Plasmodium falciparum was responsible for over 91% of infections. Children from Pitoa health district had the highest number of asymptomatic infections (45.60%) compared to those from Garoua and Mayo Oulo. The most suitable cut-off for the association between parasite densities and fever was found among children less than 24 months. Overall, parasite densities that ranged above 3,200 parasites per μl of blood could be used to define the malaria attributable fever cases. In groups of children aged between 24 and 59 months and 60 and 94 months, the optimum cut-off parasite density was 6,400 parasites per μl of blood, while children aged between 95 and 120 months had a cut-off of 800 parasites per μl of blood. In the same ecoepidemiological zone, clinical malaria case definitions are influenced by age and location  and this could be considered when evaluating malaria intervention strategies in endemic areas.Malaria endemicity in Cameroon greatly varies according to ecological environment. In such conditions, parasitaemia, which is associated with fever, may not always suffice to define an episode of clinical malaria. The evaluation of malaria control intervention strategies mostly consists of identifying cases of clinical malaria and is crucial to promote better diagnosis for accurate measurement of the impact of the intervention. We sought out to define and quantify clinical malaria cases in children from three health districts in the Northern region of Cameroon. A cohort study of 6,195 children aged between 6 and 120 months was carried out during the raining season (July to October) between 2013 and 2014. Differential diagnosis of clinical malaria was performed using the parasite density and axillary temperature. At recruitment, patients with malaria-related symptoms  and a malaria positive blood smear were classified under clinical malaria group. The malaria attributable fraction was calculated using logistic regression models. Sub-Saharan African countries remain endemic for malaria and bear the highest morbidity and mortality rates. The risk of having malaria is more pronounced in the Sahelian areas with frequent outbreaks due to seasonality of the transmission. In such areas, individuals, especially children, often harbour malaria parasites, but without clinical symptoms .The policies recommend that in health facilities, malaria should be first diagnosed by the presence of fever and a subsequent confirmatory blood test by microscopy or rapid diagnostic test (RDT). However, this is not always the case due to many challenges such as delay in delivery of certain materials such as RDT or lack of laboratory technician. These weaknesses limit malaria diagnoses to symptoms such as fever only and could contribute to an overdiagnosis of malaria cases and consequently an inappropriate use of antimalarial drugs. Defining clinical malaria cases in such endemic areas is difficult as clinical signs of the disease are nonspecific. Moreover, malaria parasitaemia which is associated with clinical symptoms does not always imply a situation of clinical malaria . Indeed,A clear and precise approach to estimate the proportion of fever attributable to malaria based on the model of the relationship between fever and parasite density has been reported , 10. Thi Plasmodium falciparum is the most prevalent parasite species and malaria is mainly transmitted by Anopheles arabiensis, An. gambiae and An. funestus [ An. pharoensis and An. rufipes play only secondary roles in the transmission of the disease [This study was conducted from 2013 to 2014 during the rainy season in three health districts (HD) in the North Region of Cameroon . Thirty-funestus . Other s disease , 13. Agr Plasmodium positive blood smear. Cohort members who dropped out were replaced. All cases were entered into the study database monthly. The cohort was followed from 2013 to 2014 during the raining season (July to October). Details of the methods used have previously been described [A cohort of over 6,195 participants aged between 6-120 months was randomly selected (through a systematic random sampling) from over 1,409 houses in 38 villages. In each health district, houses were randomly selected and all eligible children were enrolled prior to a written consent by their parents or a caregiver. In each village, cohort details of all participants  were gathered. A unique identification was assigned to each participant and corresponded to the household number and the village code. Participants were visited at home, at least once every 2 weeks, and information on the health of enrolled children was recorded and used to identify those who may be having or have experienced fever within the last 48 hours or who they suspected to have malaria. The body axillary temperature was taken with a digital thermometer and the symptoms and signs of the illness were recorded. Every child that was febrile or had been reported to be febrile was tested (TDR) and labelled thin and thick blood smears were prepared. Clinical cases were referred to the nearest hospital for free treatment. A clinical malaria case was defined as an individual with malaria-related symptoms  at the time of examination or 1–2 days prior to the examination and the presence of a Plasmodium infection as recommended by the national policy. Plasmodium species were further confirmed by nested-PCR. Briefly, when a slide was positive for Plasmodium sp., the portion of blood previously spotted on filter papers (Whatman® N°3) was subjected to Plasmodium DNA extraction using the Chelex method [ Plasmodium species was identified by nested-PCR [Thin and thick blood smears were prepared and air-dried; then, the thin smears were fixed in methanol and stained in 4% Giemsa for 30 minutes. Two independent microscopists examined the slides under a ×100 oil immersion microscope objective to obtain a species-specific parasite count. Malaria parasites were counted against 200 white blood cells (WBC) and a blood smear was declared negative after examination of 100 high power fields. The counts were expressed as the number of parasites per microliter of blood, assuming an average leukocyte count of 8,000 cells/ml of blood. For every slide where the difference in parasitaemia between the two readers was greater than five parasites per microliter of blood, a third reader had to re-examine the slide and the mean of the two closest values considered. Free malaria treatment with artesunate-amodiaquine was given to all patients referred to the nearest health centre in case ofx method  and the sted-PCR .The Cameroon National Ethics Committee (102/CNE/SE/09) gave ethical clearance. The children were enrolled into the study if their parents or caregiver provided a written consent after receiving an explanation of the study procedures.πi) represents the probability that an observation (i) with parasite density xi is a fever case. β(xi)τ corresponds to a type 3 model described by Smith [ (xi). The models were fitted using maximum likelihood. To constrain the parameter τ to be positive, the maximum likelihood was estimated for the log (τ). Parasite density cut-off for different study areas and age groups was determined by the intersection of sensitivity and specificity curves. The malaria attributable fraction was calculated as the reduction in the incidence that would be observed if the population was entirely unexposed, compared with its current exposure pattern.The attributable proportion of fever due to malaria was estimated according to the method previously described by Smith  and whicby Smith  and is a Plasmodium from those whose fever had other origin. The sensitivity and specificity of biological and molecular techniques applied for differential diagnosis were used to validate malaria case definition. This was done by an estimation of parasite cut-off densities in different settings, using the point at which the sensitivity and specificity are both highest as the optimum case definition. The prevalence of the disease was used to estimate the fraction of prevalent febrile episodes that are attributable to the presence of malaria in blood.Data were entered into Epi Info 3.5.3 spread sheet, processed and analysed using the same software. Sampled households were divided according to the health district. Individuals who lived in the same health district were pooled together to calculate the prevalence of asymptomatic malaria infections. The asymptomatic malaria infection rate was calculated as the ratio of infected individuals without symptoms over total samples. The differences in asymptomatic infection rates between districts were determined by chi-square test. Threshold for statistical significance was set at P< 0.05. Parasite thresholds were used to differentiate individuals whose fever was caused by Plasmodium infection was 40.95% .The prevalence of fever due to malaria in children was higher in Pitoa HD (22.7%) compared to Garoua (18.3%) and Mayo Oulo (15.3%), although the difference was not significant , with an overall prevalence of 19%. The prevalence of fever cases associated withs 40.95% . Among t Plasmodium falciparum was the most prevalent species in the three sites with a percentage of 91.62% and was found in coinfections, with P. malariae at very low percentage (2.37). Multiple infections consisted of the association of P.f+P.m (5.96%) and P.f+P.o (0.05%)  followed by those living in Garoua (24.61%) and Mayo Oulo (15.48%) HDs . The highest infection rate was found in Pitoa (47.83%) compared to Garoua (26.23%) and Mayo Oulo (17.53%) . Regarding the gametocytes index, this was 12.16%, 11.65%, and 7.30% in Garoua, Mayo Oulo, and Pitoa, respectively . (0.05%) .The sensitivity and specificity of various cut-off parasite densities used in the definition of clinical malaria, according to the health district, are given in μl of blood could be suitable to define malaria attributable cases in children from Mayo Oulo and Pitoa Health districts, respectively. This cut-off was highest at 12,800 parasites per μl of blood in individuals from the Garoua health district.Overall, the optimum cut-off parasite densities of 800 and 6,400 parasites per μl of blood could be used to define the malaria attributable fever cases in children less than 24 months of age. In children aged 24-59 months and 60-94 months, the parasite density of 6,400 parasites per μl of blood is suitable to define the malaria attributable fever cases. This cut-off decreased to 800 parasites per μl of blood in children aged 95–120 months.Malaria case definition by age was classified following the optimum cut-off parasite densities . The finBecause the number of fever cases encountered by health district was low, the determination of the malaria attributable fraction (MAF) by age groups in each district was not possible. Thus, cumulative MAFs by age in the three HDs were determined . From thIn endemic areas, differential diagnosis of clinical malaria cases is facing many challenges, including the increasing prevalence of asymptomatic malaria infections and the nonspecific symptoms of the disease. Because the evaluation of malaria control interventions is mainly based on the morbidity trend in the population at risk, differential diagnosis is critical for accurate impact intervention measures and for first-line treatment administration . In most Plasmodium infection. Hence, more than half of febrile children were estimated not to have malaria infection. In the context of limited access to affordable diagnostics and lack of knowledge of the causative agents of other febrile illnesses, most febrile cases are treated and managed as malaria in malaria endemic areas [It is well documented that fever symptoms commonly accompany a wide range of childhood illnesses. In general, young children are more vulnerable to infections due to their immature immunity, the different modes of exposure to pathogenic organisms and possibly the low rate of immunisation . Among tic areas . This inic areas . Plasmodium falciparum was the most predominant malaria parasite species in investigating health districts as previously described [ P. vivax was absent, its presence was reported in the South part of the country [Our findings revealed that malaria is mesoendemic in investigating study sites, with an overall prevalence of 31.15%. This prevalence did not significantly vary by health district. Although the gametocytemia index was low, the production of gametocytes within an infected human host is essential for transmitting the parasite to the mosquito. In a low transmission context, the proportion of these sexual stages and their density are seminal to understand the transmission of malaria in the population . In the escribed , 24. Alt country , 26.The study revealed a significant difference in MAFs according to health districts. This variation could be due to a difference in the level of transmission as previously described elsewhere , 27. Indμl among children aged between 94 and 120 months. Thus, 800 parasites per μl of blood were enough to cause malaria in these children. In contrary, it is well known that older children normally manage high parasite density because of their immune system, which is well developed compared to that of children under five years [The differences observed in parasite density cut-off values among children age groups could be attributed to the fact that older people can tolerate higher parasite densities than younger ones. The immunity builds up during many years, preferably after previous exposures and observed episodes could explain this , 29. Theve years , 30. Theve years . This shve years  scaled uve years . The natve years . The couve years , 36 to cHowever, the study faced some limits such as the fact that we were unable to determine the proportion of patients whose fevers were treated with antipyretic drugs prior to the visit. In fact, fever is the predominant symptom of the use of self-medication by parents for the treatment of their children in Cameroon . This maIn the Northern region of Cameroon, where seasonal malaria transmission varies spatially, clinical malaria should be defined by age and by malaria transmission intensity when evaluating interventions. The high prevalence of nonparasitaemic fever underlies the fact that fever is not a specific marker for clinical malaria; hence, there is a need to encourage health workers to perform first-line diagnosis before the administration of an antimalarial treatment. The shift in age groups at high risk of clinical episodes of malaria as observed from children under five to older age groups suggests the need to expand prevention and free treatment strategies for older children in order to cover the peak transmission months.


The effect of FEV1 on later cf-PWV and its progression was estimated using linear mixed-effects modeling. Possible explanatory mechanisms, such as mediation by low-grade systemic inflammation, common-cause explanation by preexisting cardiometabolic risk factors, and reverse-causation bias, were assessed. Poorer pulmonary function was associated with later higher cf-PWV and its subsequent progression  after adjustment for age, sex, ethnicity, heart rate, and mean arterial pressure. Decrease in pulmonary function was associated with later higher cf-PWV . There was no evidence to support mediation by circulating CRP (C-reactive protein) or IL (interleukin)-6. Furthermore, arterial stiffness was not associated with later FEV1 after accounting for cardiometabolic status. In conclusion, poorer pulmonary function predicted future arterial stiffness. These findings support pulmonary function as a clinically important risk factor for arterial stiffness and provide justification for future intervention studies for pulmonary function based on its relationship with arterial stiffness.Whether poorer pulmonary function accelerates progression of arterial stiffness remains unknown as prior observational studies have not examined longitudinal changes in arterial stiffness in relation to earlier pulmonary function. Data  were drawn from the Whitehall II cohort study. Participants completed repeated assessments of forced expiratory volume in 1 second (FEV As many as 1 in 5 of the general population may have abnormal spirometry patterns3 and impairment of pulmonary function is a risk factor for incident angina, myocardial infarction, stroke, and CVD mortality,7 making it important to identify the mechanisms underlying the association between pulmonary function and CVD. Arterial stiffness is among plausible candidates.To improve health management and facilitate cardiovascular disease (CVD) prevention, it is necessary to identify and control cardiovascular risk factors appropriately at an early stage. Among surrogate markers of cardiovascular risk, vascular function, especially aortic stiffness, which can be assessed noninvasively, is a robust predictor of CVD end points and all-cause mortality.9 showed inconsistent associations between pulmonary function and arterial stiffness, as measured by pulse wave velocity (PWV).14 Longitudinal changes in PWV in relation to pulmonary function are unclear, and it remains unknown how spirometrically-defined pulmonary function and its change affect later PWV. The mechanisms connecting pulmonary function and arterial stiffness have not been studied.Prior observational studies, including 2 prospective studies,1) with carotid-femoral PWV (cf-PWV) and its progression. In addition, we examined mechanisms other than the direct effect of pulmonary function on arterial stiffness: (1) the possible mediating role of low-grade systemic inflammation,8 (2) a possible common-cause explanation considering cardiometabolic factors as the underlying causes for both accelerated arterial stiffness and poorer pulmonary function, and (3) a potential reverse causation , among middle-aged and older men and women.To address this gap in research, we assessed the cross-sectional and 5-year prospective associations of forced expiratory volume in 1 second .Data supporting the findings of this study are available from the corresponding author on reasonable request . Details are published.16The cf-PWV measure indexed arterial stiffness. There is an inverse relationship between PWV and vascular compliance; therefore, the pulse wave travels more quickly through arteries with decreased flexibility than elastic ones. The transit time was defined as the pulse wave’s travel time between the carotid and femoral sites in the supine position, captured by applanation tonometry  based on ECG monitoring. Thus, cf-PWV (m/s) was calculated by dividing the path length, measured with a tape measure using a standard protocol, by the transit time. At each assessment, cf-PWV was measured twice unless the difference between measurements was >0.5 m/s when a third measurement was taken. The average of the measurements was used in analyses. Extensive analysis shows that within-person change in adiposity during the study did not have an artifactual effect on measurement of path length or on longitudinal change in cf-PWV.1 (L) using flow-sensing spirometry  in the standing position. The procedure was conducted by trained nurses following a standard protocol.17 FEV1 was measured ≤5×, and the maximum measurement identified. Body size, especially standing height, is directly proportional to total lung capacity. To correct FEV1 for height dependence, we divided by the square of the participant’s standing height and multiplied by the square of the sample average height: women: 1.61 m, men: 1.75 m.18 These adjusted values were used in the analyses.Pulmonary function was evaluated through FEV19Two systemic inflammatory indicators were measured at the 2002 to 2004 study phase. CRP (C-reactive protein) is an acute phase reactant: a protein produced by the liver in response to induction by IL (interleukin)-6. Serum CRP levels were measured through a high-sensitivity immuno-nephelometric assay in a BN ProSpec nephelometer, and serum IL-6 level was measured through a high-sensitivity ELISA .Unmodifiable risk factors were the following: sex, age at each phase, and ethnicity (white or not). Modifiable risk factors were body mass index, waist circumference, systolic blood pressure, mean arterial pressure, heart rate, total cholesterol, high-density lipoprotein cholesterol, hemoglobin A1c, smoking status (current smoker or not), antihypertensive medication (current user or not), and diabetic status (diabetic or not). Each item was examined during the 2007/2009 phase. Systolic blood pressure, mean arterial pressure, and heart rate were also assessed in 2012/2013 because these factors, especially mean arterial pressure and heart rate, can affect the cf-PWV measurement by shifting the form of the pulse wave. Follow-up time in years between the clinical screening in 2007/2009 and 2012/2013 was computed and divided by 5 so that a unit change represented a time difference of 5 years.1 between 2002/2004 and 2007/2009 waves of the study. Linear mixed-effects modeling was used to assess the cross-sectional and longitudinal effects of pulmonary function  on arterial stiffness , allowing for subject-specific random effects for both slope and intercept. To examine the association between FEV1 and arterial stiffness, the fixed part of the model (ignoring covariate terms) for cf-PWV measured on individual i on occasion k (=2007/2009 or 2012/2013) at time t:Cuzick nonparametric test was used to test for trend of each variable across quartiles of change in FEVik=0, and for measurements at 2012/2013, tik=time between 2007/2009 and 2012/2013 measurements. In this model, β2 shows the cross-sectional association of FEV1 with cf-PWV at 2007/2009, β1 shows how cf-PWV changes with time and β3, the coefficient for the interaction term, shows how change with time is modified by levels of FEV1. Thus, β3 indicates whether FEV1 is associated with progression of cf-PWV over time after adjusting for the effects of included covariates. In the base model, we adjusted for age, sex, ethnicity, heart rate, and mean arterial pressure. Heart rate and mean arterial pressure were also included as time-varying covariates.For measurements at 2007/2009, t1 before baseline cf-PWV at 2007/2009 and change in cf-PWV between 2007/2009 and 2012/2013 were examined by calculating residuals, regressing FEV1 at 2007/2009 on FEV1 at 2002/2004. These residuals were included in the mixed models for cf-PWV as a continuous variable and in quartiles. The advantage of using residuals, rather than absolute changes in FEV1, is that these residuals are orthogonal to, and independent of, baseline FEV1 (2002/2004). Modeling absolute changes with baseline adjustment is a questionable method.20 The mixed-effect models were estimated using maximum likelihood. Likelihood-ratio tests were conducted to test for effect modification using fixed-effect interaction terms between pulmonary function quartiles and follow-up time.The effect of changes in FEV1 and cf-PWV in terms of a hypothesized process, we estimated and tested the indirect effect of FEV1 on cf-PWV and its mediation by inflammation (CRP and IL-6) using the Baron and Kenny approach.21 CIs for the percentage attenuation were computed using the bootstrap method with 2000 resamplings.To explain the relationship between FEV1 at 2012/2013. Percentage change after adjustment estimated the role of cardiometabolic factors as a common cause of arterial stiffness and poorer pulmonary function. A sensitivity analysis was conducted to examine potential bias arising from chronic respiratory diseases. We excluded 623 participants (11.7%) at the 2007/2009 phase with diagnosis of chronic obstructive pulmonary disease, asthma, chronic bronchitis, pulmonary fibrosis, pneumoconiosis, interstitial pneumonia, pulmonary tuberculosis, or lung cancer.To investigate reverse causation, linear regression models were fitted to estimate the effect of cf-PWV at 2007/2009 on FEV22 Statistical significance was defined as a 2-tailed P<0.05. Analyses were conducted using Stata/MP Ver. 13.1 .We imputed missing data using multiple imputation methods whose models included all assessments related to participants’ vascular function together with risk factors at the 2007/2009 phase. The following auxiliary variables, associated with ≥1 variables with missing data, were included in the imputation models: systolic blood pressure, mean arterial pressure, heart rate, and forced vital capacity from adjacent phases, triglycerides, angina, myocardial infarction, stroke, CVD medication, chronic respiratory diseases, and alcohol dependence. We generated 20 imputed data sets and combined the results using Rubin rules.1 were, on average, older, male, white ethnicity, and had higher cf-PWV at the start of the follow-up. Greater decline in FEV1 was associated with being a current smoker, higher waist circumference, higher systolic blood pressure, higher mean arterial pressure, and lower heart rate. There were no associations with body mass index, total cholesterol, or hemoglobin A1c. Medication history of hypertension and diabetes mellitus were not associated with change in FEV1.Participants (N=5342) were mean age 65.4 years in 2007/2009. The cohort was 26% female and of predominantly white ethnic origin. Table 1 in 2007/2009, FEV1 in 2002/2004, and ΔFEV1 between 2002/2004 and 2007/2009) on aortic stiffness (cf-PWV in 2007/2009) and its subsequent progression (Δcf-PWV between 2007/2009 and 2012/2013). Lower FEV1 in 2007/2009 was associated cross-sectionally with higher cf-PWV in 2007/2009 after adjustment for age, sex, ethnicity, mean arterial pressure, and heart rate .Similarly, as shown in the second panel of Table 1 in 2007/2009 or 2002/2004 associated with higher cf-PWV in 2007/2009 and faster progression of cf-PWV. As previously described, whereas longitudinal decreases in FEV1 between 2002/2004 and 2007/2009 were significantly associated with higher subsequent cf-PWV in 2007/2009, FEV1 change (2002/2004 to 2007/2009) was not associated with subsequent progression of cf-PWV.As sensitivity analyses, the mixed-effect regressions were repeated after excluding participants with prevalent chronic respiratory diseases. The same associations were observed, with lower FEV1 in 2002/2004 was associated cross-sectionally with CRP and IL-6 (P<0.001). The mediation effect of inflammatory markers on the association between FEV1 in 2002/2004 and cf-PWV in 2007/2009 is shown in Table 1 in 2002/2004 with cf-PWV 2007/2009 was not attenuated after CRP adjustment. IL-6 in 2002/2004 was weakly associated with cf-PWV in 2007/2009 (P=0.013). IL-6 adjustment did not attenuate the association between FEV1 and cf-PWV. Similarly, adjustment for smoking status had negligible effect on this association .FEV1 in 2012/13 was examined before and after adjustment for cardiometabolic status  were added to the model. The association between cf-PWV and later FEV1 was not attenuated significantly, although the PWV-FEV1 association was not significant (P=0.053) in the adjusted model.The association between cf-PWV in 2007/2009 and FEVus Table . In the 1 was associated with later higher cf-PWV and its subsequent longitudinal progression after adjustment for age, sex, ethnicity, heart rate, and mean arterial pressure; (2) 5-year decrease in FEV1 was associated with higher cf-PWV, whereas the FEV1 decrease was not associated with progression of aortic stiffness; (3) there was little evidence that the association between poorer pulmonary function and later higher aortic stiffness was mediated by circulating inflammatory markers CRP and IL-6; and (4) higher cf-PWV was only weakly associated with later impairment of FEV1 after adjustment for cardiometabolic status, suggesting that there was no strong evidence for reverse causation. The current evidence supports the clinical importance of observing pulmonary function as a new marker or potential cause for aortic stiffness.This large longitudinal study provides new evidence for poor pulmonary function as a risk factor for aortic stiffness in early old age. The present findings show that (1) lower FEV23 Systemic inflammation might contribute to, or trigger, the process of remodeling within vascular walls of arteries, resulting in elastic fibers’ degradation and their replacement by collagen deposition.26 Studies focusing on low-grade systemic inflammation have typically assessed only CRP. Despite CRP being a general marker of inflammatory processes, IL-6 may be a more sensitive and appropriate marker of systemic inflammation through regulation of CRP synthesis and assumed causal association with cardiovascular end points, such as myocardial infarction.27 However, research on the relationship between IL-6 and lung function is also scarce.30 Our mediation analysis showed that the association between poorer pulmonary function and higher arterial stiffness was unchanged when CRP was used as marker of inflammation and insignificantly mediated by only 5.1%  when IL-6 was used. Therefore, we found little evidence that the effect of FEV1 on cf-PWV could be attributed to mediation by low-grade systemic inflammation, despite the plausibility of this pathophysiological mechanism. This finding was supported by sensitivity analysis, excluding people with chronic respiratory disease.We hypothesized that the association between poorer pulmonary function and higher arterial stiffness would be mediated by low-grade systemic inflammation. The human respiratory system is thought to be particularly vulnerable to environmental factors because it has a large interface with the outside world. Exposure to environmental factors can cause poorer pulmonary function with acute or chronic respiratory inflammation. This could result in low-grade systemic inflammation, which can be measured through higher blood concentration of CRP and IL-6.1, but the association was weak. After controlling for cardiometabolic factors, the cf-PWV- FEV1 association was not significant. Two inferences can be made. First, cardiometabolic status does not seem to play an important role as a common cause of higher arterial stiffness and poorer pulmonary function. Second, the prospective association may be due entirely to confounding by cardiometabolic status. In summary, the evidence that aortic stiffness is a determinant of pulmonary function in our cohort is weak.Analyses to investigate reverse causation were included in this study. Higher cf-PWV was a predictor of later lower FEV32 Alternatively, the association between pulmonary function and aortic stiffness could partly be due to shared structural and mechanical properties of the 2 organs. Elastin may be important in this context, as a protein conferring distensibility and elastic recoil to connective tissue in the aortic wall and lung. Serine proteases and matrix metalloproteinases are able to degrade elastin, contributing to loss of elasticity and potentially to correlated properties of aorta and lung.33The balance of our findings suggests pulmonary function may have direct impact on arterial stiffness. Poor pulmonary function may lead to an imbalance between demand and supply of oxygen in the arterial wall to cause aortic stiffening. Development of atherosclerotic lesions may contribute concurrently to arterial stiffening.1 and later change in cf-PWV. However, the mean change in cf-PWV over about 5 years (between 2007/2009 and 2012/2013) was <0.7 m/s; hence, the study may be underpowered to detect such a small effect.We did not find evidence to support an effect of longitudinal changes in pulmonary function on later cf-PWV change, possibly because the study lacked statistical power. If direct impact of pulmonary function on arterial stiffness is assumed, then, in theory, there should be a significant association between change in FEV1 on later aortic stiffness and its progression in early old age. Three statistical approaches were applied to examine the direct effect, possible pathway, and reverse causation. Another strength is the large study sample which included both men and women. This is one of the largest observational studies examining the association between pulmonary function and arterial stiffness, which enables us not only to have good statistical power but also to validate the reference value of FEV1 and to avoid spurious differences because of sampling error.The main strength of this study lies in its longitudinal design, including repeated measurements of pulmonary function and arterial stiffness over time, which enabled us to address causality. To our knowledge, this is the first study to assess the impact of longitudinal changes in FEV35 Simultaneous loss of flexibility in the connective tissue of both alveoli and arterial walls due to genetic factors could plausibly lead to both impaired pulmonary function and higher arterial stiffness because elastin is known to confer elasticity to both organs. In the context of common origins, the direct effect of FEV1 on cf-PWV captured in the current study cannot be separated from the effects of genetic factors or shared pathological mechanisms. Furthermore, the Developmental Origins of Health And Disease hypothesis demonstrates that adverse exposures in early life could lead to permanent structural and metabolic changes, resulting in both pulmonary and CVDs in later life.37 If impaired pulmonary function and higher arterial stiffness share early life origins, it will be also necessary to keep in mind that early life prevention could be indicated, although the evidence currently available for the shared origins is limited. In addition, within-person longitudinal variability of inflammatory markers may bias attenuation towards the null in the mediation analysis,38 although recent analyses, based on the same cohort study, examining IL-6 and CRP have suggested such variability could be a relatively minor concern.40Regarding this study’s limitations, unmeasured confounding factors might also affect the association between pulmonary function and arterial stiffness. For example, genetic factors or susceptibility might contribute to spurious associations.41Finally, the data used in this study were from middle- to older-aged, white-collar, London-based civil servants; therefore, the generalisability of this study’s findings need to be confirmed in future research including other generations, ethnicities, and occupation types. In the context of standard risk factor-CVD associations, despite marked differences in both risk factors and disease incidence, close agreement between the findings from Whitehall II and general population samples have been found.1 on later aortic stiffness, a possible indirect pathway through mediation by inflammation and reverse causation considering cardiometabolic status were investigated. These results imply that the association between poorer pulmonary function and later increase in aortic stiffness is not mediated by systemic inflammation indicated by elevated levels of CRP and IL-6. Furthermore, higher cf-PWV does not predict later impairment of FEV1 after accounting for cardiometabolic status.The present findings suggest that pulmonary function could have predictive value for future increases in arterial stiffness and its progression. To clarify the causal effect of pulmonary function measured by FEV1 may be an effective potential option for improving health management and facilitating CVD prevention.Future intervention studies are needed to clarify the preventive effect of monitoring and intervening in lung function on aortic stiffness progression. The current evidence supports the clinical importance of observing pulmonary function as a new potential cause for aortic stiffness. Considering the feasible nature of spirometry as a routine clinical examination, FEVWe thank every participant in the Whitehall II cohort study, as well as all Whitehall II researchers and support staff who make the study possible.The Whitehall II study is supported by the British Heart Foundation (RG/16/11/32334), UK Medical Research Council (K013351) and US National Institute on Aging . M. Kivimaki is additionally supported by the UK Medical Research Council (S011676), NordForsk , the Academy of Finland (311492), and Helsinki Institute of Life Science.None.


Plasmodium falciparum can help targeting control interventions towards elimination. This can be inferred from genetic similarity between parasite isolates from different sites and timepoints. Here, we imposed a P. falciparum life cycle time on a genetic distance likelihood model to determine transmission paths from a 54 SNP barcode of 355 isolates. Samples were collected monthly during the 2013 malaria season from six pairs of villages spanning 300 km from western to eastern Gambia. There was spatial and temporal hierarchy in pairwise genetic relatedness, with the most similar barcodes from isolates within the same households and village. Constrained by travel data, the model detected 60 directional transmission events, with 27% paths linking persons from different regions. We identified 13 infected individuals (4.2% of those genotyped) responsible for 2 to 8 subsequent infections within their communities. These super-infectors were mostly from high transmission villages. When considering paths between isolates from the most distant regions (west vs east) and travel history, there were 3 transmission paths from eastern to western Gambia, all at the peak (October) of the malaria transmission season. No paths with known travel originated from the extreme west to east. Although more than half of all paths were within-village, parasite flow from east to west may contribute to maintain transmission in western Gambia, where malaria transmission is already low. Therefore, interrupting malaria transmission in western Gambia would require targeting eastern Gambia, where malaria prevalence is substantially higher, with intensified malaria interventions.Malaria has declined significantly in The Gambia and determining transmission dynamics of  P. falciparum-infected individuals, probably infected during the previous transmission season, can restart the seasonal peak by infecting the local vector2. Such a transmission dynamic is probably not entirely local as human movements may contribute to it3. Human migration promotes parasite mixing, potentially impacting on the effectiveness of control interventions4. The resulting patterns of parasite flow and transmission5, genetic diversity and recombination (outcrossing) of different parasite types can be detected using new high-throughput molecular technologies and detailed field epidemiology6.The malaria burden in The Gambia has significantly declined and malaria transmission which is markedly seasonal (July-December) has become increasingly heterogeneous, with two main transmission strata—low in the west and moderate to high transmission in the eastP. falciparum and P. vivax9. Similar barcodes have been explored for establishing sibling or ancestral relationships between isolates, from which transmission paths can be inferred11. Specifically, a 24 SNP barcode for P. falciparum has enabled genetic fingerprinting, the determination of infection complexity and changes in transmission pattern13. It can be used to track the spread of clones during transmission and to determine whether an infection is local or imported14. However current phylodynamic methods applied to prokaryotic pathogens may be inaccurate for reconstructing transmission trees for a complex recombining eukaryote like P. falciparum. Indeed, recombination and other evolutionary events can exaggerate genetic distance, calling the need for new models to infer transmission links and direction from small SNP barcodes. Integrating high resolution epidemiologic surveys with genetic evidence can allow for more accurate reconstruction of transmission pathways. This will provide important information to understand ongoing residual transmission despite high coverage of control interventions.Single nucleotide polymorphism (SNP) barcodes have been developed for Plasmodium falciparum isolates were detected by varATS PCR from 12 villages across The Gambia . When including all isolates and markers, genome-wide index of association between allelic states (IAS) was 2-fold higher in western than eastern Gambia.In the 2013 malaria transmission season, 1113 Mixed allele calls represented 6.2% of retained SNPs called. The proportion of mixed allele calls for SNP loci ranged from 0% to 44%. SNP loci with mixed allele calls increased from west to east, with the highest median of 7% of loci in eastern villages. Similarly, the complexity of infection (COI) modelled from the mixed allele calls with McCOIL ranged between 1–5 clones per infection and increased from west to east 18. Identifying and determining the presence of such infections is vital for malaria elimination and requires improved diagnostic approaches for targeted intervention. On the other hand, sites with multiple shared barcodes and highly similar parasite pairs in paired villages suggest clonal expansion similar to outbreaks after the introduction of infection(s). Such situations would require approaches able to detect and target infections “imported” into The Gambia, which is surrounded by Senegal, where there is still relatively high transmission in the southern-eastern regions19. These findings are interesting given that the overall village-scale analysis showed clustering of highly similar barcodes at household level, where the risk of clinical malaria is higher in households with at least one infected individual at the beginning of the transmission season1. Therefore, approaches such as reactive household interventions with drugs or other tools may target this infection reservoir and facilitate local elimination of malaria. Such interventions are already being tried and results may help further clarify our findings20. Further establishing the chain of transmission of such imported or local outbreaks could facilitate approaches towards elimination.Genotyping malaria parasites to determine relatedness between clones and assessing the temporal and spatial relationship among them has become a standard tool in malaria epidemiological research21. Analyses can be further constrained by known case-contact information and temporal-spatial distances to refine the likelihood of a transmission event between infection pairs. As previously shown in Kenya and The Gambia, variation between parasite genotypes increases with both time and geographical distance4. Intuitively, the likelihood of a transmission event should increase with decrease genetic distance between samples. However, direct application of genetic distances to detect malaria transmission events can be affected by several factors including; human migration, mutation and recombination of P. falciparum isolates from different geographical areas22. Transmission paths can be inferred by eliminating these evolutionary confounders of relatedness using probabilistic models of genetic distance within the linear range of the relationship with evolutionary distance22. Here we employed genetic distances within 30% (the linear range of correlation with evolutionary distance), reducing uncertainties in ancestry due to recombination. Moreover, Hudson’s measure of identity-by-descent23 correlated linearly within these genetic distances, increasing confidence in connectivity and recent ancestry between pairs in detected transmission paths5. Our novel model also combined two unobserved processes; within-host and within-vector parasite dynamics and duration of infection, with chronology of sampling and travel history of sampled individuals. Transmission paths were only restricted to sample pairs (source and infected) whose chronology satisfied the parasite’s life cycle. This enabled the estimation of the direction of P. falciparum (a recombining eukaryotic infectious pathogen) flow across The Gambia. More than half of the identified person-to-person transmission pathways occurred within household and village, indicating substantial local transmission. Although some infections in the east may have originated from western Gambia, there was a higher flux of parasites from east to west, particularly at peak transmission. Therefore, interrupting transmission in western Gambia would require intensifying control efforts in eastern Gambia and probably southern and eastern Senegal, where malaria transmission is higher19.Transmission analysis can make use of genetic polymorphisms to link infectious pathogen isolates using genetic distance and phylogenetic approaches as applied for viruses and bacteria24. These can further apply recent de novo genomic variants approaches to reconstruct transmission trees25. This new whole genome approach remains largely inapplicable to most African malaria settings because of the higher complexity of infections and the poor availability of genome sequences from dense temporal and spatial samples. These difficulties explain the small number of studies investigating the relatedness and transmission pathways between malaria infections, either at small or large geographical scale in sub-Saharan Africa. Thus, SNP barcodes will continue to provide cheaper and low-density data which can be combined with our approach for transmission studies.The analysis here was limited by the relatively low proportion of samples successfully genotyped, especially during the early months of the transmission season and from the low transmission peri-urban regions near the Atlantic coast of The Gambia. Thus, latent transmission links could have been missed from low parasite density samples and missing genotypes within transmission chains. Future genotyping studies can be improved by higher resolution sampling and inclusion of genome amplification steps as demonstrated for whole genome sequencingP. falciparum isolates within households and villages, and between pairs of villages in close proximity. While this represents the local transmission hierarchy, long range transmission from high to low malaria prevalence areas were also detected. Hence, genotyping P. falciparum isolates can be used to track pathways of infection, including discriminating between indigenous and imported infections. It would be helpful to start considering a more programmatic use of these and other transmission tracking approaches in support of national malaria control programs to better target control efforts.We have shown a strong spatial hierarchy between genetic relatedness of pairs of 1. Six sites, each comprising a pair of villages, were selected. Each village pair was within 5 km and differed by prevalence of infection according to a survey carried out in November 201218. Each village was coded with a letter, from A to M  and allelic signal intensity ratios (theta). Alleles were either classified as homozygous with the reference (0), alternative (1), or heterozygous . The multilocus SNP genotype calls were aggregated for all samples and filtered to remove loci with less than 25% call rate and per sample missingness of more than 12 loci (22%). The number of retained SNPs from chromosomes 1–14 were 2, 4, 3, 6, 1, 2, 5, 4, 3, 2, 4, 2, 7, 9, respectively. The filtered dataset with maximum loci missingness of 22% included 355 subjects (32% of samples attempted) and 54 SNPs (62.7% of 86 SNPs), (Supplementary Table DNA was extracted from 3 discs of dried blood spots on filter paper using automated DNA extraction system (Qiagen). ernative , or hete30. A genotype file representing the major alleles at mixed positions was generated and used to derive summary population genetic statistics  in R statistical software (version 3.3.2). Allele frequency differences represented by average Fst over all pairs of villages was calculated with the hierFstat package. All further analyses for transmission paths were based on the “shared variant”. We retained the rarer alleles (minor variant) for isolates where loci had mixed calls. This approach considers that the presence of less common variants in same physical position between 2 barcodes maximise the likelihood of link between the infection pairs and offer additional resolution in detecting true transmission paths31. Compared to alternative approaches in which the major variants or random choice of allele at mixed positions are used, this did not significantly bias allele frequency spectra and genetic distances between pairs of isolates.In case of mixed barcodes, the complexity of infection was determined following the model in McCOIL23. Spatial distances were calculated as the geographic distance between sample sources (Km), based on their recorded latitude and longitude position. As The Gambia is uniformly low-lying, altitude was not considered in calculation of geographic distances. Temporal distance was expressed as the difference in days between sample collection dates. For all pairwise genetic distances, spatial and temporal differences were visualized as a 3D plot  between two genotypes, expressed as the percentage of the number of SNP positions with alternative alleles between pairs of barcodes for all positions without missingness in the sample pair. Additionally, relatedness was inferred by calculating barcode identity by descent (IBD) using the hmmIBD algorithmA represents the human infectious time since the previous sampling  paths and the direction from source to infected. These paths were constrained by the unobserved parasite life cycle time within the vector and length of infection between human sampling times Fig. . In the ing Fig. . It is apes Fig. . ConsideSNP genotypes of 355 isolates were fitted with 88 possible evolutionary models using a maximum likelihood approach in IQ-TREE. The TVM (transversion model) + I (invariant sites) + G4 (4 regions of variable substitution rate) was the most appropriate model by the Bayesian Information Criterion. The evolutionary distances between all pairs of isolates for the optimal tree were calculated. The evolutionary distance saturated at a genetic distance of approximately 0.3 (p-distance adjusted for missingness),  satisfied, the bivariate density was used to ascribe a probability to each possible transmission pair based on the observed genetic and temporal difference. The probability of a transmission event was zero for pairs with a predicted genetic distance outside the 95% confidence limits of the bivariate distribution. Pairwise probabilities were weighted by the probability of an observed temporal difference (based on the life cycle simulation), given that the genotype pair arose from a person-to-person transmission. A transmission probability matrix between each pair of isolates was built in this manner. To avoid ambiguity in source versus recipient pathways and vice-versa, the most likely transmission paths were chosen as the pairs with the highest probability from a maximization over columns in each row and over rows in each column of the transmission probability matrix. This resulted in a set of pathways where each source had a unique maximum likelihood for an infected subject and vice-versa. Sources with more than 2 paths to subsequent infections were classified as ‘super-infectors’  were used to draw maps and calculate summary population genetic statistics. Evolutionary models and maximum likelihood phylogenetic trees were fitted using IQTREE (version 1.5.3). MCMC simulations, graphics and modelling of evolution and transmission likelihoods were performed in MATLAB (version R2016b).Written informed consent was obtained from all participants ≥18 years old and parents/caregivers/guardians provided written informed assent for children aged 12–17 years following standardised good clinical research practice SOPs at MRCG-LSHTM. The study and protocols were approved by the Gambia Government/MRC Joint Ethics Committee (SCC 1318). All methods in this study were performed in accordance with the relevant guidelines and regulations enforce by the local EC and the MRCG-LSHTM research governance.Supplementary figuresSupplementary table 1Supplementary table 2


Plasmodium infecting humans will need to be targeted with evidence-based and concerted interventions. In this perspective, the potential barriers to achieving global malaria elimination are discussed with respect to the related diversities in host, parasite, and vector populations. We argue that control strategies need to be reorientated from a sequential attack on each species, dominated by Plasmodium falciparum to one that targets all species in parallel. A set of research themes is proposed to mitigate the potential setbacks on the pathway to a malaria-free world.Important strides have been made within the past decade toward malaria elimination in many regions, and with this progress, the feasibility of eradication is once again under discussion. If the ambitious goal of eradication is to be achieved by 2040, all species of  If malaria elimination is to be achieved by 2040, the essential infrastructure of this launchpad will need to be created within the next 3–5 years, to allow sufficient time for implementation and scale-up toward impact. Because the significant progress observed in the last decade appears to have stalled in many endemic regions,3 the time is opportune to reexamine the global strategy.In 2015, the United Nations with the Bill & Melinda Gates Foundation published a new framework for malaria eradicationPlasmodium falciparum in sub-Saharan Africa. The recent frameworks prioritize the challenges to overcome this particular malaria problem, but downplay the significance of the other five parasite species causing human malaria, which dominate the epidemiology in other regions. If global eradication of human malaria is the goal, then compared with P. falciparum, these other pathogens pose problems of greater complexity, geographic range, and technical difficulty, with fewer tools available to tackle them and more unknowns. The major challenges that arise on the pathway to malaria eradication have been stated before, by the then director of the Global Malaria Eradication Campaign:…..but it was thought useful to draw attention […] to the commonplace truth that if malaria eradication is technically possible, it is and will always remain a very serious task, and that governments should not embark on it underestimating its difficulties, or hoping that as the years pass some of the problems will automatically be cleared ….Global estimates of malaria burden are dominated by infections due to Plasmodium vivax in particular is highly prevalent across endemic Asia and South America but also occurs in Africa.5 Vivax malaria is associated with severe morbidity and with mortality, but our current clinical and public health tools for its diagnosis, prevention, treatment, and control are suboptimal in many endemic areas.6 Malaria due to Plasmodium malariae is present in most malaria-endemic settings and associated with greatly prolonged parasite carriage, severe anemia and renal impairment. In several regions in Asia, zoonotic knowlesi malaria is now the predominant cause of clinical and severe malaria.7 Similar to P. vivax, the two sympatric species of Plasmodium ovale produce dormant liver stages that can relapse months after the initial infection and are also associated with severe and life-threatening syndromes.8 Conversely, if the ultimate aim is indeed limited to eliminating the most harmful species, then the global community must reformulate and express this goal in more specific terms.Eliminating malaria one species at a time may increase the time and resources required. Rather than initially targeting the species of greatest ease to eliminate, a strategy targeting all species in parallel may be the more efficient and successful strategy. A focus on all-species elimination should not come at the expense of progress in reducing global mortality from falciparum malaria, but eradication is not just simply minimizing burden, and moreover, all species cause morbidity and mortality.9 The simple phrase “malaria eradication” obscures the complex reality of all six species of Plasmodium being driven to extinction. For a single one , a well-defined path has been charted and for a second (P. vivax), a strategic plan is rapidly evolving,2 but there are no technical agendas for P. malariae, either P. ovale subspecies or for Plasmodium knowlesi. It is recognized that current tools are inadequate for elimination10 and until now, little thought and attention has been given toward P. malariae, P. ovale sp., or P. knowlesi, and understanding of the biology and transmission dynamics of each of these species will be required. Moreover, the burden of morbidity and mortality from these species is poorly understood and almost certainly underestimated.12Eradication is a biological process whereby a living entity no longer exists in the natural world as a consequence of sustained and deliberate human interventions.13 Examples of challenges in the end game include the identification of human monkey pox infections in the final phases of the smallpox campaign, the recognition of sylvatic reservoirs of yellow fever virus, and the recent detection of canine hosts and potential aquatic reservoirs of Guinea worm.15 Furthermore, nearly all elimination efforts have been directed toward a single species of pathogen and most had the advantage of a vaccine providing sterilizing immunity, including polio and measles. Malaria raises issues of far greater complexity as it involves at least 10 intermediate host/reservoir species globally, and more than 40 major anopheline vector species. Diverse systems may need to be considered to achieve total elimination and subsequent eradication of the malarias from humanity—a one-size approach certainly will not fit all.All disease eradication programs have encountered surprising complexities in the final stages. As Bruce-Chwatt suggested decades ago, “Malaria eradication has been compared with high-mountain climbing: more obstacles appear as one approaches the summit, and at that stage every step demands an increased effort.”Four broad and interconnected ideas frame the specific scientific issues that need to be addressed for malaria elimination: diversities within a single parasite species, zoonotic and anthroponotic parasite transfers, the diverse bioecology of each parasite species, and the extensive diversities within both human hosts and vectors. Strategies for elimination and eradication that do not incorporate and address these complexities may lead to delays and even failure as each of these directly impact the themes described in the following paragraphs.16 yet in many regions where P. falciparum and other Plasmodium are co-endemic, there has been a relative increase in the proportion of infections due to the five other species, especially where PCR-based testing is performed.17Plasmodium vivax is estimated to cause approximately 14 million clinical episodes of malaria each year,12 but much larger estimates also exist.18 Once considered a benign infection, vivax malaria is now recognized to cause severe and fatal outcomes across a range of epidemiological settings.19 This parasite also causes extensive indirect mortality attributable to recurrent infections and a cumulative risk of severe anemia.20 Outside sub-Saharan Africa, P. vivax is now often the predominant cause of malaria; however, this shift in the burden of disease has yet to be appropriately weighted in research prioritization.Recent malaria control efforts have reduced significantly the global burden of malaria,P. knowlesi is the leading cause of malaria morbidity and mortality.7Plasmodium malariae contributes significant morbidity in sub-Saharan Africa and elsewhere.21 In areas of stable malaria transmission, almost 10% of clinical malaria can be attributed to P. malariae and its ability to maintain prolonged low-level parasitemias is associated with a high burden of anemia and hospitalization,23 tropical splenomegaly,24 and with the potential for irreversible kidney damage.25 The two sympatric species of P. ovale have differing epidemiology26 and are often misidentified as other species in the clinic by microscopy, suggesting underreporting. Finally, all five of these species, including P. knowlesi, have been associated with fatal outcomes in well-resourced settings.27 The common distinction between harmful and harmless Plasmodium species has been discredited by available evidence. Priority assigned to P. falciparum on this basis dangerously minimizes the clinical and public health importance of the other Plasmodium species.In Malaysia, P. knowlesi cases in Southeast Asia.28 Several zoonotic reservoirs of malaria have been confirmed,29 and most recently, a common parasite of howler monkeys Plasmodium simium,30 was found to have regular spillover into humans with initial misidentification as P. vivax in Brazil,31 from which it is nearly indistinguishable.32 Genetic evidence also suggests regular exchange of P. malariae between humans and nonhuman primates in the Venezuelan Amazon.33 Finally, possibly anthroponotic P. malariae, P. ovale, and P. vivax have been found in wild “pristine” nonhuman primate populations in Central Africa.34Over the past decade, compelling evidence has revealed continual interactions between malaria species in human and nonhuman primate reservoirs, driven largely by the discovery of widespread 9 a challenge identified during the Global Malaria Eradication era, which remains unaddressed.36 The potential burden of disease attributable to the transfer of parasites between humans and primate reservoirs is unknown, the assumption that this is negligible needs to be explored further to ensure evidence-based eradication programs.Major challenges exist in studying parasites in these primate reservoirs, and currently, there are limited tools or sampling strategies to estimate the frequency of parasite transfer between humans and primates in these populations. This lack of evidence for a stable animal reservoir of human malarias cannot be considered as evidence of absence. The geographic extents and magnitude of animal-associated malaria in humans (whether zoonotic and anthroponotic) need to be quantified to ensure that malaria eradication is an evidence-based undertaking,Plasmodium vivax has evolved an ability to sustain transmission in environments inhospitable to P. falciparum. Central to this survival strategy is the dormant liver stage (hypnozoite) capable of relapsing weeks, months, and even several years after the initial mosquito-borne infection. Furthermore, this species can persist with much lower peripheral parasitemias, with all stages of the parasite, including sexual stages (gametocytes—the parasite life stage infectious to mosquitoes), circulating before patients developing symptoms.P. vivax are able to transmit to a larger range of vectors and complete the parasite stages in the mosquito over a much wider range of temperatures than P. falciparum.37 Conversely, the sexual stages in P. falciparum infection tend to occur days to weeks after clinical presentation, allowing transmission-blocking interventions such as administration of a single dose of primaquine (PQ).38 Unlike the gametocytes of P. falciparum, those of P. vivax are inherently sensitive to most blood schizontocidal treatments and do not persist for any significant length of time following clearance of asexual parasitemia.Gametocytes of P. vivax transmission is its ability to relapse, a property that accounts for most of the subsequent recurrent infections following an initial infection.40 Hence, good coverage of P. vivax radical cure is likely to reduce transmission to a far greater extent than strategies focused solely on treatment of symptomatic patients.41 Numerous studies have shown that similar investments in malaria control measures have a large differential impact on the incidence of P. falciparum relative to P. vivax.42 In some instances, hotspots of P. vivax transmission may persist for years with a significant risk of resurgence.45 The primary reason for this is the inability to deliver safe, effective, and acceptable radical cure of the hypnozoite reservoir due to the risk of PQ-induced hemolysis in glucose 6-phosphate dehydrogenase (G6PD)–deficient patients along with the necessity of prolonged dosing (2 weeks) and attendant barriers to adherence.The main driving force of P. ovale and P. malariae, especially concerning quiescent parasite stages, should not be ignored.46Finally, although generally regarded as not posing important barriers to elimination, the highly complex and currently obscure biology and epidemiology of both species P. vivax relative to P. falciparum,47 with two important consequences. First, transmission can persist in areas with multiple potential vectors after control of the primary vector species. Second, persistent foci may be highly resilient in spite of changing ecological conditions, because of both changes in vector composition  and in biting behavior .A wider range of vectors is capable of transmitting 50 and the evidence base for many key entomological interventions outside of sub-Saharan Africa  is remarkably sparse.53 In addition, proposed interventions for areas with exophilic vectors have had limited or no impact at scale, including treated hammocks54 and topical repellants.55Many of the vector complexes outside of sub-Saharan Africa are less impacted by insecticide-treated nets because of exophagy and exophily,56 and many cryptic or presumed secondary vectors may in fact be major contributors to transmission.58 The presence of urban vectors (including Anopheles stephensi on the Indian subcontinent and the Horn of Africa59), and adaptation of Anopheles spp. larvae to polluted water both represent another potentially important barrier to elimination. Regular large-scale field surveys and concurrent laboratory studies will be required to ensure that these adaptive malaria vectors do not become both globally dispersed or resistant to interventions as Aedes spp. have become in many dengue-endemic areas.61Vector populations in sub-Saharan Africa hide unexpected complexity,62 While field work captured ample female Anopheles, the sporozoite rates of all suspected vectors were too low to support the observed malaria prevalence, and 2 years of dedicated study was required to identify miniscule jungle breeding sites for Anopheles leucosphyrus.62 Rationally targeting residual transmission may require similar levels of intensive entomology to define individual disease ecotypes.63 For subsequent targeting of these niches, housing improvements and the generally neglected, historical interventions focused around environmental modifications need be closely reexamined.66The efforts required to identify cryptic vector populations in residual transmission is highlighted by work in Borneo during the 1940s.P. vivax than in P. falciparum, because of inherently lower parasitemias, the relative insensitivity of both microscopy and rapid diagnostic tests, and the presence of undetectable parasites in the liver, bone marrow, and spleen.67 Recent studies suggest that parasite densities are approximately 5-fold lower in P. vivax infections than in P. falciparum infections,68 although the exact magnitude varies with methodology.70 This problem is compounded by the species’ transmissibility to mosquito vectors at very low levels of parasitemia, greatly increasing the size of the infectious reservoir relative to P. falciparum.71The proportion of all parasites in the human reservoir that are subpatent is far greater in 73 Even in very low-transmission settings, P. vivax genetic diversity remains significantly higher than that of P. falciparum, a likely reflection of the underlying contribution of hypnozoites,75 and such intrinsic genetic diversity provide the parasite with an increased ability to respond to challenges to its survival.76Greater genetic diversity of global parasite populations highlights the need for interventions to be tailored to specific epidemiological settings, particularly with respect to relapse patterns and incubation periods.77 The genetic diversity of host populations across P. vivax–endemic areas is extensive . Evidence suggests that P. vivax may be rapidly evolving alternate invasion pathways.78 Parasite populations have been identified capable of “invading” Duffy-negative human populations along with increasing virulence80 and genomic studies suggests that these non-Duffy antigen invasion pathways appeared very recently. Identification of novel invasion pathways in Duffy-negative individuals suggest the potential for expansion of populations to be at risk from P. vivax to include much of sub-Saharan Africa.81The impact of host genetics on risk of malaria infection is poorly understood, although there are important differences in host susceptibility to infection.P. vivax in most endemic areas because of its widespread availability, sustained efficacy, low cost, and excellent safety and tolerability profile. However, resistant strains have been identified across the vivax-endemic world, especially in Southeast Asia.82 Given widespread resistance to chloroquine and difficulties of malaria species identification, several countries have changed national policy to ACTs for all malaria infections .84 Although both P. ovale and P. malariae are assumed to be sensitive to chloroquine, data on drug susceptibility of these species are lacking.85 The dearth of research into these issues is highlighted by the total number of registered trials currently listed as “recruiting” or “completed” by species: “radical cure” for vivax  or ovale (one); “treatment” for malariae (four), and “treatment” for knowlesi .86Chloroquine remains the first-line treatment of 89 Further complexities also exist in the use of PQ. There are wide variations in prevalence and severity of G6PDd, which typically range from 1% to 30% (mean 8%) in malaria-endemic regions.90 Glucose 6-phosphate dehydrogenase deficiency in males are usually associated with an enzyme activity of less than 30% of normal levels, with some variants being 10% or lower. These “severe” (low activity) variants are highly susceptible to PQ-associated hemolytic toxicity, which may require immediate clinical management, including blood transfusion. Primaquine is also contraindicated in pregnant women as the G6PD status of the fetus is unknown, and in infants less than 6 months of age. However, these exact groups are the most vulnerable to the serious adverse health effects of recurrent vivax malaria.Primaquine, an 8-aminoquinoline, is the only currently available hypnozoiticide but causes severe drug-induced hemolysis in G6PD-deficient patients. To improve safety and tolerability, PQ is usually administered as a 14-day regimen, but this is associated with poor adherence to a prolonged course of treatment with consequent poor effectiveness.P. vivax patients be tested for G6PD deficiency before treatment with PQ; however, testing is not standard-of-care in most settings. Although rapid tests for G6PD deficiency have been developed, expansion into routine use will require considerable effort and finances. Furthermore, most qualitative G6PD assays usually return a normal result for enzyme activity greater than 30% normal, missing heterozygous females, who may have mixtures of G6PD normal and deficient red blood cells and are therefore at risk of hemolysis.The WHO recommends that all 91 More recently, an MDA campaign including PQ radical cure in Greece achieved good coverage (76%) in ≈1,200 people, but required enormous resources, and one patient required hospitalization for PQ-induced hemolysis after a false-normal laboratory-based G6PD test.92 Although high compliance can be achieved, it is a major and expensive undertaking—the Greek endeavor required multiple teams making regular household visits for all enrolled persons.92Hypnozoite carriage is asymptomatic and there are currently no diagnostics able to detect their presence—hence the only reliable way to attempt to clear out the hypnozoite reservoir in populations is through presumptive treatment to those at risk and/or mass drug administration (MDA). Mass drug administration using PQ for radical cure has been implemented at a massive scale, giving doses to millions of people in the former Soviet Republics, People’s Republic of China, and the Democratic Republic of Korea (North Korea); but differences exist in reporting and G6PDd prevalence.93 and the relative risk-benefit of PQ radical cure may vary in different epidemiological settings.94 The biological activity of PQ against hypnozoites may require the formation of reactive intermediates generated by the cytochrome P-450 isozyme 2D6 (CYP2D6). Recent clinical trials identified a spectrum of clinical responses to PQ based on the host’s CYP2D6 isoform ranging from null- to ultra-metabolizers.95 These polymorphisms are widely variable among ethnic groups, but there are currently no population-level data to assess the potential impact on PQ efficacy. One study in Indonesia found that 20 of 21 PQ treatment failures among 171 patients were indeed associated with impaired CYP2D6 genotypes and phenotypes . Interactions between parasite and host genetics may also play a role.The efficacy of PQ varies by parasite populations, which may require different dosing to prevent relapses,In summary, PQ is the only hypnozoitocidal drug now available; it has the potential for severe and fatal adverse events, major issues in patient adherence, and is contraindicated in the most vulnerable populations in need of radical cure. Hence, a safe and effective therapy for the radical cure of vivax malaria is needed. A drug currently in Phase III trials, tafenoquine, may simplify adherence, but this will require routine testing for G6PD deficiency.P. falciparum, focused on reducing the high burdens of this parasite and its associated morbidity and mortality. However, in both a practical and technical sense, none of these tools are ideally suited to either the distinct task of bringing low-level transmission to zero or dealing with all of the Plasmodium parasites and their anopheline vectors. Because all malaria species in humans contribute severe morbidity and mortality, a roadmap attacking the easiest target species first and then moving on to the next needs to be reexamined. A strategy that addresses the combination of Plasmodium species where they exist will decrease the time, resources, and energy required to achieve global malaria eradication.The diversity and complexity of the human malarias are great, presenting huge challenges to the ambitious goal of eradicating malaria by 2040. The tools currently available have been focused on Plasmodium should not come at the expense of progress in reducing global mortality from falciparum malaria; however, the goal of malaria eradication does not equate to simply minimizing disease burden. As all species can cause morbidity and mortality,8 malaria eradication must by definition encompass them all. However, if the ultimate goal is indeed limited to eliminating the most harmful species, then the global community must reformulate and express this goal in more specific terms. A more comprehensive research agenda is required to ensure continued progress (A focus on all species of progress . The cur


Plasmodium falciparum antigen histidine-rich protein 2 (HRP2) and the malaria-conserved antigen lactate dehydrogenase (LDH) for P. vivax and other malaria species. The performance of RDTs and their utility is dependent on circulating antigen concentration distributions in infected individuals in a population in which malaria is endemic and on the limit of detection of the RDT for the antigens.Malaria rapid diagnostic tests (RDTs) primarily detect  Plasmodium falciparum antigen histidine-rich protein 2 (HRP2) and the malaria-conserved antigen lactate dehydrogenase (LDH) for P. vivax and other malaria species. The performance of RDTs and their utility is dependent on circulating antigen concentration distributions in infected individuals in a population in which malaria is endemic and on the limit of detection of the RDT for the antigens. A multiplexed immunoassay for the quantification of HRP2, P. vivax LDH, and all-malaria LDH (pan LDH) was developed to accurately measure circulating antigen concentration and antigen distribution in a population with endemic malaria. The assay also measures C-reactive protein (CRP) levels as an indicator of inflammation. Validation was conducted with clinical specimens from 397 asymptomatic donors from Myanmar and Uganda, confirmed by PCR for infection, and from participants in induced blood-stage malaria challenge studies. The assay lower limits of detection for HRP2, pan LDH, P. vivax LDH, and CRP were 0.2 pg/ml, 9.3 pg/ml, 1.5 pg/ml, and 26.6 ng/ml, respectively. At thresholds for HRP2, pan LDH, and P. vivax LDH of 2.3 pg/ml, 47.8 pg/ml, and 75.1 pg/ml, respectively, and a specificity ≥98.5%, the sensitivities for ultrasensitive PCR-confirmed infections were 93.4%, 84.9%, and 48.9%, respectively. Plasmodium LDH (pLDH) concentration, in contrast to that of HRP2, correlated closely with parasite density. CRP levels were moderately higher in P. falciparum infections with confirmed antigenemia versus those in clinical specimens with no antigen. The 4-plex array is a sensitive tool for quantifying diagnostic antigens in malaria infections and supporting the evaluation of new ultrasensitive RDTs.Malaria rapid diagnostic tests (RDTs) primarily detect  Low-density malaria parasite infections are highly prevalent in low-transmission areas, often presenting in individuals with no clear malaria symptoms but with high potential for transmission of parasites –3. The a–The most widely used malaria diagnostic methods, such as blood film microscopy and rapid diagnostic tests (RDTs), have insufficient sensitivity to detect low-density parasite infections . Molecul5–Plasmodium lactate dehydrogenase (pLDH)  or e (pLDH) , 12. pLD species . C-react species , 15.P. vivax LDH and to monitor host inflammatory response via CRP.Fluorescent bead-based assays have been developed to support creation of more sensitive assays for HRP2 . A sensiThis study describes the validation of the 4-plex array. This 4-plex assay was then used to investigate the antigen concentration distributions and CRP in asymptomatic malaria infections.All specimens used in this study have been described previously , 19. WhoP. falciparum-parasitized red blood cells on day 0. On day 7, participants were admitted to the study unit, treated with an antimalarial, and observed for at least 72 h. All specimens were processed and stored at −80°C prior to any molecular and antigen testing.In Nagongera , 100 random households were selected and visited to recruit children from the ages of 6 months to 11 years, as well as their primary caregivers, into a surveillance cohort, as previously described . In a KaP. falciparum LDH and P. vivax LDH proteins were purchased from CTK Biotech , and MyBioSource . CRP was from HyTest . Monoclonal antibodies recognizing P. falciparum HRP2, P. falciparum LDH, and P. vivax LDH were obtained from commercial vendors, and the custom anti-HRP2 antibodies were produced under contract at Precision Antibody  , and recombinant bia, MD) . The detP. falciparum-parasitized red blood cells from culture-adapted strain ITG (ATCC) were used as HRP2 and pLDH reference material. P. falciparum parasites were cultured in type O+ human erythrocytes diluted at 4% hematocrit in RPMI 1640 medium supplemented with HEPES (Gibco), sodium bicarbonate (Sigma), hypoxanthine (Sigma), gentamicin (Sigma), and either 10% human serum (Interstate Blood Bank) or 0.5% AlbuMax II (Invitrogen), using a modified in vitro culture technique (d-sorbitol three times prior to harvesting them at ring stage (>99%), and the parasitemia of the culture was determined by microscopic analysis , and recombinant P. vivax LDH  were used on each ELISA kit to make standard curves.echnique . Parasitanalysis . CulturePATH  developed a partnership with Quansys Biosciences  to develop a customized human malaria 4-plex array designed to test blood specimens.http://www.quansysbio.com/assay-development/). Fourteen anti-HRP2, five anti-pan LDH, and three anti-P. vivax LDH antibodies from different vendors were evaluated in custom spotted micro wells to identify the best-performing antibody pairs for each target antigen, HRP2, pan LDH, or P. vivax LDH. The best-performing mouse monoclonal capture and detection antibodies recognizing Plasmodium antigens were selected from comprehensive checkerboard antibody pairing against recombinant proteins at 10 pg/ml for HRP2 and 1,000 pg/ml for pan LDH and P. vivax LDH. The signal-to-noise ratio was calculated with chemiluminescence signals from antigen wells and blank wells.Q-Plex technology is based on printing capture antibodies in 350- to 500-μm spots at the surface of polypropylene 96-well plate to capture target antigens  were optimized to maximize sensitivity and specificity of the assay. Calibrator concentrations were confirmed using HRP2 and pLDH concentrations within reference material measured by two commercial ELISA kits.The protocols and procedures from phase II were verified with a lot of product produced in this phase. Company verification parameters included precision, dilutional linearity, edge effect, drift effect, interference from rheumatoid factors, sensitivity, and specificity.On the day of analysis, frozen blood samples were thawed and kept on ice until use. Calibrators and samples were prepared according to the manufacturer’s protocol. After addition of 50 µl of calibrators and samples, the plate was incubated at room temperature with shaking at 500 rpm for 2 h. Plates were then washed with proprietary wash buffer using an automated plate washer. A 50-µl aliquot of detection mix containing proprietary ingredients, including biotinylated antibodies and buffer, was added to each well, and the plate was incubated with shaking for another hour and then washed again. For detection, a 50-µl aliquot of horseradish peroxide (HRP)-conjugated streptavidin solution was added to each well and then incubated with shaking for 30 min. After a final wash, a 50-µl aliquot of chemiluminescent substrate solution was added to each well and the chemiluminescent intensity from the array spots in each well was immediately measured using the Q-View Imager Pro (Quansys Biosciences) at an exposure time of 300 s.P. falciparum cultures and P. vivax-infected blood, were used to assess dilutional linearity. Accuracy and precision were determined using a set of specimens spiked with exogenous HRP2, P. vivax LDH, and CRP at three different concentrations within the assay dynamic range. Diagnostic sensitivity and specificity were determined with clinical specimens from Myanmar and Uganda, described previously (P. vivax infection (n = 94); specimens with P. falciparum infection (n = 106); and negative specimens (n = 197). The 4-plex array was also performed on IBSM challenge study specimens  is the lowest concentration of analyte that can be detected in a diluted liquid specimen with a specific degree of probability. LLD was calculated using the following formula: LLD = 2 × (standard deviation of negative-control pixel intensities before negative well subtraction) × lower limit of quantification (LLOQ)/(difference between pixel intensity of lowest standard and negative control). The LLOQ and upper limit of quantification (ULOQ) are defined as the lowest calibrator point and the highest calibrator point, respectively, for which the concentration can be back-calculated on the nonlinear regression curve with 80% to 120% accuracy and a coefficient of variation (CV) of less than 30%. The prozone effect was also evaluated as presence of false-negative or false-low results due to presence of excess amounts of antigens or antibodies in immune reactions .P. falciparum culture specimens (samples 1 through 3) and two P. vivax-infected clinical specimens (samples 4 and 5) in 2-fold dilution  in sample diluent. Each dilution was treated as a test sample. Dilutional linearity was determined by accuracy, investigating the relationship between expected theoretical concentration and the observed concentration.Dilutional linearity of the 4-plex array was assessed by diluting five high-concentration blood specimens, including three P. vivax LDH or CRP at the expected concentrations , and they were evaluated by measuring each of the biomarkers in 20 tests over 2 weeks or in a single day, respectively. The precision of the method at each concentration was reported as CV%, expressing the standard deviation as a percentage of the mean calculated concentration. The accuracy was determined by expressing the observed concentration as a percentage of the expected concentration.Interassay and intra-assay precision and accuracy were determined by repeated analysis of the control samples spiked with recombinant HRP2 and x axis and the sensitivity (the true positive rate) on the y axis. At each cutoff value, sensitivity and specificity were calculated with their confidence interval. The cutoff value to yield a specificity of 98.5% was selected. Cutoff values in protein concentration were extrapolated from a long-term calibrator curve using GraphPad Prism 6.0 software .Each clinical specimen was tested both neat and diluted 100-fold unless noted otherwise. To determine the optimal cutoff values for malaria antigens in pixel intensity for each biomarker, receiver operating characteristic (ROC) analysis was conducted by plotting the 1-specificity  on the P values of ≤0.05 were considered significant. Statistical analyses were performed using GraphPad Prism 6.0 software.Statistical analyses were performed to summarize data descriptively, and included means, standard deviations, and CV%. All tests for significance were two sided, and Plasmodium antigens  and CRP was developed by redesigning a previously described 2-plex array .A sensitive and quantitative multiplexed immunoassay that can simultaneously detect three ex array . InitialCheckerboard analysis of paired antibodies was performed to select antibody pairs with the highest signal-to-noise ratio . The array was optimized to maximize specificity and sensitivity and limit cross-reactivity from soluble interfering biomaterials. The final antibodies and calibrators used in the 4-plex array are shown in Table S4 in the supplemental material.To determine the minimum detectable concentration and the range of quantifiable concentrations for each biomarker, the calibrator curve for each biomarker was established using the 5-parameter logistic fit. The accuracy and precision of each calibrator were examined from 12 runs. The assay results showed the acceptable range of 80% to 120% for the back-calculated concentrations of the upper 6 to 7 serial dilution points for each biomarker . For this calibrator evaluation, LLD, LLOQ, and ULOQ values for each biomarker are shown in Dilutional linearity of the array was examined to determine if assay results are proportional to level of dilution in assay diluent. Each of the assay results were within the acceptable range of 80% to 120% for sample dilution of up to 128-fold .The results of intra-assay precision demonstrated limited variation in the mean analyte concentrations of spiked specimens, with CV% within the acceptance criterion of 10% or less, as shown in P. vivax LDH. To extend quantitative range for the malaria antigens, each of the clinical specimens was screened neat and at 100-fold dilution. Infection status in each specimen was determined by two different molecular tests as described previously  values from each ROC curve for the malarial biomarkers were 95.8% or greater  gives a crude estimation of the association between parasite density and protein concentrations. The results revealed that HRP2 showed poor correlation with parasite density , whereas pan LDH showed good correlation to parasite density . Importantly, a wider variation of HRP2 levels were shown in specimens within the ranges of 0.1 to 100 parasites/µl, but pan LDH and P. vivax LDH levels in P. falciparum-positive specimens and P. vivax-positive specimens showed a small variation throughout the given ranges of parasite density  for pan LDH in P. falciparum-positive specimens, and 0.003 pg/parasite  for P. vivax LDH in P. vivax-positive specimens . The amount of P. vivax LDH per circulating parasite was statistically significantly lower for P. vivax parasites than the pan LDH for P. falciparum parasites (P < 0.0001).The amount of circulating HRP2 and pLDH per parasite was calculated at the low range of parasite density (0.01 to 100 parasites/µl). The median values were 0.049 pg/parasite  for HRP2 in P. falciparum 3D7 parasites on day 0 and treated on day 7 with an antimalarial drug. From days 0 through 11, 238 specimens from these volunteers were tested by both qPCR and the 4-plex array. The mean concentration for each malaria antigen was compared to the mean parasitemia as measured by qPCR. Both HRP2 and pan LDH antigens had similar kinetics, with an increase in concentration toward day 7 and a subsequent decrease posttreatment (R2 = 0.853), while HRP2 showed poor correlation (R2 = 0.241)  infections were investigated. IBSM allows malaria infection to be done in a controlled environment . Sixteenreatment . However= 0.241) . The impP = 0.053) . However, median values of CRP from the HRP2-positive subgroup (P < 0.01) and pan LDH-positive subgroup (P < 0.005) of P. falciparum specimens were statistically significantly higher than that of the noninfected group . The same was not observed for P. vivax.CRP levels of noninfected individuals were compared to levels in infected individuals to assess the relationship between CRP levels and low parasite density malaria infection. There was no significant difference in the CRP levels of the two groups as determined by qPCR . The effects of other potentially interfering factors, such as rheumatoid factor, hepatitis C, toxoplasmosis, human African trypanosomiasis, dengue, leishmaniasis, Chagas disease, and schistosomiasis have yet to be investigated.Two significant challenges encountered in the development of the quantitative assays for pLDH and HRP2 were the observation of the prozone effect in the HRP2 assay and the wide variability in the reactivity of antibodies to different sources of pLDH. First, the prozone effect resulting in dampening signal at high HRP2 concentrations that caused narrowing of the dynamic range of this assay. This phenomenon has also been observed in HRP2-based RDTs –32. This–Good positive correlation of pLDH concentration and parasite density was observed in specimens collected from Uganda and Myanmar, but the HRP2 concentration correlated poorly with parasitemia for the same specimens. These results were confirmed in the IBSM study . Good co34–P. falciparum were equivalent. However, P. vivax LDH levels were statistically lower in P. vivax compared to pan LDH levels in P. falciparum samples when normalized for parasite count. The poor sensitivity observed in RDTs for P. vivax malaria is likely the compound result of poor assay reagents, low parasite densities in infection, and relatively low availability of antigen per parasite. In future studies, the root cause may be resolved through quantitative proteomic analysis via matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS). Development of more sensitive tests for P. vivax malaria will need to take these considerations into account.The HRP2 assay achieved a lower LOD than that of the pLDH assay, possibly as a result of the availability of antibodies with better binding kinetics and larger numbers of epitopes per molecule. Despite this, within the constraints of the number of clinical specimens tested for this study, it was observed that HRP2 and pan LDH levels in P. falciparum infections with confirmed antigenemia (either HRP2 or pan LDH). The increase was not statistically significant in confirmed parasite infections for the same sample set. Likewise, no significant differences were observed in CRP levels for P. vivax infection by parasitemia or antigenemia. Further investigation with a larger sample set is necessary to understand the association between CRP levels and low parasite density infections.CRP is often used as an inflammatory biomarker that is indicative of malaria parasitemia and other infections associated with febrile illness , 15. In The 4-plex array is a useful tool for further characterizing low density infections, which prevail in settings approaching elimination and have significant transmission potential. The tool is also essential to validate the performance of new ultrasensitive RDTs and to inform their impact in malaria elimination strategies , 19, 37.


Plasmodium falciparum parasites from their blood showed genetically identical microsatellite and allele patterns. Both had severe malaria. It took more than a week before the diagnosis was made. If symptoms are indicative and there is a plausible exposure, malaria should be considered even if patients have not travelled to an endemic area.Two cases of presumably airport-acquired falciparum malaria were diagnosed in Frankfurt in October 2019. They were associated with occupation at the airport, and  Here, we report two cases of malaria that have been diagnosed outside an endemic area. Both cases occurred in conjunction with occupation at an airport, indicating that a vector-competent mosquito was imported by air traffic. The parasites found in both cases were found to have shown genetically identical microsatellite and allele patterns.Plasmodium falciparum with a parasitaemia of 7% was demonstrated microscopically  scan showed signs of cerebral swelling without necessity for neurosurgical intervention and acute kidney failure occurred . The patient was transferred to the University Hospital Frankfurt the next morning and treatment was continued with five doses of artesunate 2.4 mg/kg intravenously and oral follow-up treatment with atovaquone/proguanil  for 3 days. He made a full recovery after 16 days of inpatient treatment. Until 40 days after discharge, he remained well and free of symptoms.Similarly to case 1, he did not have any history of recent travel to malaria endemic areas, nor had he previously had blood transfusions or underlying illnesses. He was also working at Frankfurt Airport, and on the same night shift as Case 1 but he did not recall any insect bites.P. falciparum was suspected in both patients and whole blood samples were sent to the National Reference Centre for Tropical Pathogens at the Bernhard Nocht Institute for Tropical Medicine (BNITM) in Hamburg, Germany. In order to identify possible clonality of the two P. falciparum strains, we used polymerase chain reaction (PCR)-based parasite typing targeting the microsatellite PfRRM of the rif gene family [var gene family [P. falciparum strains. Fragment length analysis was performed using the 3130 XL Genetic Analyzer with GeneMapper analyser software version 4.1 .An autochthonous infection with e family , the Dufe family  and the e family . The marGenetic typing of the two samples showed identical DNA patterns for PfRRM and DBLα respectively . FurtherOn 11 October 2019, the public health authority of the city of Frankfurt contacted the employer of the patients and the airline responsible for the service hall. A list of people who worked in this aircraft service hall during the previous 4 weeks and who were on sick leave was provided; nine people were identified and contacted by phone. One of them reported mosquito bites approximately 10 days prior to the interview, but none had any symptoms of malaria.Anopheles mosquitoes was found. There were no further cases of malaria in the following 2 weeks in the region. The Robert Koch Institute  was asked to screen all infectious disease notifications in the Frankfurt Airport region for malaria cases without a travel history to endemic areas between 25 August and 20 October. None was reported.The airline confirmed the presence of airplanes returning from three falciparum malaria endemic countries in sub-Saharan Africa in the service hall in which both patients had worked during 21 days before the symptom onset (27 September). During this period, one of them noticed mosquito bites. Considering the day temperatures of more than 20 °C during the time of likely exposure, company rooms, the aircraft service hall and the surrounding areas were inspected for possible mosquito breeding sites. No stagnant water as a possible source of reproduction of (imported) Here we describe two cases of falciparum malaria that were presumed to have been acquired at Frankfurt Airport. While 896 imported malaria cases to Germany were reported in 2018  there haP. falciparum infections in employees of an airport in a non-endemic country that were found to be genetically identical. The simultaneous onset of symptoms also suggests a common source of the infections. P. falciparum strains in the two cases showed identical allele and microsatellite patterns using PCR-based fingerprinting. In our opinion, the autochthonous transmission of malaria parasites in central Europe remains highly unlikely. In the two severely ill cases described here, the time from symptom onset until the final diagnosis was long (9 and 12 days), a fact which has also been previously reported in airport malaria [To our knowledge, there were no previously documented cases  malaria ,14. ThisAnopheles species (e.g. A. plumbeus) showed susceptibility for P. falciparum in experimental infections but no natural parasite transmission has been proven so far [A. plumbeus was suspected, however [Anopheles species in the specific region around Frankfurt. The population of A. plumbeus in the Frankfurt area is thought to be small [P. falciparum by an infected imported mosquito seems to be highly likely. Considering sporadic reports of autochthonous malaria especially in southern Europe in the last years [Indigenous n so far ,17. In t however . We did be small . In the st years , it is i