MB) was the first synthetic antimalarial to be discovered and was used during the late 19th and early 20th centuries against all types of malaria. MB has been shown to be effective in inhibiting This systematic review followed PRISMA guidelines. Studies reporting on the efficacy and safety of MB were systematically searched for in relevant electronic databases according to a pre-designed search strategy. The search (without language restrictions) was limited to studies of humans published until February 2017.P. falciparum gametocyte reduction and synergy with ACT. MB treatment was associated with mild urogenital and gastrointestinal symptoms as well as blue coloration of urine. In G6PD-deficient African individuals, MB caused a slight but clinically non-significant haemoglobin reduction.Out of 474 studies retrieved, a total of 22 articles reporting on 21 studies were eligible for analysis. The 21 included studies that reported data on 1504 malaria patients (2/3 were children). Older studies were case series and reports on MB monotherapy while recent studies were mainly controlled trials of combination regimens. MB was consistently shown to be highly effective in all endemic areas and demonstrated a strong effect on P. falciparum malaria in areas outside Africa and against P. vivax malaria. Adding MB to ACT could be a valuable approach for the prevention of resistance development and for transmission reduction in control and elimination programs.More studies are needed to define the effects of MB in CRD42017062349).This study is registered at PROSPERO (registration number The online version of this article (10.1186/s12916-018-1045-3) contains supplementary material, which is available to authorized users. Malaria remains the most important parasitic disease in humans . CombinaMethylene blue (MB) is a water-soluble dye, which has been used for a long time in industry and medicine , 9. The MB is a registered drug in most countries for various indications, such as the treatment of both acquired and hereditary methaemoglobinaemia, the prevention of ifosfamide-induced encephalopathy in human cancer management, the prevention of urinary tract infections, the intraoperative visualisation of nerves and endocrine glands as well as of pathologic fistulae, and the sterilisation of transfusion blood , 15–23. Plasmodium falciparum glutathione reductase was identified as a new drug target [P. falciparum in culture, in the mouse model and in rhesus monkeys [P. falciparum and P. vivax [P. falciparum [The interest in MB as an antimalarial drug was reactivated when g target , 32–34, g target . Like itg target –38. Indeg target , 10. Theg target . MB has  monkeys , 41. MB P. vivax , 43. In lciparum –48. MB hlciparum . The aimThis systematic review follows the PRISMA  guidelines .All available and accessible data published until 28 February 2017 on the efficacy and/or safety of MB in the treatment of malaria were considered. Studies reporting data on MB given to humans of any age as monotherapy or in combination with other antimalarials and irrespective of drug formulations and sample size were included. In vitro and animal studies with MB, reports on the use of MB in humans for indications that were not malaria and review papers were excluded.Standard electronic databases were searched for scientific papers on the subject, but also hand searches were done in scientific books of major libraries. The following databases were searched for studies published in any language: Medline, Embase, BIOSIS, Cochrane Central Register of Controlled Trials, Cochrane Library, Web of Science, and the China National Knowledge Infrastructure and the Chinese Biomedical databases. The search used combinations of the terms “malaria” and “methylene blue” as both medical subject headings and key or free text words and included a broad range of derivations to ensure as wide a search strategy as possible. A list of the detailed search strategy used is available online as Additional file Endnote and duplicates removed. Each paper was assessed in two phases: first by screening title and abstract, and then by full-text review to ensure it met the inclusion criteria. The first assessment was done by two reviewers (MN and NG). The second assessment was done by GL and OM. An additional reviewer (AJ) settled any discordance between the reviewers.The electronic reports identified were imported into the reference manager Predetermined study characteristics were defined for extraction and documentation by two of the investigators (GL and OM), first independently and then in a consensus procedure. Reports selected were stratified according to study type. The primary outcome was the reported cure rate. Secondary outcomes were fever and parasite clearance rates, the effect on malaria gametocytes and adverse events (AEs).Two of the investigators (GL and OM) evaluated the quality of included studies in a consensus procedure. The risk of bias of randomised controlled trials (RCTs) was evaluated by the tool described in the Cochrane Handbook for Systematic Reviews of Interventions . The risQuality criteria for case series and case reports were developed from MINORS and the 2016 Critical Appraisal Checklist of the Joanna Briggs Institute for case reports and case series –54. SeveFigure n = 47), studies were not retrievable despite exhaustive efforts due to very old date (n = 38), manuscripts provided insufficient information (n = 32), preclinical studies (n = 5), MB was not used for treatment of malaria (n = 4), and patients were not treated with MB (n = 3).The most common reasons for exclusion were as follows: the manuscripts were reviews or comments or guidelines (n = 7), Asia (n = 3), the Americas (n = 3) and Europe (n = 8) and reported data on 1504 malaria patients (2/3 were children). The majority of reports were historical studies from around the year 1900 (n = 15), one was from the year 1949, and the remaining (n = 5) were published after the year 2000. The historical studies reported on malaria types diagnosed microscopically and/or clinically, which were not always well specified . The more recently conducted controlled trials reported microscopically well determined outcomes on falciparum malaria (n = 5) and on vivax malaria (n = 1).The 21 studies included are summarised in Table n = 5) or case series (n = 10), while six studies were non-randomised controlled trials (n = 3) or RCTs (n = 3) from more recent years.Fifteen studies were historical case reports (Summaries of treatment regimens are shown in Table Tables n = 8), vomiting of treatment (n = 3), low dose of MB (n = 6) and unknown (n = 19). In a more recent trial, MB monotherapy in 60 West African adults with falciparum malaria (a fixed dose of 780 mg for 3 days) showed a 100% cure rate when MB was given for 7 days compared to a 85% cure rate when MB was given for a shorter time period; this difference was close to being statistically significant [In MB monotherapy studies conducted around the year 1900, 337/373 (90%) of malaria cases were reported to be cured. Reported potential reasons for treatment failure were: unsuccessful quinine pretreatment (nificant .n = 391) showed superior efficacy compared to control regimens (n = 207). In the first trial, the cure rate of MB (4 mg/kg per day) plus CQ (56%) was slightly but non-significantly higher compared to that of CQ alone (46%) [Like MB in the combination treatment, a controlled study on vivax malaria among US prisoners from 1949 reported a cure rate of 6/9 (67%) for MB plus IQ and of 3/3 (100%) for MB plus IQ plus quinine . In the ne (46%) . A subsene (46%) . A third plus CQ % was sline (46%) .P. falciparum parasite was non-significantly shortened in children receiving AS-AQ-MB compared to the AS-AQ group (medians 43.7 vs 47.0 h) [Parasite clearance with MB appeared to be rather slow. In African adults treated with MB monotherapy, 9% were still parasitaemic on day 3 . In chil 47.0 h) . Fever w 47.0 h) , 56, 58.Gametocyte clearance following MB-based treatment was investigated in two RCTs. The first study was a secondary analysis of data from an RCT in children. Compared to AS-AQ, both MB-containing regimens were associated with significantly reduced gametocyte carrier rates during follow-up days 3, 7 and 14 . The secTreatment of malaria with MB was consistently associated with green-blue discoloration of urine. AEs concerning the urogenital system (urethritis) and the gastrointestinal system (vomiting) were reported to be associated with MB treatment. These AEs were more frequently reported with higher doses of MB , 69, 75.Despite some AEs being clearly associated with MB treatment, one study reported no differences between study groups in acceptance rates by parents and caregivers of children .This is the first review within the last 100 years to attempt systematically to collect and analyse all the data that have been published on the effects of MB in the treatment of human malaria. As MB was used for this indication globally in the late 19th and early 20th centuries, some early reviews were published at that time. In 1904, a meta-analysis of 425 malaria cases from 11 publications concluded that MB was effective in 85% of patients .In the early 20th century, MB was gradually replaced by new synthetic antimalarials with different characteristics and finally without colouring properties . ScientiP. falciparum [P. falciparum strains, triggering the hypothesis that combining MB and CQ might overcome resistance [The revival of MB as an antimalarial drug candidate began in 1995, at the height of the development of resistance against existing antimalarials, in three biochemical laboratories , 35, 38.In our analysis of 15 case reports and case series, 339/373 (91%) of patients were cured, which provides evidence for the high efficacy of MB monotherapy in the treatment of malaria. Treatment failures were attributed to previously unsuccessful quinine treatment, vomiting and a low and/or short MB dosing regimen, which helps to explain early controversies on whether quinine or MB was more effective against malaria –65, 77. In the recent studies, MB was usually given in combination with other antimalarials and tested in RCTs. In an area of high CQ resistance, MB-CQ was more effective than CQ alone but not sufficiently so , 60. ComP. falciparum parasites and reduced P. falciparum gametocytes [P. falciparum gametocyte reduction [Despite this limitation, other properties of MB are notable and promising. Adding MB to an ACT reinforced the particular beneficial effects of the artemisinins, i.e., it further accelerated the elimination of asexual etocytes –57. The etocytes . This sueduction , 45, 79.eduction . HoweverIn the historical studies, MB was usually given orally and often in high doses and for prolonged periods of time, both in children and in adults, and without reports of major safety problems. During World War I for example, some European soldiers received more than 400 g of MB over several weeks without major side effects, apart from moderate urogenital symptoms . BraziliAEs shown to be associated with MB treatment are mild gastrointestinal symptoms, which may manifest as vomiting, and mild urogenital symptoms, which usually manifest shortly after drug intake. This has consistently been reported, both in historical studies and in more recently conducted RCTs. Pure MB powder or MB dissolved in water has a very bitter and metallic taste. The gastrointestinal symptoms are clearly influenced by the formulation of MB, which suggests it is better to use taste-masked formulations. As the World Health Organization is now also recommending solid formulations for small children, mini-tablets, which should also be taste-masked, will be preferred over liquid formulations . MB giveMB is on the list of drugs potentially dangerous for patients with G6PD deficiency, but the clinical importance of this is still controversial , 84. In Reassuringly and despite the characteristic AE profile, MB has been well accepted by patients and caregivers of children in West Africa, which was also supported by an anthropological study in Burkina Faso . HoweverThe strength of this review is that it includes all studies ever conducted with MB or MB-containing regimens in humans with malaria irrespective of study design and sample size and irrespective of the language of the report. Moreover, it can be considered as beneficial that a number of authors of this review have been the investigators of all recently conducted studies on this topic. Limitations of this review are the lower quality of the historical studies included compared to recent RCTs, the availability of three reports only in an abbreviated form from other sources , 91, 92 P. falciparum, it shows synergy with the artemisinin component in rapidly clearing the parasites and it is very effective in reducing the gametocytes and consequently mosquito transmission. MB is usually well tolerated and accepted with mild and regularly self-limiting gastrointestinal and urogenital symptoms, which are the main AEs. To avoid vomiting, the drug should be given in a taste-masked formulation and/or together with food. In historical studies, nutmeg has been shown to be effective in moderating the frequent occurrence of urethritis, which should be evaluated in future studies. Whether the small effect of MB on the haemoglobin development is of clinical significance needs to be monitored in future large-scale studies including regions with more severe forms of G6PD deficiency compared to African populations.This review shows that MB has substantial antimalarial activity against all types of malaria in various endemic areas and, in combination with other antimalarials, against falciparum malaria in Africa. Although MB alone appears to act rather slowly against the asexual parasites of P. falciparum infections, a useful partner for triple combination therapy regimens with the goal of protecting the artemisinin component of the ACT and of reducing the spread of drug-resistant parasites, and a potentially valuable partner drug for mass drug administration in malaria elimination programs. Further studies should investigate the efficacy, safety and community acceptance of different ACT regimens in combination with MB against falciparum malaria in different areas inside and outside Africa, while well-designed pilot studies should investigate the effects of MB and MB-containing combination regimens as a treatment for vivax malaria.MB appears to be a potential alternative to PQ for reducing post-treatment infectivity in Additional file 1:Search strategy. (DOCX 12 kb)Additional file 2:N = 474). (PDF 329 kb)A list of all the records screened after duplicates were removed (


Plasmodium infection is crucial for prompt malaria treatment and surveillance. Microscopic examination has been widely applied as the gold standard for malaria diagnosis in most part of malaria endemic areas, but its diagnostic value has been questioned, particularly in submicroscopic malaria. In this study, the diagnostic performance of microscopic examination and nested polymerase chain reaction (PCR) was evaluated to establish optimal malaria diagnosis method in Myanmar.Accurate diagnosis of A total of 1125 blood samples collected from residents in the villages and towns located in Naung Cho, Pyin Oo Lwin, Tha Beik Kyin townships and Mandalay of Upper Myanmar were screened by microscopic examination and species-specific nested PCR method.Plasmodium falciparum or Plasmodium vivax infections, which were missed in microscopic examination. Microscopic examinations also either misdiagnosed the infected Plasmodium species, or did not detect mixed infections with different Plasmodium species in 31 cases.Among the 1125 blood samples, 261 samples were confirmed to be infected with malaria by microscopic examination. Evaluation of the 1125 samples by species-specific nested PCR analysis revealed that the agreement between microscopic examination and nested PCR was 87.3% (261/299). Nested PCR successfully detected 38 The nested PCR method is more reliable than conventional microscopic examination for the diagnosis of malaria infections, and this is particularly true in cases of mixed infections and submicroscopic infections. Given the observed higher sensitivity and specificity of nested PCR, the molecular method holds enormous promise in malaria diagnosis and species differentiation, and can be applied as an effective monitoring tool for malaria surveillance, control and elimination in Myanmar. Plasmodium species and quantify parasitaemia levels at a low cost [Plasmodium species infections and differentiate the infected species with high specificity and sensitivity [Plasmodium species other than Plasmodium falciparum and Plasmodium vivax.Accurate diagnosis of malaria is important for providing realistic estimates of malaria burden and preventing misinformed interventions , 2. For low cost . Howeverlow cost . To overlow cost –9. Polymsitivity –13. Nevesitivity . Rapid dsitivity –17. Due sitivity , 19. MorPlasmodium species associated with human infection, P. falciparum, P. vivax, Plasmodium malariae, and Plasmodium ovale, are considered to occur in Myanmar [Plasmodium knowlesi infections have also occurred in some areas, predominantly as a co-infection with either P. falciparum or P. vivax [Plasmodium species and mixed infections with different malaria parasites using these two diagnostic methods. Moreover, information on the diagnostic efficiency of these diagnostic methods is also highly limited.The incidence of malaria in Myanmar has decreased remarkably in recent years, but malaria is still one of the major public health concerns in the country. Myanmar accounts for approximately 77% of the estimated cases and 79% of the estimated malaria deaths in the Greater Mekong Subregion . Moreove Myanmar , 26. PlaP. vivax . The comPlasmodium species and submicroscopic infections. These results suggest that molecular diagnostic approach is more reliable than microscopic examination for the accurate diagnosis of Plasmodium species as a part of the malaria surveillance programs in Myanmar.In this study, the accuracy of routine microscopic examination was analysed by comparing to species-specific nested PCR detection method. PCR method produced conflicting results, particularly in cases of mixed infections with different p < 0.001). A slide was considered negative if no parasite was observed in a count of 500 leucocytes.Between August 2013 and December 2015, field surveys for malaria were conducted in towns and villages located in the regions of Naung Cho, Pyin Oo Lwin, Tha Beik Kyin townships and Mandalay in Upper Myanmar  from a finger prick were spotted on Whatman 3 MM filter paper  and allowed to air dry for subsequent molecular analysis.This study was approved by the Ministry of Health, Myanmar, and by the Ethical Review Committee of Inha University School of Medicine, Korea. Informed written consent and permission (in the case of children under 18 years of age) were obtained from each patient or a parent/legal guardian.2O. Nested PCR specific to the five human malaria parasite species  was performed using a pair of primers targeted to the 18S rRNA gene described previously [P. knowlesi was detected by PCR using a set of primers specific to the 18S rRNA gene [Plasmodium species was analysed with species-specific amplicon sizes. Randomly selected PCR products for each Plasmodium species were also sequenced to confirm the accuracy of the amplified products.Genomic DNA was isolated from each spotted blood filter paper by using the QIAamp DNA Blood Mini Kit . Genomic DNA was eluted with 100 μl of sterile Heviously , 6. P. kRNA gene . For theP < 0.05 was considered statistically significant.The 95% confidence intervals (CI) were calculated as appropriate and the value of P. falciparum mono-infections, 94 cases (36.0%) were P. vivax mono-infections, and 31 cases (11.9%) were P. falciparum and P. vivax mixed infections. There was a case (0.4%) confirmed as a P. malariae mono-infection.Thin and thick blood smears from a total of 1125 residents including 325 suspected malaria patients were examined for the presence of malaria parasites using microscope. The results of the microscopic examination identified 261 cases  as positive for malaria infection . The 261 positive cases were corresponded to the samples detected by microscopic examination, but the 38 positive cases were negative in the microscopic examination. The specific Plasmodium species were identified as follows: P. falciparum (140 cases), P. vivax (99 cases), P. malariae (1 case), P. ovale curtisi (2 cases), and mixed infection with P. falciparum and P. vivax (52 cases), mixed infection with P. falciparum and P. malariae (4 cases), and mixed infection with P. vivax and P. ovale curtisi (1 case)  or P. vivax (15 cases) infections. These results suggest that the molecular approach is more sensitive and reliable than microscopic examination, particularly in the case of mixed infections and submicroscopic infections.The results of the nested PCR analysis were different from those of the microscopic examination, and an overall comparison of the results for these active malaria detection methods is summarized in Table Plasmodium species is not only important for establishing a successful treatment regimen, but also for designing effective malaria control measures in endemic regions where multiple species of malaria parasites exist. Misidentification of the Plasmodium species could cause a severe public health concerns since it could extend the parasite clearance times and lead to recrudescence and drug resistance [P. knowlesi, have been shown to exist in Myanmar [Plasmodium species and prompt treatment are essential to malaria control and elimination in the country. Therefore, studies are needed to assess quality and impact of these diagnostic methods for optimal malaria diagnosis setting and nationwide malaria surveillance outcomes.Accurate diagnosis of the sistance –31. Conv Myanmar –27, 32, P. falciparum and P. vivax mixed infections highly increased with nested PCR analysis. Misdiagnosis using microscopic examination technique can easily occur, particularly in the case of mixed infections where one parasite species is dominant over the others [P. falciparum and P. vivax in a single human host results in parasitic suppression through interspecies inhibition [P. falciparum or P. vivax super-infection. Although it still remains controversial whether mixed infections involving P. falciparum or P. vivax and other Plasmodium species are beneficial or harmful [In this study, 1125 blood samples collected from residents in malaria endemic areas in Upper Myanmar were analysed using microscopic examination and species-specific nested PCR. Overall malaria incidence as determined by microscopic examination and nested PCR method were relatively similar, with a coincidence of 87.3%, but important discrepancies between the microscopic examination and nested PCR analysis were also identified. The role of microscopic examination as the gold standard for malaria diagnosis has been questioned due to false negative results at low parasitaemiae levels (less than 20–30 parasites/μl of blood) and frequent errors in species identification in mixed infections . The lowe others . It has hibition –43. The  harmful , 45, acuP. falciparum and P. vivax, has been recognized an important obstacle to controlling malaria since asymptomatic carriers do not seek treatment for the infection, so that they constitute a reservoir available for new infections. A significant proportion of P. falciparum and P. vivax infections are asymptomatic or subclinical in malaria endemic countries [P. falciparum and 15 cases of P. vivax), which were not detected by microscopic examination, were identified by molecular detection method. These results suggest that the importance of detection of asymptomatic infections with submicroscopic level in Myanmar for successful malaria control and elimination in the country. Aggressive approach to detect asymptomatic carriers followed by prompt treatment would be required.Asymptomatic infection, particularly in the cases of ountries –52. RepePlasmodium species in the regions studied. Based on the results from the nested PCR analysis, overall malaria infection rate in the residents in the studied endemic areas was 26.6% (299/1125). It has been estimated that total malaria cases confirmed either by microscopy examination or by RDT in Myanmar were 420,808 in 2010 [P. falciparum and P. vivax infections  and the ratio of P. falciparum/P. vivax infections was 70% [P. falciparum and P. vivax accounted for most of the PCR-confirmed cases in this study. But the ratio of P. falciparum/P. vivax was not as high as previously reported and a high proportion of mixed P. falciparum and P. vivax infections (17.4%) were identified. It is assumed that P. vivax is becoming predominant species in the studied areas with the recent decrease of falciparum malaria cases. Asymptomatic and submicroscopic infections by the two Plasmodium species were also a great concern. The prevalence of P. malariae and P. ovale was low, but single as well as mixed infections involving either P. falciparum or P. vivax were identified. Distribution of P. malariae and P. ovale in Myanmar has been reported previously [P. ovale cases  were reported in Pyin Oo Lwin area [P. malariae and P. ovale are endemic in Upper Myanmar, though the prevalence is not high. P. malariae and P. ovale infections can be easily missed to diagnose by microscopic examination, primarily due to quite low parasitaemia of the parasites [Plasmodium species, it is more difficult to differentiate the parasites from a background with large numbers of parasites of other species [P. malariae and P. ovale infections that were not detected or misread in microscopic examination were successfully identified by nested PCR methods in this study. High rates of P. knowlesi co-infection with other malaria parasites also has been reported in North-Eastern Myanmar [P. knowlesi mono- or co-infection were detected in this study. It is worthy to realize the limitation of PCR-based molecular diagnosis methods to detect parasite DNA, especially in very low level of parasitaemia. A routine extraction process of parasite DNA from dried blood filters could result in loss or dilution of parasite DNA for successful amplification [Plasmodium species with high specificity and sensitivity. Molecular diagnostic approach is highly recommended for the accurate diagnosis of Plasmodium species as a part of the malaria surveillance and elimination programs in Myanmar.The results of this study also provide partial epidemiological information regarding the prevalence of  in 2010 . Most of was 70% . Consist6% 299/11. It has eviously . In part6% 299/11. It has arasites , 56. Mor species , 56. In  Myanmar , but no fication . Neverth.6% 299/15. It hasfication –39, PCR-Plasmodium species in the studied areas co-endemic for multiple Plasmodium species. Nested PCR not only detected all microscopy-positive samples, but also detected single or mixed infections and submicroscopic infections that were missed or misread by microscopy. Given the observed higher sensitivity and specificity associated with the nested PCR technique, molecular detection method holds enormous promise for malaria diagnosis and can be used as effective monitoring tools for malaria surveillance, control and elimination in Myanmar. Submicroscopic and asymptomatic infections also highlight the urgent need to develop new public health strategies and appropriate community-based interventions for the effective control and elimination of malaria in Myanmar.This study has shown that microscopic examination does not reliably distinguish


Plasmodium species infections are asymptomatic with densities too low to be detectable with standard diagnostic techniques. The importance of such asymptomatic plasmodium infections in malaria transmission is probably related to their duration and density. To explore the duration of asymptomatic plasmodium infections and changes in parasite densities over time, a cohort of participants who were infected with Plasmodium parasites was observed over a 2-year follow-up period.A substantial proportion of Plasmodium falciparum and Plasmodium vivax infections and changes in parasite densities over a study period of 24 months.In this open cohort study, inhabitants of four villages in Vietnam were invited to participate in baseline and subsequent 3-monthly surveys up to 24 months, which included the collection of venous blood samples. Samples were batch-screened using ultra-sensitive (u)PCR (lower limit of detection of 22 parasites per mL). Participants found to be infected by uPCR during any of these surveys were invited to join a prospective cohort and provide monthly blood samples. We estimated the persistence of P falciparum monoinfections, 507 (27%) were P vivax monoinfections, 463 (25%) were co-infections with P falciparum and P vivax, and 225 (12%) were indeterminate species of Plasmodium. The median duration of P falciparum infection was 2 months (IQR 1–3); after accounting for censoring, participants had a 20% chance of having parasitaemia for 4 months or longer. The median duration of P vivax infection was 6 months (3–9), and participants had a 59% chance of having parasitaemia for 4 months or longer. The parasite densities of persistent infections oscillated; following ultralow-density infections, high-density infections developed frequently.Between Dec 1, 2013, and Jan 8, 2016, 356 villagers participated in between one and 22 surveys. These study participants underwent 4248 uPCR evaluations (11·9 tests per participant). 1874 (32%) of 4248 uPCR tests indicated a plasmodium infection; 679 (36%) of 1874 tests were P vivax and P falciparum infections are common in this area of low seasonal malaria transmission. Infections with low-density parasitaemias can develop into much higher density infections at a later time, which are likely to sustain malaria endemicity.Persistent largely asymptomatic The Wellcome Trust, Bill & Melinda Gates Foundation. Plasmodium spp infections remain asymptomatic, and most of these cannot be detected by light microscopy because of low parasite densities.Plasmodium vivax parasitaemias and more than 70% of all Plasmodium falciparum parasitaemias.In the past 20 years, it has become apparent that in low-transmission settings such as southeast Asia many Persistent, asymptomatic plasmodium infections carry health risks for the infected individual including chronic anaemia, increased risks of maternal and neonatal mortality, impaired immune competence resulting in co-infections with invasive bacterial diseases, and cognitive impairment.Evidence before this studyPlasmodium species infections up to Oct 29, 2017. We searched PubMed using the MeSH terms “plasmodium”, and the  terms “persistence” or “duration” and “oscillation” and “dynamics” restricted to cohort studies or trials. 143 papers included in the search terms of which 14 addressed the persistence and oscillations in parasite densities. In high-transmission settings, repeated malaria infections induce partial immune responses in the human host, rendering most plasmodium infections asymptomatic in older children and adults. In low-transmission settings such as southeast Asia, the proportion of asymptomatic carriers is much larger than previously appreciated. Although substantial evidence from high-transmission settings suggests that persistence of untreated asymptomatic Plasmodium falciparum infections is an important source of transmission, this is not known for low-transmission settings.We searched PubMed and Google Scholar for studies on persistence and oscillations of Added value of this studyP falciparum, Plasmodium vivax, or both, who were followed up for 24 months and documents the frequent persistence of infections over extended periods. The study shows large fluctuation in parasite densities during a single episode, oscillating by orders of magnitude over time, with a substantial proportion of participants developing symptomatic disease.The limitations of contemporary cohort studies on the persistence of plasmodium carriage include the challenges of frequent sampling over longer periods, as well as the limited sensitivity of conventional diagnostic tools  to detect very low-density parasitaemias. We used an ultrasensitive quantitative PCR method, which has a much lower limit of detection. Our study reports on a cohort of asymptomatic participants infected with Implications of all the available evidencePlasmodium spp reservoir could have a crucial role in sustaining malaria transmission in low and seasonal transmission settings and can explain the seasonal malaria increase after the dry season. Screening interventions that rely on standard diagnostic tools such as rapid diagnostic tests will not identify a large proportion of the asymptomatic low-density plasmodium reservoir. Targeting the entire asymptomatic plasmodium reservoir with antimalarial treatment is important to accelerate malaria elimination.The submicroscopic, asymptomatic Here, we explore the persistence of plasmodium infection parasitaemia and density levels over a 2-year study period in a large cohort of asymptomatic malaria-infected participants in Vietnam.P falciparum infections. Villagers found to be infected with P falciparum, P vivax, mixed P falciparum and P vivax, or unidentified Plasmodium spp during baseline mass drug administration or follow-up surveys were invited to join the study.This was an open cohort study nested in a village randomised trial in Vietnam to evaluate the effect of mass drug administrations on the prevalence of P falciparum malaria incidence in Dak O commune was 25 cases in 1000 people and the P vivax malaria incidence was 17 cases in 1000 people.P falciparum malaria incidence was 24 cases in 1000 people and P vivax malaria incidence was seven cases in 1000 people.Mass drug administrations were done in two villages in Dak O commune, Binh Phuoc province  and two villages in Phuoc Ha Commune, Ninh Thuan province  in Vietnam. In 2015, the annual Ethical approval for the study was received from the Institute of Malariology, Parasitology and Entomology in Ho Chi Minh City (185/HDDD), the Institute of Malariology, Parasitology and Entomology in Qui Nhon, and the Oxford Tropical Research Ethics Committee (1015-13). Individual written informed consent was obtained from those aged 16 years and older and parental or guardian consent was obtained for children younger than 16 years.P falciparum malaria from study villages. They were embedded in several malaria elimination strategies including community engagement, improved case management, and vector control strategies. The drug regimen consisted of three monthly rounds  of three once per day treatment doses of oral dihydroartemisinin [DHA] and piperaquine (7 mg/kg dihydroartemisinin and 55 mg/kg piperaquine phosphate) combined with a single low dose of oral primaquine (15 mg or 0·25 mg/kg). Frequency and timing of the mass drug administration rounds was based on the modelled maximum effects in transmission reduction and the post-treatment prophylactic effect of piperaquine, which is around 30 days for sensitive strains.P vivax relapse.The mass drug administrations were done with the aim to accelerate the elimination of The surveillance period was 24 months; directly preceding the mass drug administration and then in intervals of 3 months , P vivax (microsatellite marker 3.502), and Plasmodium malariae (18s rRNA).Plasmodium spp). We defined a high-density infection based on observed distributions as any infection with more than 200 000 parasites per mL.Blood samples were stored in cooling boxes in the field and then transported within 6 h to the local laboratory for further sample processing. Sample processing for the quantitative uPCR included separation of plasma, buffy coat, and packed red blood cells, which were frozen and stored at −80°C. The frozen samples were shipped to Ho Chi Minh City, Vietnam for DNA extraction and quantitative uPCR. Detailed description, evaluation, and validation of the high-volume ultrasensitive quantitative uPCR methods have been reported previously.Plasmodium species can transit between a monoinfection, a mixed species infection, and an infection in which the Plasmodium species cannot be identified. To follow the progress of infections over time, we grouped P falciparum monoinfections with mixed infections  and unidentified species occurring before or after a P falciparum infection. P vivax monoinfections were grouped with P falciparum and P vivax mixed infections and unidentified species. The use of group definitions results in an overlap between the P falciparum and the P vivax group infections  of 1874 were P falciparum infections, 507 (27%) were P vivax infections, and 463 (25%) of 1874 were co-infected with P falciparum and P vivax. The species of the remaining 225 (12%) of 1874 could not be identified.Between Dec 1, 2013, and Jan 8, 2016, 356 participants were recruited for this study. The cohort included 356 participants with parasitaemia; the median age was 35 years (IQR 25·3–45·3) and 238 (67%) participants were male . The cohP falciparum monoinfections, but not for the other species, had significantly higher parasite densities at the time of treatment (mean 652 497 per mL) than untreated cohort members . Cohort participants with an increasing number of episodes of parasitaemia were more likely to receive treatment (test for trend p<0·0001). Participants who tested positive only once for P falciparum had a 33% chance of being treated, whereas participants with 12 positive episodes had an 88% chance of being treated once or more. 178 (50%) of 356 individuals participated in all three rounds of the mass drug administration, 82 (24%) in two rounds, 50 (14%) in one round, and two (1%) did not participate in the mass drug administration.Of 356 cohort members, 218 (61%) remained afebrile, 94 (26%) had one episode of fever, 31 (9%) had two episodes, eight (2%) had three episodes, and five (1%) had four fever episodes during the 24-month study period. Parasite densities of febrile participants were higher than non-febrile participants , or unidentified species during the 24-month study period ; and after accounting for censoring, participants had a 20% chance of having parasitaemia for 4 months or longer. There was no correlation between the age of participants and the duration of episodes . The longest episode in the P falciparum group lasted for 11 months and the duration of the 10% longest persistent episodes ranged between 3 and 11 months. The median duration of P vivax was 6 months (IQR 3–9), and participants had a 59% chance of carrying detectable parasites for 4 months or longer. The longest P vivax group episode lasted for 20 months; the duration of the 10% most persistent episodes ranged from 5 to 20 months . The proportion of participants with long durations of plasmodium episodes (>5 months) was not significantly different in male and female participants (p=0·09). The age of participants with episodes of 6 months or longer were not significantly different in age from participants with episodes of shorter duration .The median duration of 0 months . After aduration . Higher P falciparum or P vivax infections are shown in P falciparum densities varied with the duration of infection  and increased to log 6·5 (3·8–9·5) in individuals who had P falciparum for 6 months. P vivax densities increased from log 4·3 to log 6·5 in individuals who had parasitaemia for 2 months or 3 months or longer, but decreased to log 4·6 (4·3–8·2) in individuals who had P vivax for 5 months.Parasite densities oscillated over time, and ultralow-density infections frequently evolved into higher density infections. 16 (7%) of 222 untreated infections changed to a density of more than 200 000 parasites per mL. Variations in parasitaemia over time in two individuals with persisting nfection . The medP falciparum and P vivax infections in a cohort of villagers in Vietnam. The median duration of P falciparum and P vivax infections was 1 month, but 9% of participants with P falciparum infections and 46% of participants with P vivax infections had infections lasting 6 months or longer. Overdispersion is a common feature of infectious disease transmission with a small proportion of the population contributing a large proportion of transmission. In the study area in Vietnam, the malaria transmission season is from September to March of the following year. Transmission is almost absent during the intervening months, so persistent carriage of parasites for 6 months or longer in a proportion of individuals could bridge this period and sustain malaria endemicity. The parasite densities of persistent infections varied widely (over six orders of magnitude); high-density infections developed after ultralow-density infections, which are undetectable by traditional diagnostic methods . This pattern was observed for both P falciparum and P vivax infections, with parasite densities varying between 102 and 108 times over time. A significant correlation between parasite density and duration of infection was observed for P falciparum and P vivax. The probability that a high-density infection was detected during persistent infections increased with the number of observations. 7% of the untreated ultralow infections escalated into high-density infections that threaten the host, as well as the community because of their transmissibility. An untransmissible infection at one time could become a transmissible infection weeks later. It is difficult to imagine that malaria parasites have evolved sophisticated mechanisms to ensure long-term persistence in their hosts if this did not translate into a transmission advantage.In this study, we found frequent persistent P vivax infections is well known. P vivax hypnozoites trigger relapsing infections that can be cleared by radical cure, which must include a full course of an 8-aminoquinoline . Although the persistence of P falciparum infections is less well described, especially in low-transmission settings, there is an extensive literature from malaria therapy of neurosyphilis suggesting that persistent infections for longer than a year are not unusual.P falciparum infections of up to 13 years have been reported.The extended persistence of P falciparum and P vivax infections, most notably studies of malaria therapy,Plasmodium spp infections, and therefore did not have immunity. The initial inoculation of P falciparum or P vivax in malaria therapy tended to result in high parasite densities, which patients controlled with or without antimalarial treatment . Conversely, the Vietnamese study population has been previously exposed to plasmodium infections potentially resulting in premunition. In this pre-exposed population, ultralow-density infections can persist undetected for extended periods followed by wide oscillations above 10 million parasites per mL.Previous studies of persistent This study has several limitations. First, cohort members were sampled at monthly intervals. Parasite density changes that occur more frequently could have been missed. Ultralow-density infections can take 2–3 weeks to reach high densities detectable by standard diagnostic tools. Thus, most large oscillations in parasite densities can probably be detected by monthly surveys. Second, the preceding period of carriage could not be assessed in individuals with parasitaemia identified at the first survey, which could have caused underestimation of the period of carriage reported in this study. Third, based on the available data, it is not possible to exclude serial reinfections as a cause of the observed persistence in parasitaemia. However, this is unlikely in a very low endemicity setting such as the study sites. Genotyping was not possible due to insufficient quantities of parasite nucleic acid. Fourth, treatment of symptomatic participants required censoring as artemisinin-based combination therapies permanently clears susceptible schizontocytes. Treatment-seeking behaviour, prescription practices, and adherence to follow-up vary between communities and regions limiting the generalisability of the study findings.P falciparum infections than this study. However, in the Pailin study, the number of P falciparum-infected individuals was low (n=32), and initial parasitaemias were lower . Undocumented treatment can also not be excluded in the Pailin study site.Plasmodium spp infections depends on the evasion of adaptive immunity by antigenic variation over time.P falciparum in Pailin province, with only a few parasite strains circulating, might increase the likelihood of strain-specific immunity in the host, facilitating parasite clearance without antimalarial treatment.20 years ago, Farnert and colleagues found daily changes in parasite densities determined by microscopy over a 14-day follow-up period in rural Tanzania.P falciparum and P vivax infections with oscillating densities was observed in this study population over 2 years of follow-up. The submicroscopic, asymptomatic plasmodium reservoir might have a crucial role in sustaining transmission of malaria and probably explains the seasonal malaria increase after the dry season in settings like southern Vietnam. Screening interventions that rely on standard diagnostic tools such as RDTs will not identify most of the asymptomatic low-density plasmodium reservoir. Even highly sensitive tests will not identify all parasitaemic individuals. Targeting the asymptomatic plasmodium reservoir is likely to be important to accelerate malaria elimination.In conclusion, a pattern of persistent This online publication has been corrected. The corrected version first appeared at thelancet.com/infection on April 19, 2018