Plasmodium falciparum apical membrane antigen 1 (AMA1) elicited strain specific efficacy in Malian children that waned in the second season after vaccination despite sustained AMA1 antibody titers. With the goal of identifying a humoral correlate of vaccine-induced protection, pre- and post-vaccination sera from children vaccinated with the AMA1 vaccine and from a control group that received a rabies vaccine were tested for AMA1-specific immunoglobulin G (IgG) subclasses  and for antibody avidity.A malaria vaccine based on P. falciparum parasite density measured for each child as area under the curve were used to assess correlation between IgG subclass antibody titers and parasite burden.Samples from a previously completed Phase 2 AMA1 vaccine trial in children residing in Mali, West Africa were used to determine AMA1-specific IgG subclass antibody titers and avidity by ELISA. Cox proportional hazards models were used to assess correlation between IgG subclass antibody titers and risk of time to first or only clinical malaria episode and risk of multiple episodes. Asexual AMA1 vaccination did not elicit a change in antibody avidity; however, AMA1 vaccinees had a robust IgG subclass response that persisted over the malaria transmission season. AMA1-specific IgG subclass responses were not associated with decreased risk of subsequent clinical malaria. For the AMA1 vaccine group, IgG3 levels at study day 90 correlated with high parasite burden during days 90–240. In the control group, AMA1-specific IgG subclass rise and persistence over the malaria season was modest and correlated with age. In the control group, titers of several IgG subclasses at days 90 and 240 correlated with parasite burden over the first 90 study days, and IgG3 at day 240 correlated with parasite burden during days 90–240.Neither IgG subclass nor avidity was associated with the modest, strain-specific efficacy elicited by this blood stage malaria vaccine. Although a correlate of protection was not identified, correlations between subclass titers and age, and correlations between IgG subclass titers and parasite burden, defined by area under the curve parasitaemia levels, were observed, which expand knowledge about IgG subclass responses. IgG3, known to have the shortest half-life of the IgG subclasses, might be the most temporally relevant indicator of ongoing malaria exposure when examining antibody responses to AMA1.The online version of this article (10.1186/s12936-019-2637-x) contains supplementary material, which is available to authorized users. A malaria vaccine is regarded as an essential tool towards the global eradication of malaria, and short of this ambitious goal, an effective blood stage malaria vaccine would prevent disease and death , 2. PredA adjuvant system (FMP2.1/AS02A) in Malian children. Children who received the vaccine had strain-specific protective efficacy and had increased AMA1 titers that were sustained for 24 months; however, no clinical efficacy was observed in the second season after vaccination, even though titers remained elevated [A previous clinical trial tested a blood stage malaria vaccine composed of a recombinant apical membrane antigen 1 (AMA1) formulated with the AS02elevated . The shoTotal immunoglobulin G (IgG) is comprised of IgG subclasses named in order of relative abundance, IgG1, IgG2, IgG3, and IgG4 . DiffereAnother humoral immune response characteristic that might influence protection is binding strength of antibodies to their targets, or avidity. This measure of intrinsic antibody function may distinguish effective from ineffective responses when total immunoglobulin quantities are equivalent. Avidity has been shown to differ across malaria transmission settings and to be associated with different clinical presentations , 16. LowThe hypothesis explored was that increased levels of cytophilic antibodies (IgG1 or IgG3) in malaria vaccine recipients and increased antibody avidity would be associated with protection from clinical episodes of malaria.P. falciparum malaria from July to November with a peak of up to 60 infective mosquito bites per person per month in August or September [A, which comprises recombinant AMA1 based on the 3D7 P. falciparum strain formulated with GSKBio’s adjuvant system AS02A, or unadjuvanted rabies vaccine as a control, as three vaccinations given intramuscularly 1 month apart. Participants were followed for 2 years. The primary endpoint was a clinical episode of malaria, defined as fever (axillary temperature of ≥ 37.5 °C) with P. falciparum density of ≥ 2500 parasites/mm3 visualized on a thick blood smear. Efficacy was measured 6 months after final vaccination, or day 240. The primary analysis was of the first clinical malaria episode in an individual; a secondary analysis included multiple episodes in an individual. Exploratory efficacy endpoints included cumulative asexual P. falciparum parasite density measured for each child as the total area under the curve (AUC).The study was conducted in Bandiagara , a rural town in Mali, West Africa that has intense seasonal transmission of eptember . A randoeptember . BrieflyA recipients and 10 controls who received rabies vaccine were randomly selected from all 400 participants for whom the previously measured day 90 total IgG titer was in the 25th–75th percentile for their respective groups . Avidity and IgG subclass ELISAs were performed for samples from day 0, 90, and 150 for these subjects.Ten FMP2.1/AS02A recipients and 30 controls who had not already been included in the pilot study and from whom sera from appropriate time points were readily available  were randomly chosen from the study population. Evaluable data was obtained from 85 vaccinees and 29 controls. Blinding to vaccine assignments was maintained until all assays were complete. IgG subclass ELISAs were performed on samples from days 0, 90, and 240.90 FMP2.1/AS02A, a merozoite surface protein-1 vaccine [A pool of sera from fifteen semi-immune adults obtained during peak malaria season who participated in a Phase 1 clinical trial of FMP1/AS02 vaccine , was useNinety-six-well round-bottom Immulon 2 plates  were coated with 50 µg of FMP2.1 diluted in phosphate buffered saline (PBS) to a total volume of 100 µL per well and incubated at 37 °C for 3 h. Plates were washed with PBS 0.05% Tween-20 (PBST) six times by a Biotek ELx405 automated plate-washer , blocked with 10% nonfat milk by mass  in PBS, and incubated at 4 °C overnight. After washing, two-fold serum dilutions were tested in duplicate in PBS 0.05% Tween-20 with 10% nonfat milk (PBSMT). Plates were incubated for 2 h at 37 °C and then washed. For avidity assays, 100 µL of 5 M urea in PBST or 100 µL of PBST were added to wells and allowed to incubate at room temperature for 30 min. After washing, 100 µL of horse radish peroxidase (HRP)-conjugated goat anti-human IgG diluted with PBSMT to 1:10,000  for avidity assays or subclass-specific HRP-conjugated sheep-anti-human secondary antibodies  diluted in PBSMT at concentrations of 1:400, 1:200, 1:250, and 1:100 for IgG1, IgG2, IgG3, and IgG4, respectively, were added to all wells. Plates were incubated at 37 °C for 1 h and then washed. For developing, 3,3′,5,5′-tetramethylbenzidine  was allowed to equilibrate to room temperature and 100 µL was added to each well. Plates were placed in the dark for 15 min. The reaction was stopped with 100 µL per well of 1 M phosphoric acid, and optical densities (OD) were read by a Spectramax M2 plate-reader  at 450 nm. Titers were calculated from linear regression curves created from positive control standards run on each plate, and defined as the reciprocal of the serum dilution that produced an OD of 0.2 above the blank. Avidity index was calculated as the ratio of the titer of the urea assay to the PBST assay. Cytophilic ratios were calculated as the sum of IgG1 and IgG3 titers divided by the sum of IgG2 and IgG4 titers.10-transformed IgG subclass titers, cytophilic ratios, avidity indices as well as differences between time points for these variables were compared using a two-tailed two-sample student’s t-test; a Kruskal–Wallis test was used for comparisons among age strata.Log10-transformed IgG subclass antibody titer as the explanatory variable, day of episode as the censoring variable, and survival to episode as the response variable. The models were fit with and without age as a covariate. The log-transformed titer was used to reduce the influence of extreme values. Subjects who had a clinical malaria episode or any parasitaemia  before day 90 were excluded from the models, as the day 90 antibody responses would reflect both vaccination and infection. As a post hoc assessment of statistical power, for the AMA1 vaccine group, minimum detectable hazard ratios were calculated that took into account the number of clinical malaria illnesses (events) and standard deviations of each subclass IgG titer. The study had 80% power to detect a significant association between IgG subclass titer and hazard of clinical malaria episode when the hazard ratio varied from 1.61 to 4.03 (for risk factors), or from 0.62 to 0.25 (for protective factors), depending on the subclass titer and time point.For analysis of time to first or only clinical malaria episode, Cox proportional hazards models, using the Firth penalized likelihood to reduce bias , were fi10-transformed day 90 IgG-subclass titer with clinical malaria [For analysis of multiple episodes, a Cox proportional hazards gap time model with common coefficients for each episode, using the Firth penalized likelihood function, was used to assess the association of the log malaria .P. falciparum parasitaemia measured as AUC was estimated using the trapezoidal rule for each participant from day 90 to day 240 (AUCd90–240) and for control volunteers from time of enrollment to day 90 (AUCd0–90). As specified in the statistical analysis plan of the study protocol, all recorded episodes of parasitaemia were included regardless of symptom presence or absence, and parasite density was assumed to decline linearly to zero 3 days after a treated malaria episode [d90–240 for the malaria vaccine group and the control group and AUCd0–90 for the control group, and Spearman’s rank correlations were calculated.Asexual  episode . IgG subStatistical analysis was performed using SAS software, version 9.4 for Windows ; GraphPad Prism version 6.0 for Windows ; and R version 3.4.4 with RStudio version 1.0.136 and packages tidyverse, ggplot2, ggbeeswarm, scales, and grid. Results with a P-value of less than 0.05 were considered statistically significant. No adjustment was made for multiple comparisons.In children immunized on days 0, 30 and 60 with either the AMA1 vaccine or rabies vaccine as a control, avidity levels did not change significantly from day 0 to day 90 or from day 90 to day 150 and did not differ when comparing 10 AMA1 vaccinees to 10 children from the control group . In addition, to ensure that each day 90 subclass titer reflected AMA1 vaccination only and not exposure to natural infection including asymptomatic parasitaemia, a model that excluded all parasitaemias before day 90 was also explored in the AMA1 vaccine group (n = 49). For all models tested in the malaria vaccine and control groups, no log-transformed subclass titer predicted decreased time to first or only clinical malaria episode Table .Table 2CPlasmodium falciparum parasite densities measured as area under the curve (AUC) were calculated to study correlations between parasite burden and IgG subclass levels. In the malaria vaccine group, not including those with clinical malaria episodes between days 0 and 90 (n = 66), the AUCd90–240 correlated positively with IgG3 at day 90 and IgG3 at day 240. In contrast, AUCd90–240 did not have a statistically significant correlation with any IgG subclass at any time point in the control arm when those with clinical malaria episodes between days 0 and 90 were excluded (n = 22)  and then waned by day 240, but remained ≥ 1 log10 higher than pre-vaccination antibody responses. In contrast, over the malaria transmission season, the rabies vaccine control children had a steady increase in IgG subclass antibodies that remained 1–2 log10 lower than the antibody responses in the AMA1 vaccine group, representing the naturally occurring immune response to a blood stage malaria antigen over a malaria transmission season. In the control group, IgG subclass titers correlated with increasing age, reflecting cumulative acquisition of immunity. In the AMA1 vaccine group, these effects were not observed, presumably because the adjuvanted vaccine increased titers to all subclasses globally, such that the youngest children’s titers approached the levels of the oldest children. Indeed, children who received FMP2.1/AS02A had a several-fold lower baseline than adults residing in the same area, but after vaccination they had total IgG titers that were similar in magnitude to those of adults who received the same vaccine [P. falciparum exposure.In this analysis of IgG subclass antibodies elicited in response to an AMA1 vaccine, the vaccine elicited robust, ≥ 2-log vaccine , 23—thusThe original hypothesis of this study was that increased levels of cytophilic antibodies (IgG1 or IgG3) in malaria vaccine recipients would be associated with protection from clinical episodes of malaria. However, increased IgG subclass titers were not associated with decreased risk of clinical episodes among children who received the AMA1 vaccine. By examining antibody responses in the control group, the effect of natural malaria exposure on subclass IgG titers was explored; however, no associations between day 90 (peak season) IgG subclass titers and risk of subsequent clinical malaria illness were identified.P. falciparum and subclass immunoglobulin titer was assessed by estimating the parasite burden for each volunteer, expressed as AUC parasitaemia. In the AMA1 vaccine group, for children who did not have clinical malaria episodes within the first 90 days (n = 66), AUCd90–240 correlated with increased IgG3 at the day 90 and day 240 time points, suggesting that IgG3 levels in the AMA1 vaccine group correlate with exposure to parasites. One explanation is that increased IgG3 levels at day 90 indicate ongoing malaria exposure and counterintuitively predict infection in the ensuing 150 days. This correlation was not seen in the control group; the smaller sample size of the control arm might have limited the power to detect this association.The relationship between exposure to d0–90 (rather than AUCd90–240), an exposure interval not explored in the AMA1 vaccine group because vaccination occurred within the 0–90 day interval. In a pre-planned exploratory analysis of the original vaccine trial, median AUC was lower in vaccines than controls, suggesting an overall reduction in parasite burden that did not translate into a statistically significant reduction in the hazard of clinical malaria episodes [d0–90 support further evaluation the utility of AUC as a clinically relevant readout of malaria exposure. The finding that IgG3 at day 90 correlated with AUCd90–240 in both the AMA1 vaccine and control arms suggests that IgG3, which has the shortest half-life of the IgG subclasses, might be the most temporally relevant indicator of ongoing exposure. Thus IgG3 may be worthy of exploring as a biomarker for surveillance to detect populations experiencing ongoing or very recent exposure to help target malaria elimination interventions.When the association between IgG subclass titer and AUC parasitaemia in the control arm without censoring subjects who had clinical infections in the first 90 study days was examined, correlations were identified between IgG subclass titers and parasite burden attributable to natural exposure. These correlations were observable because of the larger sample size (n = 29 vs. n = 22) and examination of AUCepisodes . In the In other infectious diseases, humoral immune responses have served as correlates of protection , 25. In A vaccine across all subclasses, and are among a handful of descriptions of antibody subclass and avidity responses to a candidate malaria vaccine. Although a correlate of protection based on antibody subclass or avidity was not observed for this vaccine, antibody subclass levels or avidity could be associated with protection for other malaria vaccines that exert broader efficacy. The lack of correlation of avidity or antibody subclass titer with protection despite the FMP2.1/AS02A vaccine eliciting strain-specific efficacy, and the lack of correlation of a growth inhibition assay with protection [Overall, the results from this study demonstrate the robust humoral response elicited by the FMP2.1/AS02otection  suggest otection , and traotection , 39.IgG subclass titers were not associated with vaccine-induced protection from subsequent malaria illness. However, older age correlated with increased IgG subclass titers in the control arm and not in the AMA1 vaccine arm, which reflects IgG subclass response to natural infection in the former and the adjuvant effect in the latter. IgG subclass titers were a marker of exposure that correlated with AUC parasitaemia levels. An association between IgG3 and subsequent parasite burden suggests that of the IgG subclasses, IgG3 may be the most temporally relevant indicator of ongoing malaria exposure when assessing AMA1 responses, and worthy of further exploration as a biomarker for surveillance in support of elimination efforts.Additional file 1. AMA1 IgG subclass titers, total IgG titers, and cytophilic ratios for AMA1 vaccine and control groups.Additional file 2. Effect of age strata on IgG subclass titer.Additional file 3. Spearman’s Rank correlations of AMA1 IgG subclass titers grouped by time point and vaccine group.


In resource-poor settings, transfused children often experience recurrence of severe anemia (SA) following discharge from hospital. This study determined the factors associated with recurrent severe anemia (RSA) among previously transfused Ugandan children aged less than 5 years.A case-control study was conducted in five hospitals in Uganda from March 2017 to September 2018. We prospectively enrolled 196 hospitalised children who had been transfused for severe anemia 2 weeks to 6 months prior to enrollment. Of these, 101 children (cases) were re-admitted with a hemoglobin [Hb] level of ≤6 g/dL and required transfusion; and 95 children (age-matched controls) were admitted for other clinical illness with a Hb > 6 g/dL. Children known to have sickle cell anemia, cancer, or bleeding disorders were excluded. Clinical and laboratory evaluation were done. Conditional logistic regression adjusted for age, was used to determine factors associated with RSA.p-value = 0.015). Risk factors  for development of RSA were: hemoglobinuria ; sickle cell anemia – newly diagnosed ; history of earlier previous transfusions  and malaria infection .The median time (IQR) between the earlier transfusion and enrollment was 3.5 (1.9–5.7) months for cases, and was 5.0 (2.9–6.0) months for controls (Malaria chemoprevention, follow up visit for Hb check after discharge from hospital and sickle cell screening among previously transfused children represent practical strategies to prevent and identify children at risk for recurrent severe anemia. The cause of hemoglobinuria in children merits further investigations. Recent studies have indicated that up to 10% of children who receive blood transfusion for severe anemia (SA) return to health facilities with recurrence of severe anemia within three months of discharge, while others die at home [Malaria infection in the immediate post-discharge period has been identified to be a major contributing factor to RSA in children . Other dA better understanding of the risk factors for development of RSA can identify children at highest risk and can enhance the benefits of blood transfusion in children , 6. We cThis was an age-matched study with a ratio of 1:1 between cases and controls.The study was conducted at Jinja, Masaka, Hoima, Mubende and Kamuli hospitals, in Uganda. The first four are all public regional referral hospitals serving east-central, south-central, north-central, and western sub-regions of Uganda respectively, while Kamuli mission hospital is a private not-for-profit hospital located in eastern Uganda. Each of the hospitals has a pediatric ward with an in-patient bed capacity ranging from 30 to 80.We enrolled children aged 2 months to 5 years with prior severe anemia (cases and controls) that required a blood transfusion, which was given > 2 weeks but < 6 months prior to enrollment in the present study. Cases were defined as children who at the time of study enrollment were re-admitted to the hospital with a hemoglobin level of ≤6 g/dL and required blood transfusion. Controls were defined as children who at the time of study enrollment were being seen for other clinical illness as an inpatient or outpatient, and had a hemoglobin level of > 6 g/dL. Cases and controls were matched using an age range of ±12 months. Children known to have sickle cell anemia (SCA), cancer, bleeding disorders or whose anemia was caused by trauma, were excluded.Phiri KS et al 2008 [The sample size of 196 was estimated using Open EPI calculator; using malaria as the main risk factor with a prevalence of 29.3% among cases and 12.1% among controls, according to a study by Clinical evaluations included the past medical history , socio-family history and a detailed physical examination and anthropometric measurements . Socio-demographic data collected included; sex, age in months, age and occupation of caregiver, number of household members, number of children in the household, number of meals per day, among others. A structured case-report form was used to record study variables.Poorana et al [A blood sample of 2 ml collected in an EDTA tube was taken (for cases - taken off at pre-transfusion). Hemoglobin was measured using a point-of-care device . ABO typing and Rhesus blood grouping was done using commercial reagents available at the hospital blood banks. Complete blood count (FBC) tests were performed using the Mindray automated haematology analyzer . Sickle cell status  was determined by capillary hemoglobin electrophoresis assay . For patients returning within two months since previous transfusion, hemoglobin electrophoresis was deferred for at least two months. HIV serology was tested using HIV-1/2 test strips . Urine analysis was done using URS-10 T reagent test strips . Malaria thick-smear was stained using field stain A and B. Malaria rapid diagnostic test (RDT) was done using SD Bio-line malaria Ag P.f/Pan test strips . A reticulocyte count (%) was performed from a fresh (within 2 h) sample, using a thin smear stained with new methylene blue, and the reticulocyte production index – RPI (corrects for the degree of anemia) was calculated using the method of na et al . Some teThe presence of asexual forms of plasmodium species on a thick malaria smear or a positive malaria RDT defined the diagnosis of malaria, while hemoglobinuria was defined by both a history of passage of dark or red-colored urine, and confirmed evidence of ‘blood’ at urine dip-stick. Sickle cell anemia and sickle cell trait were defined as the presence of Hb-SS and Hb-AS, respectively. Suspected bacteremia was a clinical diagnosis backed by laboratory evidence neutrophilia on FBC. Mean cell volume (MCV) < 70 fL defined microcytosis, while MUAC of ≤12.5 cm defined malnutrition  .p-values are presented from conditional logistic regression adjusted for children’s age as matching variable. The stepwise backward model building technique was followed to identify significant factors after adjusting for factors with p values < 0.2 for consideration into the multivariable model. A p < 0.05 was considered statistically significance. All p-values presented are two sided.Data were entered into EPI-DATA version 3.1 software package  and analysed using STATA v14.0 . We computed descriptive statistics and present, medians (interquartile range), proportions for the demographic characteristics by case or control status. Association between categorical variables was assessed using odds ratios and statistical significance determined using the McNemar test. Means of symmetrical continuous variables were compared using the paired t-test. The difference in median time from earlier transfusion to enrollment was evaluated with a Wilcoxon rank sum test. 95% test-based confidence intervals for odds ratios and p-value = 0.015) .A total of 101 cases and 95 age-matched controls enrolled in the study were included in the analysis. Kamuli study site had slightly more cases than controls  was HIV infected.p-value < 0.2 at bivariable analysis  were entered into a stepwise backward conditional logistic regression model. After controlling for all ten variables, we found that hemoglobinuria, sickle cell anemia (Hb-SS), history of earlier previous blood transfusions (in the period > 6 months), and malaria diagnosis were each significantly associated with RSA  that of females.There were 10 (23.3%) cases and 1 (2.6%) control female participants compared to 28 (48.3%) cases and 4 (6.9%) control male participants with hemoglobinuria respectively. However at logistic regression, the interaction between hemoglobinuria and sex was not statistically significant. The odds of a participant with hemoglobinuria being male were; AOR = 1.27 and malaria are risk factors for recurrent severe anemia. In addition, history of other previous admissions, and Artemisinin-based combined therapy (ACTs) use prior to admission on the current illness are independently associated with RSA. In contrast, mother’s age ≥ 29 years and a diagnosis of malarial anemia at the most recent admission seem protective. These findings are comparible to the findings of It is worth noting that 75.0% of cases had a diagnosis of malarial anemia at the most recent prior admission – in the immediate past six months, before returning with RSA. This underscores the role of malaria in the etiology of severe anemia among children in malaria endemic areas . AlthougOlopot-Olupot P et al did not find this to be so.This study has found hemoglobinuria (defined by history of passage of dark or red-colored urine and the presence of blood at urine dip-stick) to be associated with RSA. This syndrome whose cause is not well understood has recently been documented to be common in the eastern region of Uganda. The syndrome has been associated with SA and positive malaria RDT  but not with sickle cell disease or G6PD-deficiency . AlthougSimilarly, in our case-control although we did not perform G6PD assays, we found that the odds of a participant with hemoglobinuria being male were not statistically significant.The potential relationship between hemoglobinuria and prior use of ACTs and/or other genetic factors in the causation of RSA need to be confirmed. Other reports have shown the possible role of ACTs-related delayed hemolytic anemia in causing RSA .Phiri KS et al 2008.Although the reticulocyte production index was not statistically lower among cases, the potential role of insufficient erythrocyte production predisposing to RSA cannot be excluded for reasons of the fewer numbers we tested. What also remains unknown is how malarial anemia at the most recent admission may be protective against RSA, yet malaria itself is implicated in causing both SA and RSA. This paradoxical finding has also been reported by Phiri KS et al where HIV infection was associated with RSA, we found only one participant – a control to be HIV positive. This may be explained by the marked progress made with regard to elimination of mother to child transmission of HIV in Uganda [Nutritional status as measured by the MUAC and socio-economic factors such as occupation of caregiver, highest education level of mother, number of children in the household, number of meals per day, among others were not associated with recurrent severe anemia. Although this study did not have power to evaluate them, there is evidence to suggest that these factors among others may play a significant role in causing both SA and RSA , 14. Conn Uganda .In summary, recurrent severe anemia among previously transfused children in Uganda occurs after about three months, and is related to hemoglobinuria, sickle cell anemia, history of earlier previous transfusions and malaria infections and/or re-infection.The current list of factors associated with RSA may not be complete. One uncertainty may be the role of socio-demographic factors such as occupation of caregiver, number of household members, number of meals among others that our study did not have power to evaluate. Similarly, the fewer numbers tested for variables such as reticulocyte production index further limits the power.NCT02671175) is currently testing the hypothesis that malaria is a key factor in the cause of post-discharge mortality and morbidity in children with severe anemia. The results of that trial are eagerly awaited. However, there is need to address the other risk factors for RSA; such as screening for SCA among all children with SA and a follow up visit between 2 weeks and 3 months for Hb check after discharge from hospital (the best timing for this check remains yet to be determined). Generally, the problem of recurrent severe anemia among children merits further investigation, including areas such as the cause of hemoglobinuria and its potential relationship with prior ACTs use.Evidence based interventions are needed to prevent and mitigate the problem of recurrent severe anemia among children. The post-discharge malaria chemoprevention trial (


The acquisition of immunity to malaria by an individual depends on their age and the number of infectious mosquito bites they have received. Related research article Rodriguez-Barraquer I, Arinaitwe E, Jagannathan P, Kamya MR, Rosenthal PJ, Rek J, Dorsey G, Nankabirwa J, Staedke SG, Kilama M, Drakeley C, Ssewanyana I, Smith DL, Greenhouse B. 2018. Quantification of anti-parasite and anti-disease immunity to malaria as a function of age and exposure. eLife7:e35832. doi: 10.7554/eLife.35832.In a league table of infectious diseases, malaria would rank above all others in how often it causes infections and the number of parasites in infected hosts. In areas that are particularly favourable for malaria transmission, a single person can be infected more than 1,000 times per year . Each inAlthough every new infection boosts immunity to further infections, this comes at a high price: in some areas malaria can kill up to 5% of children before their fifth birthday . Adults Rodriguez-Barraquer et al. looked at data from 773 children aged between six months and 10 years old in three Ugandan villages. Each child was monitored for up to three years in an attempt to understand the relationships between their age, how often they were infected, the number of parasites in their blood, and the number of parasites required to cause symptoms of malaria. A child was considered to have developed a case of clinical malaria if they had a body temperature above 38°C, and if the density of parasites in their blood was high enough to be seen under a microscope. Alongside clinical follow-up, there was an accompanying effort to catch mosquitoes in the children’s houses. This allowed exposure to be measured in terms of the number of infectious mosquito bites the children received per year.The villages had varying levels of malaria, with children experiencing an average of 22 bites and two cases of clinical malaria every year . There wRodriguez-Barraquer et al. analysed data on two aspects of immunity – anti-parasite immunity (which reduces the number of parasites in the blood) and anti-disease immunity  developed immunity more efficiently than children in villages with moderate transmission rates (who received three times more bites). The average child coming to the health clinic in the low transmission village had both a lower parasite density and a lower body temperature than their counterpart from the moderate transmission village.This is a counter-intuitive but statistically robust finding. If replicated elsewhere, it has important implications. Traditionally it has been assumed that as transmission rates decline, so does immunity, presenting an obstacle to malaria control . HoweverThe latest analysis also suggests future avenues of research. Asymptomatic infections, and those that do not reach sufficiently high density to be seen under a microscope, could be detected using PCR (polymerase chain reaction) techniques. Exposure could be measured more accurately by using genotyping to count the number of parasite clones circulating in the host . MeasureLevels of immunity are not just dependent on age and exposure, but also on the cumulative number of episodes of clinical malaria that an individual has experienced . Other d


Plasmodium falciparum and, to a lesser extent to Plasmodium malariae and Plasmodium ovale. Plasmodium vivax is thought to be absent in Nigeria in particular and sub-Saharan Africa in general, due to the near fixation of the Duffy negative gene in this population. Nevertheless, there are frequent reports of P. vivax infection in Duffy negative individuals in the sub-region, including reports from two countries sharing border with Nigeria to the west (Republic of Benin) and east (Cameroon). Additionally, there were two cases of microscopic vivax-like malaria from Nigerian indigenous population. Hence molecular surveillance of the circulating Plasmodium species in two states (Lagos and Edo) of southwestern Nigeria was carried out.Malaria in Nigeria is principally due to Plasmodium species as well as sequencing of a portion of the promoter region of the Duffy antigen receptor for chemokines. Samples positive for P. vivax were re-amplified several times and finally using the High Fidelity Taq to rule out any bias introduced.A cross-sectional survey between September 2016 and March 2017 was conducted. 436 febrile patients were included for the present work. Venous blood of these patients was subjected to RDT as well as microscopy. Further, parasite DNA was isolated from positive samples and PCR diagnostic was employed followed by direct sequencing of the 18S rRNA of P. falciparum was, as expected, the major cause of infection, either alone 85.5% , or mixed with P. malariae 6.3% (16/256) or with P. vivax 1.6% (4/256). Only one of the five P. vivax isolates was found to be a single infection. DNA sequencing and subsequent alignment of the 18S rRNA of P. vivax with the reference strains displayed very high similarities (100%). Remarkably, the T-33C was identified in all P. vivax samples, thus confirming that all vivax-infected patients in the current study are Duffy negative.Of the 256 (58.7%) amplifiable malaria parasite DNA, P. vivax in Nigeria in Duffy negative individuals. Though restricted to two states; Edo in South–South and Lagos in South-west Nigeria, the real burden of this species in Nigeria and sub-Saharan Africa might have been underestimated, hence there is need to put in place a country-wide, as well as a sub-Saharan Africa-wide surveillance and appropriate control measures.The present study gave the first molecular evidence of The online version of this article (10.1186/s12936-018-2588-7) contains supplementary material, which is available to authorized users. Malaria is a principal infectious disease that continues to be a worldwide cause of mortality in endemic countries such as Nigeria. Concerted malaria intervention effort has led to a substantial gain in the reduction of global malaria cases and mortality to 41% and 62%, respectively, since 2000 .Plasmodium infections in individuals with high level of parasitaemia, however parasite detection in individuals carrying low parasite density can be challenging [P. ovale from P. vivax) [Malaria diagnosis remains a big challenge in malaria control and elimination and performance of different diagnostic tools differ in different epidemiological settings . Microscllenging , thus em. vivax) , 7, lowe. vivax) , 9, and . vivax) , are allPlasmodium species, with polymerase chain reaction (PCR) being the most frequently used method [In recent times, various molecular techniques have been used to detect d method –13. Addid method –17 as wePlasmodium species, infecting humans [P. falciparum is the most widely spread in Africa and causes the most severe form of the disease worldwide [P. falciparum. This unalloyed attention to falciparum malaria control interventions should be looked into as a result of the following reasons: (i) vivax malaria  is less flexible to manage and may survive for a prolonged period as a result of the hypnozoite stage, thus serving as a reservoir of infection [P. vivax has a larger global distribution, and the number of people at risk of contracting it is more than that of P. falciparum. Hence, its incursion into sub-Saharan Africa (SSA) gives it a much wider distribution from a global perspective [Of the five g humans , P. falcorldwide . Hence, nfection , (ii) P.spective , (iii) lspective .Plasmodium vivax malaria has been presumed absent in SSA due to the fact that the Duffy antigen, the erythrocyte receptor for P. vivax merozoite invasion, is not expressed on the red blood cells of most populations in SSA, as revealed by the seminal work of Miller and colleagues, therefore are thought to be protected against vivax malaria [P. vivax receptor [Plasmodium vivax is predominantly found in Asia, Latin America and the Horn of Africa where the majority of the population is Duffy positive [P. vivax from vivax-like malaria from Africa in non-human primates. However, some scientists have opined that the source of vivax malaria in SSA could be from the different migrational pattern especially along trade pathway [P. vivax to/from Africa need further detailed study. Nevertheless, it seems more than one of the presumptions might be at play here. malaria . Althougreceptor . The proreceptor , 25. Plapositive . Recent positive , 26–37. positive , 39 supp pathway . Althougvivax-infected Duffy positive individuals serving as reservoir of infection for the Duffy negative individuals [P. vivax [Two things are at play from the above: ividuals ; and theP. vivax , 36.Studies conducted in the northern part of Nigeria attempting to characterize Duffy blood groups estimated 19.2% and 5.6% of the general population and pregnant women, respectively, to be Duffy positive , 41, thuP. falciparum accounts for > 95% of malaria infection, while P. malariae and P. ovale are estimated to be responsible for < 5% of infection [P. vivax points to the case of a pregnant Nigerian female, visiting the country briefly from Italy where she resides and was diagnosed of mixed vivax infection [In Nigeria, nfection , 2. To dnfection , and othnfection , 44. Addnfection .P. vivax isolates in the study areas, and to determine the DARC status of P. vivax-infected patients.This study was designed to confirm the different species detected by RDT and microscopy using PCR as the gold standard, to sequence the Samples were collected from Lagos and Edo states, both of which are located on the western and southern part of Nigeria respectively. Lagos state shares a border with the Republic of Benin. In Lagos state, samples were collected from four different Local Government Areas (LGAs) namely Eti-osa (06°26′N 003°29′E), Ibeju (06°26′N 003°56′E), Kosofe (06°28′N 003°22′E) and Ikorodu (06°33′N 003°35′E) LGA, while in Edo state, samples were collected from two LGAs: Oredo (06°19I′N 5°34′E) and Ikpoba Okha (06°16′N 5°68′E).Lagos state is hypo-endemic in most part with a 1.9% prevalence rate in children age 6–59 months , 45. ThiEdo state on the other hand is meso-endemic with a prevalence rate of 35% in children age 6–59 months , 45, 47.Anopheles gambiae sensu stricto (s.s.) and Anopheles funestus s.s [Generally, the rainfall pattern of the two states has little or no variation with an annual rainfall of 1400–1800 mm and a short break called “August break’. There are two climatic conditions predominant in both study locations, the dry season (lasting from November to March) and the wet season (from April to October), with a temperature range of 30–38 °C . Malariastus s.s , 50, 51.The study was a cross-sectional investigation involving patients presenting clinical symptoms of malaria and visiting any of the various hospitals in both study sites between September 2016 to March 2017. The inclusion criteria were patients that were ≥ 2 years of age, clinical symptoms of malaria detected by a febrile condition of ≥ 37.5 °C while the non-inclusion criteria were being pregnant and having complicated infections.P. falciparum specific HRP2 RDT kits . Those found positive by this initial diagnostic test were further screened by microscopy and subsequently by nested PCR. From the pool of samples that were negative by both RDT and microscopy, 136 were randomly selected by taking every fifth, seventh or tenth negative samples (depending on the study location and sample size) and were all subjected to PCR , which only detects the histidine-rich protein 2 (HRP-2) of P. falciparum, was used to carry out a rapid diagnosis following the manufacturer’s instruction, and those samples found positive by this preliminary test were processed further.Venous blood collected from each consenting/assenting patients was kept in ethylene diamine tetra acetic acid (EDTA) containers corresponding to a patient’s unique identity for RDT and subsequent microscopy and PCR analysis. Following the manufacturer’s instruction, Care StartWBCs = number of white blood cells (fixed to 500).Preparation of thin and thick blood films and determination of parasites density followed a previous protocol . Briefly® filter paper  and allowed to dry at room temperature. Each filter paper was kept in a zip-locked sachet with silica gel, stored at room temperature (or at − 20 °C where available) and transferred to Parasitology and Mycology laboratory, Aristide Le Dantec University Hospital, Dakar, Senegal. Following the manufacturer’s instruction, DNA was extracted from three 3 mm punches of DBS using the QIAmp DNA Blood Mini Kit  and eluted in a 100 µl final volume and stored at − 20 °C until ready for use.Blood spots from positive (RDT and/or microscopy) as well as negative (few selected RDT and microscopy) samples collected from consenting patients were made on WhatmannPlasmodium spp. Both PCR targeted the 18 subunit ribonucleic acid (18S rRNA) for both the genus as well as species using previously described protocol [P. vivax was extracted and re-amplified 3 times in order to reconfirm that they are P. vivax following same protocol as above. Additionally, all P. vivax isolates were re-extracted and -amplified using the High Fidelity fusion Taq  to negate any bias likely to be introduced during the amplification process. Clinical isolates of P. falciparum, P. malariae, P. ovale and P. vivax (PCR amplified and confirmed by sequencing) and sterile distilled water were used as the positive and negative controls respectively to validate the nested PCR approach. PCR products in the DNA of a patient’s specimen corresponding to 205 bp , 144 bp , 800 bp  and 120 bp (P. vivax) were confirmed as positive for the respective species . Request was also made for purification of PCR products prior sequencing. For each fragment, sequencing was carried out from both the 5′ and 3′ directions (2× coverage). Consensus sequences from both the forward and reverse sequences were generated using the BioEdit computer software. Multiple sequence alignment of the 18S rRNA of the vivax isolates and the human Duffy gene was done with BioEdit. Sequences generated from the current study were aligned with their respective reference sequences; P. vivax (SAL-1 accession number U03079.1) and accession number NG_011626.30 for the Duffy gene. All references were retrieved from the NCBI website (https://www.ncbi.nlm.nih.gov) by employing the BLAST search.In order to validate the different species identified by PCR in gel electrophoresis, the 18S rRNA gene of P < 0.05 was considered statistically significant.All other data aside the sequences were analysed using Statistical Package for Social Sciences (SPSS) version 21.0 . Furthermore, the performance of each diagnostic test method was calculated by means of sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) using nested PCR as a gold standard. Kappa’s statistics was used to test the level of agreement between the diagnostic tools. For ease of cartographic representation of the diversity of mixed species originating from the different study areas, Eti-Osa and Ibeju were grouped as Lagos Island-owing to their location on the part of Lagos state surrounded by the Lagos lagoon and extending into the Atlantic ocean while Oredo and Ikpoba-Okha were grouped as Edo due to their supposed boundary sharing on the western part of Ikpoba-Okha.From the overall 2376 surveyed study subjects, 436 were included for this study with 39.2% 171/436) of them being from Oredo LGA. The mean age of the study population was 23 years (with a range of 1–85 years). The sex ratio of the overall study population was 0.57  as detected by PCR while the remaining 22 were false positive. Hence, the sensitivity of microscopy was higher (83.7%) than its specificity (52.5%)  was very high (87.1%) while the chances of it being picked as truly negative; negative predictive value (NPV) was very low 57.2%). However, the reverse was the case for microscopy, with the NPV being higher (87.8%) than the PPV (44.1%). Assessing the level of agreement using PCR as the gold standard showed that RDT has a weak agreement (Kappa’s statistic = 0.18) with PCR while microscopy shows no agreement (Table %. HowevePlasmodium 18S rRNA was amplifiable in 58.7% (256/436), 110 from Edo and 146 from Lagos. Majority of the infections were due to P. falciparum either as single infection 85.5% , or mixed with P. malariae 6.3% (16/256), P. vivax, 1.6% (4/256) or with P. ovale 1.2% (3/256). Plasmodium malariae single infection accounted for about 4.7% (12/256) of the total positive samples. Only one of the five P. vivax isolates was found to be a single infection and only one isolates (0.4%) was found with triple infections of P. falciparum, P. malariae and P. vivax. It is interesting to note that all of the P. vivax sample were from microscopically negative slide detected both in Nigeria and Senegal by a trained and WHO level one microscopist respectively.Of the 436 samples collected, the Plasmodium species, Edo state has the highest diversity of mixed infection with all species mono infection  conspicuously represented in addition to mixed P. falciparum/P. malariae, P. falciparum/P. ovale and P. falciparum/P. vivax infection. This is followed by Kosofe and Lagos Island with P. falciparum, P. malariae mono infections as well as P. falciparum/P. malariae mixed infections. The least diversed site by species composition is seen in Ikorodu . The sequences of the 18S rRNA genes, accompanied by multiple sequence alignment with its reference sequences (U03079.1) showed perfect homology (100% similarity)  or heterozygote with the occurrence of double T and C peak. A single peak of C-nucleotide was found at the − 33rd position of the Duffy gene promoter region in both the forward and reverse direction of the sequences (2× coverage) .The sequences of the Duffy gene were trimmed to 620 bp which is more than sufficient to cover the − 33rd nucleotide position. The − 33C mutation was found in all ge) Fig. , indicatP. vivax in Duffy negative individuals, all of which have implications in malaria control programmes.This study assessed the performance of three diagnostic tools in detecting malaria from six endemic settings with varying levels of transmission in Nigeria. Analysis from this study provides the rate of false positives, false negatives as well as the confirmation of PfHRP2 antigen or even a deletion of it entirely from the isolate [P. falciparum of other studies was quite lower than what was obtained here [Firstly, with regards to the false negative rate, an appreciable number of the sub-microscopy carriers (33/136 randomly selected samples detected as negative both by RDT and microscopy) were not treated as they were wrongly diagnosed as negative both by RDT and microscopy. The failure of RDT to detect these individuals as positive on the one hand, could be due to low levels of target  isolate –58 ident isolate . Even wi isolate , 58, 61.ned here , 62, thiPlasmodium species was used to detect single and mixed infections. The study reveals the dominance of P. falciparum both as single or mixed infection with P. malariae, P. vivax or P. ovale. This elevated prevalence of P. falciparum among the overall study population as well as in previous studies from Nigeria [P. ovale mono infection. Cases of mixed P. falciparum and P. vivax infection have been found to lead to significant severe malaria in Australia as well as in Asia [P. malariae and P. ovale mixed  infections respectively might have been underestimated by microscopy in this current study. This is because P. ovale infect reticulocytes which happens to be also among the stage parasitized by P. falciparum. This could have resulted in the mis-identification observed in the current findings [P. ovale and P. falciparum in this present study is in agreement with findings from other parts of Nigeria [In the current study, nested PCR targeting the 18S rRNA gene of  Nigeria , 43, 63  in Asia , 65. Addfindings . The co- Nigeria , 43 wherP. vivax from individuals residing in both Lagos and Edo state of Nigeria. Corroborating the PCR outcomes is the result obtained from the sequencing of P. vivax amplicons and subsequent alignment with the SAL-1 reference strain which gave a perfect homology (100% similarity) with its reference strain, suggesting high reliability of molecular techniques as confirmatory diagnostic tool. Before now, the reports of P. vivax connected to Nigeria are those of a traveller [P. vivax-like malaria [P. vivax detected in the traveller who visited Nigeria originated from Nigeria or it was a relapse of P. vivax, since it is known to cause relapse as a result of the dormant hypnozoite stage weeks or even months after treatment [P. vivax in Nigeria, it is however, not surprising as it has been detected elsewhere in SSA where it was thought to be initially absent [P. vivax infection [Interestingly, the present study report the first molecular evidence of raveller  as well  malaria , 44. Wetreatment  is unknoy absent , 35, 37 nfection , 55.An. gambiae and An. funestus [P. vivax malaria in a study carried out in East Africa [P. vivax can be successfully transmitted from neighbouring African countries especially given the fact that cases of P. vivax have been reported from the Republic of Benin [P. vivax infection due to the near fixation of the Duffy-negative gene in the African population [Additionally, the two dominant vector species responsible for malaria transmission in Nigeria-funestus  have beet Africa , it therof Benin  and Camepulation  should bP. vivax infected patients revealed all of them to be homozygous Duffy negative with a single C-nucleotide peak. The present findings taken together with outcomes from similar studies in other Africa countries suggest that P. vivax might be evolving and adapting new strategies in infecting Duffy-negative individuals, just as similar adaptive strategy was observed in the simian malaria parasite-Plasmodium knowlesi now infecting humans [Interestingly, multiple sequence alignment of the DARC-coding gene of the nine g humans .P. vivax mono and mixed infection with disease clinical outcomes as studies from Asia in both P. vivax mono and mixed infection  [P. vivax in Nigeria. Additionally, though the study was conducted in core areas inhabited by indigenes, the exact travel history of those with P. vivax could not be ascertained, whether they had previously visited a country with circulation of vivax malaria is uncertain. Therefore, there is need for a country-wide survey to ascertain the burden of P. vivax as well as mixed malaria infection in order to put in place adequate control measures.An important limitation of this study is the inability to link ciparum) , 65, 68 P. falciparum [P. vivax to this, will ultimately be a daunting task in the country’s struggle to control malaria. Therefore, the National Malaria Control Programme should initiate a country-wide epidemiological survey of all the circulating species of Plasmodium using very sensitive diagnostic techniques such as PCR and plan control intervention accordingly.Given the fact that Nigeria accounts for 27% of the global malaria burden mostly due to Additional file 1: Table S1. PCR protocol and cycling conditions used for malaria diagnosis of P. falciparum, P. malariae, P. ovale and P. vivax.Additional file 2: Table S2. Primer sequence, PCR protocol and cycling conditions used for Human Duffy antigen receptor for chemokines.


After declining steadily during the early part of the twenty-first century, the worldwide incidence of malaria has recently reached a plateau . FurtherPlasmodium falciparum [P. falciparum in some regions of Africa, India and Peru due to parasite gene deletions [A significant proportion of asymptomatic malaria cases are caused by incipient or otherwise low-level infections that are not readily detectable via microscopy . RDTs malciparum . Furthereletions ,6, raisieletions . These aeletions .Volatile compounds are an under-explored class of potential diagnostic biomarkers that could conceivably be employed for population screening to detect malarial infection. It is possible, for example, to envision the development of an easy-to-use portable device for detecting malaria-specific biomarkers that could be readily deployed in areas with limited medical infrastructure. Recently, several studies have explored the ability to detect human malaria infections via volatiles from the skin and breath, and their results suggest that volatile biomarkers may hold significant potential for the reliable detection of infected people, including those with asymptomatic infections. These promising results make a volatile diagnostic for malaria a realistic option, particularly with regard to population-wide diagnostic screening and monitoring aimed at malaria eradication.The possibility of using volatile organic compounds as a method of disease diagnosis has been under investigation for some time, with progress made in identifying volatile biomarkers for various types of cancer, lung diseases and neurogenerative diseases . In somePlasmodium parasites in vitro [Plasmodium parasites using models based on six or fewer compounds.Recent studies on human malaria have examined volatiles from the skin ,13,15 anin vitro ,19. Eachin vitro ,16,17. TOne of the two studies that analyzed breath volatiles reported that a model using four compounds  identified asymptomatic infections in a clinical trial with 100% sensitivity , while tPlasmodium in vitro [Plasmodium in vitro [Taking a broader view, it is promising that each of these recent studies – which employed diverse collection and analysis techniques and examined individuals from clinical trials as well as field populations – found differences in volatile emissions associated with malaria infection status while those employing predictive models reported an ability to identify malaria-infected individuals with relatively high sensitivity ,13,15–17in vitro ,19, toluin vitro ,18 and tin vitro ,16, are in vitro ,10. The Despite the promise of using volatiles as a malaria diagnostic, a number of questions remain to be addressed. In particular, two key challenges need to be overcome before volatile-based diagnostics can be considered a viable alternative to existing methods: detecting informative diagnostic compounds that are robust despite other sources of variation and developing a device capable of doing so under real-world conditions.Overcoming the first challenge will entail the precise characterization of potential volatile biomarkers identified by recent and future studies, and determining whether they are sufficiently robust to face off broad-scale interindividual variation. Since no study to date has identified a single compound whose levels reliably predict infection status, any diagnostic method will likely have to assess the presence of a more complex disease signature involving several compounds. A further complication is that, aside from the few compounds mentioned above that have been reported from multiple populations, studies exploring the effects of malaria on human volatiles have generally identified different compounds as important predictors of infection status. In a recent field study based on skin volatiles , the mosPlasmodium species – as well as by mixed infections of multiple species – which might have different effects on volatile emissions. Most studies of malaria associated volatiles to date have focused on P. falciparum; however, a recent study [P. falciparum, P. malariae and P. ovale – suggesting that volatile biomarkers of infection may be robust to variation in parasite species.Human odors exhibit high intra- and interindividual variation , and it nt study  reportedTo date, data showing volatile shifts associated with malaria infection have come only from isolated studies carried out on individuals participating in clinical trials or from field sampling of local populations in restricted geographic areas, with these finding only a handful of characteristic compounds to be significant across multiple studies ,13,15–17The second challenge identified above entails the development of a practical diagnostic assay that can be used under field conditions. Unless further progress in refining the volatile signature of malaria is able to narrow the list of compounds required for reliable classification of disease status to one or two, this will likely require a device capable of recognizing and analyzing complex volatile signatures in the field. The technological platforms most suited to detecting multiple compounds at specific relative thresholds are biomimetic crossreactive sensor arrays, referred to in the literature as electronic noses, which are a technique currently used to detect a wide variety of diseases . SensorsThe development of a volatile-based diagnostic device may fulfill the need for a sensitive and noninvasive technique that could be used on a large scale in malaria-endemic countries. Although it is too early to tell if this method can be practically adapted for widespread use in the field, initial laboratory trials and localized field studies have identified characteristic volatile biomarkers that can in principle be used to predict malaria status with high sensitivity. Although many of these characteristic compounds vary between studies, there are several common compounds that provide a strong starting point for further investigation.Plasmodium species, or at which levels these might need to be tailored. With 2016 being the first year since 2000 in which malaria cases did not show a decline, the development of alternative technologies is a high priority to ensure that reservoirs of malaria can be fully treated to prevent ongoing transmission.The two most important challenges to overcome are the effects of variation on malaria volatile signatures due to methodological, genetic and environmental factors, and the difficulty of applying volatile diagnostic methods easily and affordably in the field. A clear priority for future work is to research different populations with comparable methods and analysis to previous studies to see if the same compounds can detect a malaria volatile signature worldwide, regardless of variation in population, environment and


Plasmodium infections are common in malaria endemic countries, but very little studies have been done in Senegal. This study investigates the genetic diversity and complexity of submicroscopic P. falciparum infections among febrile patients in low transmission areas in Senegal.Submicroscopic P. falciparum infections and characterized for their genetic diversity and complexity of infections using msp-1 and msp-2 genotyping.Hundred and fifty blood samples were collected from febrile individuals living in Dielmo and Ndiop  between August 2014 and January 2015, tested for microscopic and sub-microscopic P. falciparum infections were 19.6% and 25% in Dielmo and Ndiop, respectively. K1 and 3D7 were the predominant msp-1 and msp-2 allelic types with respective frequencies of 67.36% and 67.10% in microscopic isolates and 58.24% and 78% in submicroscopic ones. Frequencies of msp-1 allelic types were statistically comparable between the studied groups (p>0.05), and were respectively 93.54% vs 87.5% for K1, 60% vs 54.83% for MAD20 and 41.93% vs 22.5% for RO33 while frequencies of msp-2 allelic types were significantly highest in the microscopy group for FC27  and 3D7 . Multiplicities of infection were lowest in submicroscopic P. falciparum isolates.Submicroscopic P. falciparum carriage among patients in the study areas, and that submicroscopic P. falciparum isolates had a lower genetic diversity and complexity of malaria infections.The study revealed a high submicroscopic  The scale-up of malaria control interventions has substantially reduced malaria burden and transmission across several malaria endemic countries over the past 15 years with the number of malaria infections and malaria-related mortality that dropping significantly . In factPlasmodium infections in both febrile and asymptomatic subjects [Plasmodium infections sourced for 20–50% of all human-to-mosquito transmissions when transmission reaches very low levels [Plasmodium infections for ongoing malaria transmission and highlighted the relevance of addressing such infections.All malaria control programs have passive surveillance systems thatidentify and treat individuals with light microscopy (LM) and/or rapid diagnostic test (RDT)-confirmed malaria diagnosis at health facilities. In most settings, quality assured LM and RDT effectively diagnose the majority of symptomatic patients, thus guiding treatment. However, with the continuous decline of malaria transmission and progress towards elimination, both LM and RDT lack sufficient sensitivity to detect low-density parasitaemia, leading to an underestimation of parasite prevalence and malaria-related morbidity where infections are mostly subpatent in many areas ,4,5. Thesubjects ,5,6,7. Sw levels , and a mw levels . TogethePlasmodium carriage has been reported from different regions of the globe in febrile and asymptomatic individuals including Senegal [P. falciparum genetic diversity and population structure with a low transmission intensity correlated with a lower genetic diversity of Plasmodium isolates [Submicroscopic  Senegal ,10,11. W Senegal ,13,14; p Senegal . The intisolates ,17,18.P. falciparum isolates in Senegal. In this study, the prevalence of submicroscopic P. falciparum infections was explored among febrile patients during the malaria transmission season in low transmission areas in Senegal. In addition, genotyping of msp-1 and msp-2 polymorphic markers was used to examine the genetic diversity and complexity of submicroscopic P. falciparum infections.To date, very little data is available on the genetic diversity of submicroscopic Samples used in this study were collected from patients living in Dielmo and Ndiop Senegalese villages, which have been extensively described. Written informed consent was obtained from adults and legal guardians of minors participants as part of the ongoing malaria surveillance programs in the two villages,21,22,23Plasmodium parasites carriage. Genomic DNA (gDNA) from mono-infected P. falciparum positive samples were genotyped using msp-1 (block 2) and msp-2 (block 3) polymorphic markers as described previously [P. falciparum infections between microscopic and sub-microscopic parasites populations.For this study, 150 patients from Dielmo (N = 102) and Ndiop (N = 48) presenting with febrile syndromes and attending Dielmo and Ndiop health facilities between August 2014 and January 2015 were enrolled. Venous blood (5 mL) were collected for determination of microscopic and submicroscopic eviously  to invesPlasmodium infections were determined by light microscopy examination of thin and thick smears systematically prepared on site at the health facilities. Blood smears were prepared, air-dried, fixed in methanol and stained with Giemsa for 20–30 minutes. At least two hundred oil-immersion fields were examined at 100X magnification before a slide was considered negative. A second confirmatory reading was performed later at the Institute for Research and Development, and the results herein presented refer only to concordant data between the two readings.Microscopic Plasmodium spp was further investigated at the laboratory by qPCR to distinguish microscopic from submicroscopic Plasmodium infections. To this end, genomic DNA (gDNA) isolated using QIAamp DNA Blood Mini Kit  according to manufacturer’s instructions was subjected to a two-step real time PCR with genus- and species-specific primers targeting the Plasmodium Cytochrome B gene as previously described [P. falciparum, P. malariae, P. ovale and P. vivax infected patients were used as positive controls in all assays, and water and DNA from malaria negative individual served as negative controls to ensure lack of contamination [The presence of escribed ,26. DNA mination .Plasmodium carriage was defined as an infection with any Plasmodium detected by qPCR but not by microscopy.Submicroscopic P. falciparum isolates was carried out by nested PCR amplification of the two highly polymorphic regions of msp-1 (block 2) and msp-2 (block 3) genes as reported previously [msp-1  and two allelic families of msp-2 (FC27 and 3D7) have been reported elsewhere [Genotyping of both microscopic and submicroscopic eviously ,28. Primlsewhere . AmplifiP. falciparum isolates with at least one msp allele out of the total number of isolates was used to calculate the frequency of each msp allelic family. The total number of fragments detected in msp-1 or msp-2 was divided by the number of isolates positive for the same marker to define the number of genotypes per infection or mean multiplicity of infections (MOI) as described earlier [The number of  earlier ,30.R statistical software version 3.3.1 was used for data analysis . DistribDemographic details of the 150 febrile patients enrolled for the study are given in P. falciparum by microscopy while qPCR-based molecular screening detected P. falciparum DNA in 47.33% (71/150) of samples. The latter comprised all 31 microscopy positive samples and 40 microscopy negative samples . The specific prevalence of submicroscopic P. falciparum carriage among the enrolled population was 26.66% (40/150).Of the 150 blood samples screened for the presence of malaria parasites, 20.66% (31/150) were positive for P. falciparum-infected patients subsequently analyzed in this study for their genetic diversity and complexity of P. falciparum infections are detailed in The demographic characteristics of microscopic and submicroscopic P. falciparum isolates were successfully genotyped for both msp-1 and msp-2 polymorphic markers and their respective three  and two (3D7 and FC27) allelic families were detected at various proportions in both groups. The proportions of positive msp-1 and msp-2 samples were 80.66% (25/31) and 90.32% (28/31) for the microscopy group and 87.5% (35/40), and 70% (28/40) for the submicroscopy group. The genetic diversity of microscopic and submicroscopic P. falciparum isolates was compared using mean numbers of msp-1 and msp-2 genotypes instead of the total number of genotypes to rule out the potential impact of the highest number of submicroscopic P. falciparum isolates on the genetic diversity. This revealed comparable mean numbers of msp-1 genotypes between microscopic and submicroscopic P. falciparum isolates   and 1.64 vs 0.65 for 3D7   . In cont = 0.02) . These omsp-1 and 3D7 and FC27 allelic families of msp-2 were compared between microscopic and submicroscopic P. falciparum isolates. K1 and 3D7 allelic families were the respective predominant msp-1 and msp-2 allelic types in both microscopic and submicroscopic P. falciparum isolates  between microscopic and submicroscopic P. falciparum isolates and were respectively 93.54% vs 87.5% for K1, 60% vs 54.83% for MAD20 and 41.93% vs 22.5% for RO33  accounted for 16.12% and 15% of msp-1 positive isolates from microscopic and submicroscopic isolates respectively with no statistically significant difference   .msp-1 allelic types, the frequencies of msp-2 specific allelic types were significantly higher in microscopy-detectable P. falciparum isolates than submicroscopic ones and were respectively 41.93% vs 10% for FC27  and 61.29% vs 32.5% for 3D7   . Dimorphisolates .msp-1 and msp-2 allelic families were also analyzed with respect to two arbitrary defined age groups: ≤ 15 years and > 15 years  were significantly more abundant  in P. falciparum isolates from patients >15 years in both studied groups  was found to be 2.12 and 1.60 respectively for msp-1 and msp-2 in the microscopy group  showed no significant difference neither with th msp-2 .msp-1 and msp-2, and complexity of submicroscopic P. falciparum infections in isolates from patients in Dielmo and Ndiop, two low-transmission areas in Senegal.To date, very few studies have investigated the genetic structure of submicroscopic malaria parasites populations circulating in endemic countries. Our study aimed at filling this major gap and evaluated the genetic diversity, allelic frequency of P. falciparum carriage among febrile patients in both villages, thus highlighting the limitations of LM to accurately diagnose Plasmodium infections when approaching elimination. Controlled malaria infection studies have shown that most patients experienced fluctuations of parasite densities under and over the microscopic detection threshold, thus leading to alternative periods of missed and detectable submicroscopic infections [The study revealed a high proportion of submicroscopic fections . The redPlasmodium infections constitute a challenge to control program aiming to achieve elimination, and should be tackled with the deployment of more sensitive point-of-care diagnostics.The importance of low-density infection as a public health concern is supported by studies that have shown that mosquitoes feeding on individuals with parasite-negative blood smears can become infected with malaria . MoreoveP. falciparum isolates compared to microscopic isolates. An explanation to this could be due to the parasite densities in the submicroscopic infections, which are low and less likely to amplify. However, the high proportions of positive msp-1 and msp-2 samples observed in both microscopy and submicroscopy groups strongly suggest that parasite densities were not the only cause of the lower genetic diversity observed in submicroscopic P. falciparum isolates. The predominance of K1 and 3D7 for msp-1 and msp-2 observed in this study was also demonstrated in other studies from West Africa including Burkina Faso [msp-1 allelic families but lower frequencies in the msp-2 allelic families among the sub-microscopic group is interesting. This could be explained by the highest discriminatory power of msp-2 polymorphic marker over msp-1 marker, thus justifying the widest use of msp-2 marker many genetic diversity studies and to distinguish recrudescence versus re-infection in drug resistance studies[The study also revealed a lower genetic diversity of submicroscopic ina Faso ,36, Coteina Faso and Gabonina Faso , as wellina Faso ,39. The e studies,40,41,42msp-1 and msp-2 loci, the means MOI was lowest in submicroscopic P. falciparum isolates, thus supporting a direct relationship between mean MOI and parasite density as reported in several others studies [msp-2 typing based on its highest discriminatory power have shown a significant decrease of mean MOI with increasing age and the age-effect was most marked in subjects older than 17 years [msp-1-based mean MOI increased with age in both studied groups contrasting with msp-2-based mean MOI, which decreased with age of patients in both studied groups. The latter finding agrees with a study in Senegal where the number of genotypes decreased with age in those aged 15 and above [For both  studies ,44,45. P studies ,47. Howe studies ,48. Stud17 years ,49. In tnd above . The finnd above ,51 sinceP. falciparum parasites is explained by the fact that only one fragment was generated from most of the positive samples while some of the microscopic P. falciparum parasites produced two or three amplicons.The low MOI observed in this study, in particular for the submicroscopic msp-1 and msp-2 amplified products lacks sufficient sensitivity to resolve very small size difference between different bands, which thus constitute a limitation of the study. Alternative methods such as genotyping using capillary electrophoresis or whole genome sequencing are preferred and offer better resolution to distinguish parasite populations.Agarose gel electrophoresis of P. falciparum isolates in febrile patients. This establishes a correlation between the genetic diversity, MOI and parasitaemia as described by others [P. falciparum isolates.Taken together, the results reported in this study indicate a low genetic diversity and MOI of submicroscopic y others ,53,54 anP. falciparum carriage among patients and a low genetic diversity and MOI associated with submicroscopic P. falciparum infections.The findings revealed a high proportion of submicroscopic Plasmodium parasites along with the investigation of human genetic factors that contribute to the maintenance of low-density P. falciparum parasites are critically needed to guide targeted interventions on the road to malaria elimination.A deeper understanding of the genetic characteristics of low-density submicroscopic


Parameters of arterial stiffness such as pulse wave velocity (PWV) were recently proposed as independent risk factors of cardiovascular events. We analyse three PWV parameters in the large population-based study LIFE-Adult to identify risk factors, normal and reference values.r(ba/bf) = 0.6, r(ba/cf) = 0.46, r(bf/cf) = 0.59). Age and blood pressure are the dominant determinants of PWV parameters explaining > 18% of variability. Sex was only relevant for bfPWV and cfPWV. All further analysed cardiovascular and other risk factors are of minor importance. We provide age-dependent percentiles for the population  and for the subgroup of normotonic individuals. All percentiles show a strong increase with age. The difference between normotonic and all individuals is small for younger age groups but increases up to 1 m/s for elderly subjects.Brachial-ankle (ba), brachial-femoral (bf) and carotid-femoral (cf) PWV assessment was performed using Vicorder device. 8509 participants aged 19–80 were analysed. PWV parameters were moderately correlated (Our study confirms and further underpins the strong impact of age and blood pressure on arterial stiffness and the relatively weak contribution of other factors, supporting an independent role of arterial stiffness in cardiovascular disease development. Age-dependent reference and normal values were provided on the basis of the so far largest study sample facilitating the implementation of PWV assessment in clinical practice. Due to better compliance, handling and stronger association with age and blood pressure, baPWV could serve as an alternative to cfPWV. Follow-up data are required to estimate the clinical significance of specified PWV cut-offs. Cardiovascular disease (CVD) represents the leading cause of morbidity and mortality worldwide . Among mDifferent parameters of pulse wave velocity (PWV) are used to assess arterial stiffness, among others: brachial to ankle (baPWV), carotid to femoral (cfPWV) and brachial to femoral PWV (bfPWV). Currently, cfPWV is considered the gold standard because of its reliability –11. A laAs another option, bfPWV measurement focuses more on central components of the arterial tree, but avoids the uncomfortable neck cuff. Pulse wave acquisition of bfPWV has the advantage of showing no venous artefacts and the measurement is well tolerated even by children . AssessmWhile previous studies demonstrated that age and hypertension , 23, 24 In the present paper, we used data from the LIFE-Adult study, a large population-based study conducted in the city of Leipzig, Germany, to analyse and compare the three different parameters of pulse wave velocity cfPWV, baPWV and bfPWV, all measured by Vicorder device. Additionally, we examined the influence of CV risk factors on PWV. Provision of normal and reference values in dependence on age for each PWV parameter was another major objective of our study.Analyses were performed in the LIFE-Adult study, a single centre, population-based cohort study conducted by the Leipzig Research Centre for Civilization Disease (LIFE). Its objective is to investigate prevalence and early onset markers of civilization diseases, with a major focus on atherosclerosis and vascular disease. Between August 2011 and November 2014 a total of 10,000 participants, with ages ranging from 18 to 80 years underwent an extensive assessment programme including structured interviews, questionnaires, physical and instrumental examinations and biospecimen collection. Details of the study can be found elsewhere .Arterial stiffness was determined by pulse wave velocity and pulse wave analysis. In the present analysis, only participants with at least one brachial-ankle or one carotid-femoral PWV measurement were included, resulting in a total sample size of 8509 subjects. Brachial-femoral PWV assessment was available for 3904 of these individuals (see below).To determine reference values we analysed all individuals. To determine normal values, we analysed a subgroup of 3099 normotonic participants. Probands were included in this subgroup when blood pressure was optimal or normal (systolic BP < 130 mmHg/diastolic BP < 85 mmHg) according to the ESH/ESC guidelines  and theiAll PWV measurements were performed as previously described, using the oscillometry-based Vicorder device  . ValiditPWV was determined as the ratio of pulse travel distance to pulse transit time (PTT) derived from 2-point diastolic pulse wave analysis. PTT was determined from foot-to-foot real time shift between simultaneous 2-point-recorded pulse wave curves using an in-built cross-correlation algorithm based on the peak of the second derivative of the pressure curve. Pulse waves were recorded upon automatic cuff inflation to approximately 60 mmHg over at least 10 pulsations. Furthermore, central systolic and diastolic blood pressure and augmentation index were assessed applying device-specific brachial pulse wave analysis. All travel distances were measured separately for each assessment using a flexible tape. In case of significant android fat distribution, a slide caliper was used [n = 3954). Since variance of bfPWV was significantly higher than those of baPWV and cfPWV, we decided to average these two values. Measurements are discarded if the difference of first and second measurement is > 5 m/s (baPWV), > 10 m/s (cfPWV) and > 25 m/s (bfPWV), respectively. This choice is based on a practical, data driven approach resulting in roughly homogeneous distributions of first and second measurements around the diagonal (data not shown). Resulting intra-individual reliabilities assessed in terms of the concordance correlation coefficient (CCC) are high  and are in excellent agreement to those observed in our former study [Data regarding acceptability and global time requirements for the Vicorder devices were collected and published elsewhere in detail . All PWVp < 0.001).In summary, the following sample sizes for the different PWV parameters are available: 8483 for baPWV, 8460 for cfPWV and 3904 for bfPWV. Obesity status is a strong predictor for an unsuccessful PWV assessment. For example, regarding cfPWV, assessment was unsuccessful in 2.3% of non-obese but in 10.4% of obese subjects  after resting for at least 5 min. The first measurement was discarded and the second and third measurements were averaged to obtain the blood pressure.All participants provided a medical history and underwent anthropometric measurements. Prevalent diabetes was determined by fulfilling at least one of the following conditions: HbA1c > 6.5%, the use of hypoglycaemic medications or self-reported diabetes.Medication was categorized according to ATC code lists. For anti-hypertensive drugs, we summarized the codes C02, C03, C07, C08, and C09 as recommended .Data are expressed as mean (± SD) or median (interquartile range) for continuous variables or number (proportion) of participants for categorical variables. Statistical analysis was performed using IBM SPSS Statistics, version 24.0 . All PWV modes, the laboratory parameters as well as weight and body mass index (BMI) were logarithmically transformed to approximate normal distributions.t test. Linear correlations were determined using Pearson’s r correlation coefficient and linear regression analysis was performed to evaluate the association between each PWV parameter and other clinical covariates. Multiple regression analysis was performed using a stepwise regression approach. In each step, a variable is considered for addition to  or subtraction from  the set of explanatory variables (forward–backward model selection as implemented in SPSS). This method was chosen to identify independent factors determining PWV. To avoid collinearity issues we only considered SBP as representative for blood pressure for this kind of analysis.Differences between two groups were compared by the independent samples gamlss package of the statistics software R (http://www.r-project.org) was used for that purpose. Since bf-mode showed the highest variance with less than half of the measurements available for the modes cf and ba, quantile regression was stabilized by winsorizing values above 60 m/s. 11 such outliers were removed.Means (± 2SD) and medians  of each PWV parameter were calculated according to age category for the normotonic subpopulation (see above) and for the entire study population, to deduce normal and reference ranges of PWV. We also applied quantile regression to obtain smooth reference curves. The n = 4365) of the subjects were women. Anthropometric, hemodynamic and laboratory data of all 8509 participants are shown in Table Mean age of participants was 57.3 ± 12.4 years (range: 19–80), and 51.3% (r = 0.60).Figure r = 0.25 for baPWV compared to r = 0.12 and r = 0.14 for bfPWV and cfPWV respectively). Remarkably, in contrast to common expectation, smoking status correlated negatively with both, baPWV and bfPWV.Linear regression analysis between PWVs and other clinical variables showed that anthropometric, hemodynamic, cardiac, renal and metabolic variables, atherosclerotic risk factors and medications were univariate associated with all PWV parameters Table . Only Anϱ = 0.23).The results of stepwise multiple regression analysis revealed several independent variables associated with PWV parameters. Age is by far the strongest independent contributor to PWV variability  followed by systolic blood pressure , respectively, four  parameters contribute relevantly Table . BesidesAll PWV parameters continuously increase with age. We arranged all ages in groups of decades and present box plots of PWV parameters Fig. .To obtain normal and reference values for the three different PWV parameters, we calculated means ± 2SD and medians with the 2.5 and 97.5th percentile for each age category in (1) the whole study population  and (2) the subgroup of normotonic subjects . Results are shown in Table Age-dependent percentile curves for normal and reference values of PWV parameters are displayed in Fig. Cardiovascular disease as the leading cause of mortality and morbidity worldwide is predicted to continue to increase in the coming years. Therefore, easy to handle, economical and widely applicable non-invasive assessments of early vascular damage are of great clinical significance. The objective of the present study was the evaluation of the three different PWV parameters baPWV, bfPWV and cfPWV in the large epidemiologic setting of the LIFE-Adult study. We compared these parameters, analysed corresponding influencing factors and established normal and reference values.Although cfPWV is considered the gold-standard, baPWV has recently emerged to be a more feasible procedure promising better patient compliance. During our examinations we experienced that cfPWV assessment was sometimes hampered due to intolerance towards the neck cuff or in case of obesity, where the cuff did not fit around the neck or the upper thigh.Strong positive correlations between cfPWV and baPWV  as well In line with several previous reports, we observed that age and blood pressure are by far the most important factors influencing PWV parameters, explaining large parts of the overall variance , 31, 38.Though smoking is strongly associated with peripheral vascular disease and arteriosclerosis, the impact on arterial stiffness has been reported inconsistently so far. Jatoi et al. showed that smoking status correlates positively and duration of smoking cessation negatively with cfPWV . HoweverGiven the large evidence of PWV in predicting CV events but lack of strong dependence of PWV parameters on CV risk factors other than age and hypertension, we hypothesize that the prognostic value of PWV may be related to a process of arterial ageing or life time exposure of increased blood pressure independent of traditional CV risk factors . In thisIn these guidelines, a fixed threshold of 10 m/s was proposed for cfPWV regardless of age , 40. HowRegarding baPWV, only reference values in an Asian cohort are available so far . CompareSeveral limitations of the present study are of note. The age group < 40 years is underrepresented in our study and corresponding PWV percentiles are, therefore, estimated with lower accuracy. The same holds for bfPWV for which only 3904 measurements are available in total. Again, follow-up data regarding future CV events are required to provide reasonable cut-offs of PWV parameters for intervention. Moreover, PWV assessment is significantly more difficult in obese subjects resulting in higher percentages of missing values. This especially applies for cfPWV.In conclusion, we performed the so far largest population-based study of parallel assessment of three PWV parameters. We confirmed that age and blood pressure are the main determinants of PWV and showed that all other factors are of minor importance. We estimated normal and reference values for the three different PWV parameters in dependence on age facilitating the implementation of PWV assessments in ongoing and future clinical practice to improve cardiovascular risk stratification. Since baPWV reveals better handling and stronger association with age and blood pressure, it could serve as an alternative to cfPWV.