Plasmodium falciparum culture  and Plasmodium vivax infected samples . Precision was defined as the %CV of the assay results at each level of parasitemia and the %CV value for our system was lower than that for microscopic examination for all densities of parasitemia. The limit of detection analysis showed 95% probability for parasite detection was 0.00066112%, and a high correlation was observed among all three methods. The sensitivity and specificity of the system was both 100% (n = 21/21) and 100% (n = 50/50), respectively, and the system correctly identified all P. vivax and P. falciparum samples. The automated microscopic malaria parasite detection system offers several advantages over conventional microscopy for rapid diagnosis and parasite density monitoring of malaria.Rapid diagnosis and parasitemia measurement is crucial for management of malaria. Microscopic examination of peripheral blood (PB) smears is the gold standard for malaria detection. However, this method is labor-intensive. Here, we aimed to develop a completely automated microscopic system for malaria detection and parasitemia measurement. The automated system comprises a microscope, plastic chip, fluorescent dye, and an image analysis program. Analytical performance was evaluated regarding linearity, precision, and limit of detection and was compared with that of conventional microscopic PB smear examination and flow cytometry. The automated microscopic malaria parasite detection system showed a high degree of linearity for  Malaria is one of the most dangerous infectious diseases and causes 229 million infections and 409,000 deaths worldwide, according to the World Health Organization (WHO) estimates . Early aMicroscopic examination of Giemsa-stained peripheral blood (PB) smears is considered the gold standard for malaria diagnosis. Implementation of other diagnostic methods, including rapid diagnostic tests (RDTs) ,4 and poParasite density serves as one of the diagnostic criteria for severe malaria infection and its monitoring is important for the diagnosis and treatment of malaria. Parasite density is associated with patient prognosis ,8,9. ParParasitemia determination by microscopic examination has some limitations in that the method is labor-intensive, requires expert staff, and is highly dependent on the performance of the observer, leading to variability and inconsistency in results ,14,15. TTo overcome these problems, researchers have explored automated malaria detection and parasitemia assessment techniques based on the recognition of digital images using artificial intelligence algorithms to enhance the efficacy of the microscopic examination of malaria parasites. Previous studies have mostly focused on the development of a computer vision algorithm to analyze the microscopic images of Giemsa-stained thin or thick blood smears for malaria diagnosis ,21,22,23Plasmodium falciparum culture and malarial parasite-infected samples and was compared to that of conventional microscopy and flow cytometric parasitemia determination.Here, we describe an automated microscopic detection system for malaria detection and parasitemia determination. The system includes the following four elements: a fluorescent dye for malarial parasite staining, a plastic chip, an automated microscopy platform, and an image analysis algorithm for malaria detection and parasitemia determination. Although the standard method for malaria diagnosis is Giemsa staining, fluorescent staining for malaria is very fast (about 15 min), and has yielded better sensitivity and accuracy owing to the higher contrast of the malarial parasite against the background and lower stain variability. After optimization of test conditions, the analytical performance of this new system was evaluated using P. falciparum and 18 Plasmodium vivax) and 50 control samples were collected at the Korea University Guro Hospital from June 2017 to July 2020. We included samples of patients who were suspected of malaria but tested negative as control samples. Both malaria positive and negative samples included in the study were diagnosed by conventional microscopy and confirmed by PCR. All samples were collected as ethylenediaminetetraacetic acid (EDTA) anticoagulated venous blood specimens and were processed immediately. Parasitemia of P. falciparum positive patient samples ranged from 0.05% to 4.75% and the parasitemia of P. vivax positive patient samples ranged from 0.03% to 0.22%. The study was conducted in accordance with the guidelines of the Declaration of Helsinki, and was approved by the Institutional Review Board of Korea University Guro Hospital .A total of 21 malaria positive samples . The whole blood sample obtained from a healthy volunteer without malaria infection was also diluted to a hematocrit level of 3%. The resuspended malaria culture and diluted whole blood sample were mixed and the adjusted to various parasite concentration . Dilution of malaria culture to various parasite concentrations was immediately carried out and parasite concentrations in each diluted sample were measured by microscopic examination of Giesma-stained thick and thin PB smears immediately before and after dilutions. To assess the lower limit of detection (LOD), lower parasitemia samples were prepared by serial dilution , and the parasite concentrations were calculated accordingly.2EDTA tubes . Parasitemia was determined by counting parasites per 200 or 500 WBCs, depending on the number of parasites. Parasitemia was calculated on the basis of the patient’s WBC count. The number of parasites per μL was divided by the patient’s RBC count to calculate the % infected RBCs. In a thin PB smear, the percentage of infected RBCs was estimated after counting a minimum of 20 fields of the thin film [The conventional microscopic examination for malaria diagnosis was performed by the Giemsa-stained thick and thin PB smears under 1000× magnification. Thick and thin PB smears were prepared on glass microscopic slides using venous blood collected into Khin film ,28.Parasitemia was estimated with flow cytometry using SYBR Green I and CD235a-PE. In brief, the malaria-infected RBCs were washed with PBS, and 50 μL of the washed sample was incubated with 25 μL of 1:1000 diluted SYBR Green I and CD235a-PE for 15 min at room temperature. Before analysis, the stained samples were washed twice with PBS and resuspended in 1 mL of PBS. For analysis, the RBCs were gated by the forward light scatter (FSC) versus side scatter (90°) (SSC) dot plots, and over 5000 events were acquired for each dot plot. The green and red fluorescence  was used to determine malaria-infected RBCs. Parasitemia was estimated by analyzing scattergrams from the computer software and by calculating the infected RBC percentage among the gated RBCs from FL2.2  × 252 (D) × 360 (H) mm in size and weighs 8 kg A. The op2 B. The CCThe plastic chip  is a disposable chip made of a plastic optical material, polymethylmethacrylate (PMMA). The chip is 75 (W) × 25 (D) mm in size and 1.65 mm in height and contains two microfluidic channels of dimensions 27 (W) × 7.4 (D) × 0.05 (H) mm C. Each cP. vivax may be similar in size when stained using SYBR Green I. For the differentiation of small lymphocytes and gametocytes of P. vivax, we applied APC-conjugated monoclonal antibodies against the CD45 antigen . The type and concentration of the fluorescent dye was determined through an optimization process. In total, 10 μL of the blood samples was incubated for 15 min at room temperature with 10 μL of 1:1000 diluted SYBR Green I and 5 μL of three fluorochrome-conjugated monoclonal antibodies against the CD45 antigen, and 25 μL of the sample was dispensed into the plastic chip for analysis.The malaria parasites were initially stained using SYBR Green I , a fluorescent dye that stains nucleic acid and other nucleic acid-containing structures such as leukocyte nuclei were also stained as well as the malaria parasites. Some small lymphocytes and gametocytes of P. falciparum and P. vivax. For malaria diagnosis and parasitemia calculation, the light source was switched between green, blue and red in one field of view and three images were obtained for each light source. RBCs and malarial parasites were detected and counted from the images obtained for the green and blue light sources, respectively.Automated system for malaria detection and parasitemia determination was developed and validated using microcopist confirmed image set of RBC detection and segmentation algorithm was developed based on the Circle Hough transform algorithm and RBCs were classified according to the detected radius, local maxima, and gradient threshold. For malarial parasite detection, the median filter and adaptive histogram equalization techniques were applied for noise removal and enhancement of image resolution, respectively. The algorithm segmented the parasite signal using connected-component labeling, and classified as the malaria signal depending on the detected area. If a signal suspected to be for malarial parasite matched the coordinates of an RBC in the image obtained from the green light source, it was considered as an infected RBC and the number of these signals was divided by the number of RBCs for parasitemia determination. If the signal suspected to be for malarial parasite was not located within the RBC but CD45 negative, then the signal was considered as malaria parasite, most likely gametocyte A. The imP. vivax is known to have larger infected RBCs, more frequently observed with Ameboid trophozoites, and exhibits a larger chromatin of parasites than P. falciparum [P. falciparum and P. vivax samples.lciparum ,11. Howep < 0.05 was considered significant.Linearity was evaluated using simple linear regression between the observed value for parasitemia measured by the automated microscopic malaria parasite detection system and the expected value for parasitemia as measured by microscopic examination of PB smears. Precision was assessed as the percentage coefficient of variation (%CV) at each parasitemia level obtained from repeated measurement, where the %CV was calculated as the ratio of the standard deviation to the mean multiplied by 100. Probit analysis was performed for LOD evaluation using low-parasitemia samples (ranging 0.00009% to 0.005%). The parasitemia measurements by all three methods were compared and analyzed using Pearson’s correlation tests. A value of n = 21/21) and 100% specificity (n = 50/50). The automated microscopic malaria parasite detection system correctly identified P. falciparum (3/3) and P. vivax (18/18) using the proposed classification algorithm.The validation of automated microscopic malaria parasite detection system was achieved using a new set of images obtained from patient samples and malaria culture as described above, independent of the images used for image analysis algorithm validation. A total of 71 samples, including 21 malaria-positive (12 samples at diagnosis and 9 follow-up samples) and 50 malaria-negative samples were collected. When the results obtained by conventional microscopy were considered as true, the automated microscopic malaria parasite detection system showed 100% sensitivity  and P. vivax-infected patient samples (percentage infected RBCs ranging from 0.03% to 0.22%). The linearity of the flow cytometric method was also evaluated using P. falciparum culture. For P. falciparum culture, the automated microscopic malaria parasite detection system showed a high degree of linearity compared to microscopy, with a coefficient of determination (R2) of 0.958 (p = 0.005). Flow cytometry also revealed high linearity . The automated microscopic malaria parasite detection system showed similar results for P. vivax infected patient samples   .P. falciparum culture and from 0.03% to 0.22% for P. vivax infected patient samples. Evaluation of precision of P. falciparum detection by the automated microscopic malaria parasite detection system revealed a %CV value ranging from 7.33% to 34.67%. Microscopic examination of PB smears and flow cytometry showed %CV values ranging from 9.41% to 37.07% and 6.98% to 19.29%, respectively. Among the three methods, flow cytometry was the most precise technique for all levels of parasitemia. The automated microscopic malaria parasite detection system was more precise than microscopic examination of PB smear for all levels of percentage infected RBCs . At least one infected RBC counted per sample was considered as a positive result. The automated microscopic malaria parasite detection system showed a positive rate of 100% above 0.0010% but 90% at a concentration of 0.000625%, 80% at a concentration of 0.00050% and 30% at a concentration of 0.0003125%. Probit analysis indicated that 95% probability LOD for parasite detection was 0.00066112% .A total of 30 measurements of the diluted whole blood samples from healthy volunteers, as blank samples, were made to assess limit of blank (LOB). In all blank samples, the percentage infected RBCs was 0%, resulting in an LOB value of 0%. Probit analysis was used for LOD assessment. The LOD for the automated microscopic malaria parasite detection system was estimated by measuring 10 aliquots for each density of parasitemia using P. falciparum culture and found a high correlation between the automated microscopic malaria parasite detection system and microscopic examination , the automated microscopic malaria parasite detection system and flow cytometry , and microscopic examination and flow cytometry . Comparison between the automated microscopic malaria parasite detection system and microscopic examination using P. vivax-infected patient samples also showed a good correlation, but the value was lower than that assessed using the P. falciparum culture .We compared the results of percentage infected RBCs determined by different malarial parasite counting methods using In the present study, we developed an automated microscopic malaria parasite detection and quantitation system. We propose this system for measuring malarial parasitemia and density monitoring in the clinical setting. After development and optimization of the system, we evaluated its performance in terms of linearity, precision, and LOD.The sample preparation process for automated microscopic malaria parasite detection system is very fast, and the instrument is easy to use. The estimated manufacturing cost of instrument is about USD 18,000 and the running cost is less than USD 3 per test. The automated microscopic malaria parasite detection system is a platform built using a fluorescent dye for malaria parasite staining. The standard method for malaria diagnosis is Giemsa staining but fluorescent staining for malaria has yielded better sensitivity and accuracy . Moreove2 value of 0.931 and 0.958). The %CV value varied depending on the parasitemia level, but the automated microscopic malaria parasite detection system showed better performance in terms of precision for both P. falciparum and P. vivax samples. The difference in %CV values was noted, especially in samples with low parasitemia. In case of samples with low parasitemia, microscopic examination could take 20 to 30 min for monitoring parasite density, and it is likely that the automated technology will yield more consistent results, thereby overcoming the variability of results obtained by microscopic examination of PB smears. The performance of flow cytometry was better than that of the automated microscopic malaria parasite detection system for all evaluated aspects, but application of flow cytometry for parasitemia estimation in the clinical setting has many limitations such as difficulty in accessibility and high cost of the instrument as well as each test run.The performance of the automated microscopic malaria parasite detection system was compared with that of microscopic examination as well as flow cytometry. The results of the automated microscopic malaria parasite detection system showed a good fit to the expected values with a high degree of linearity (Rn = 80) may further improve its sensitivity. This LOD seems better than those of the previously developed computer image-based automated malaria diagnostic systems and malaria RDTs. The LODs of the World Health Technology autoanalyzer and the Global Good Fund prototype have been estimated to be 140 and 100 parasites/μL, respectively [The determined LOD for the automated microscopic malaria parasite detection system was 0.00066112%. Considering that the normal RBC count range is 4,500,000/μL to 5,000,000/μL, the LOD value can be estimated to be about 30 parasites/μL to 33 parasites/μL. However, increasing the number of scanning images in the automated microscopic malaria parasite detection system over the standard scan , suggesting that automated microscopic malaria parasite detection system may improve the sensitivity of malaria detection as compared with the conventional microscopic method. Taken together, automated microscopic malaria parasite detection system is a rapid, easy-to-use, and accurate method for the detection and monitoring of malaria infection and may serve as a valuable tool in clinical practice and research studies.


Plasmodium infection than healthy individuals. Herein, the study was aimed at verifying the hypothesis that increased fasting blood glucose (FBG) promotes parasite growth as reflected by increased parasite density.Adults with diabetes mellitus (DM) in malaria-endemic areas might be more susceptible to Plasmodium sp. Infection were measured in peripheral blood samples photometrically and by polymerase chain reaction (PCR)-assays, respectively. Daily physical activity and weather data were documented via smartphone recording. For the complex natural systems of homeostatic glucose control and Plasmodium sp. life cycle, empirical dynamic modelling was applied.Seven adults without DM were recruited in rural Ghana to determine the relationships between FBG and malaria parasite load. Socio-economic data were recorded in questionnaire-based interviews. Over a period of 6 weeks, FBG and Plasmodium sp. infection (parasite density: 240/µL). In this participant, convergent cross mapping (CCM) for 34 consecutive days, showed that FBG was causally affected by parasite density (p < 0.02), while the reciprocal relationship was not discernible (p > 0.05). Additionally, daily ambient temperature affected parasite density (p < 0.01).At baseline, four men and three women  showed a median FBG of 5.5 (5.1–6.0 mmol/L); one participant had an asymptomatic Plasmodium sp. density. Longer observation periods and larger samples are required to confirm these findings and determine the direction of causality.In this study population living in a malaria-endemic area, time series analyses were successfully piloted for the relationships between FBG and The online version contains supplementary material available at 10.1186/s12936-022-04076-y. Infectious diseases such as malaria, HIV, and tuberculosis have long been the main contributors to morbidity and mortality in sub-Saharan Africa (SSA) . It is ePlasmodium sp. density have been investigated in adults.At the same time, non-communicable diseases (NCDs) have gained considerable importance globally and in SSA, including an “epidemic” of diabetes mellitus (DM) . WorldwiPlasmodium falciparum infection was reported among Ghanaian adults with T2DM in urban Ghana [Anopheles gambiae due to diabetes-related alterations in olfactory signals and thus, experience increased exposure to infectious bites [P. falciparum virulence in the blood of uninfected type 1 and type 2 diabetics[Previously, a 46% increased odds of an Ghana . While tus bites , 15. Secus bites . In addidiabetics. Third, diabetics. Fourth,diabetics, which idiabetics, which hPlasmodium parasite density. Here, the term causality is employed in relation to statistics and time series. A modern method for testing causal relationships was used [Therefore, the major goal of this study was to establish the causal relationships between FBG concentrations and malaria parasite density using time series analysis of a prospective observational pilot study, among adults in rural Ghana. The specific objectives were (i) to determine the time-varying associations between FBG and parasite load, and (ii) to identify causal interaction between FBG and was used , which, was used , is tailFrom September to October 2019, a prospective observational pilot study with seven adults who resided in Agogo, Ashanti Akim North District, Ashanti Region, Ghana, was conducted. Subsistence farming, trading, and mining are the main income sources in this region . EligiblThe study protocol adhered to the principles laid down in the Declaration of Helsinki and was reviewed and approved by the Committee on Human Research, Publication and Ethics of the School of Medical Sciences/Komfo Anokye Teaching Hospital, Kwame Nkrumah University of Science and Technology, Kumasi, Ghana (CHRPE/AP/507/18). All participants gave written informed consent.+ RT device , providing plasma equivalents of FBG in mmol/L. Malaria parasites were microscopically identified on Giemsa-stained  thick and thin blood films. Parasite density was quantified by expert microscopists who examined microscopy fields of the thick film corresponding to 500 white blood cells (WBCs); the average WBC count was set as 8000/µL. Thin films served for species differentiation. In addition, venous whole blood samples were collected on filter paper . DNA was extracted using the QIAmp DNA Blood Mini kit . Plasmodium species and sub-microscopic infections were then identified by semi-nested multiplex PCR assays [Plasmodium infections, crossing point (Cp) values of quantitative real-time PCR analyses were used, using commercially available primers and probes for Plasmodium falciparum, Plasmodium vivax, Plasmodium ovale, and Plasmodium malariae  on a Roche LightCycler 480 device (https://lifescience.roche.com). In that, a high Cp value corresponds to a low parasite load, and vice versa. Plasmodium infection was defined by the more sensitive PCR assay in the absence of current fever (axillary temperature ≥ 37.5 °C) and was not treated during the present study. Clinical malaria was defined as microscopically visible parasites plus current fever and was treated according to Ghana Health Service guidelines.FBG was measured in finger-prick fasting blood samples applying a portable HemoCue Glucose 201R assays . For furDemographic and socioeconomic data were documented in face-to-face interviews by trained study personnel and comprised age (years), gender (m/f), residence (Agogo or else), known DM status (yes/no/unknown), known malaria status (yes/no/unknown), level of formal education (none/primary/secondary/tertiary/other), current occupation (subsistence farming/trading/business/public servant/unemployed), and the number of household’s physical assets (list of 11 items). Further, meteorological data and physical activity levels were assessed as factors potentially influencing both, FBG  and para2.In addition, body weight to the nearest 100 g and height to the nearest cm were assessed in duplicates, using the Person Scale DT602  and the statometer SECA 213 , respectively. Body mass index (BMI) was calculated as weight (kg) over squared height (m) and is expressed in kg/mThe baseline characteristics of the study population are presented as median and range for continuous variables and as absolute numbers for categorical data.P. falciparum density served as the affected variable (here: Cp as a proxy measure).For establishing causal links between FBG and malaria parasite load, linear statistical methods such as correlation analysis were inappropriate. In fact, the metabolic processes regulating blood glucose and parasite density over time are inherently non-linear . Also, lE) for each potential causal combination  in CCM analysis was determined following the procedure of Deyle and colleagues [ρ lagged 1-time step for the largest possible number of points in the surrogate time series. Further, all-time series were normalized to zero mean and unit variance. Due to the paucity of observation points, a leave-one-out cross-validation was performed [R [For significance testing, 100 surrogate time series that were comparable to the measured data but fulfilled the null hypothesis of no relationship between the two, time series variables were generated , followilleagues . This coerformed . Two misormed [R .2, and four individuals had a BMI ≥ 25.0 kg/m2 One woman presented with a history of gestational diabetes. The baseline axillary body temperature was in the normal range for all participants . Also, the baseline FBG was in the normal range .Table P. falciparum infection at low parasite density (baseline: 240 parasites/µL). This infection was asymptomatic throughout the study period. As shown in Fig. Only one individual had microscopically visible For the one participant with asymptomatic malaria infection, Cp values remained stable when FBG ranged between 4.5 and 5.5 mmol/L. The time series of FBG and parasite density Fig. a, b indiTo test the robustness of the CCM analysis in this dynamical system of FBG homeostasis and regulation of parasite density, additional relationships with relevant covariables were examined. Regarding causal links with Cp values, weather parameters and body temperature were assessed for the participant with asymptomatic malaria infection. The mean daily ambient temperature was 25.2 °C (SD = 1.9 °C) with the maximum recorded being 31 °C and the minimum 23 °C. The results of the CCM analysis for air temperature and Cp values in this participant are shown in Fig. Regarding causal relationships with FBG, the duration of physical activity (min/day) and step count (steps/day) among all study participants were analyzed. As shown in Table In this prospective observational study in rural Ghana, the causal relationships between the time series of FBG and measures of malaria parasite load was analysed. During the study, only one out of seven participants had an asymptomatic malaria infection at low parasite density. In this participant, CCM results showed that Cp values (as a measure of submicroscopic parasite density) affected FBG concentrations. But the effect of FBG on Cp could not be detected. In addition, temperatures affected parasite density, and physical activity affected FBG.Plasmodium sp. lacks the ability to store energy in the form of polysaccharides such as glycogen. Therefore, the parasite relies on an exogeneous glucose supply [The results agree with the fact that e supply –42. Withe supply , giving e supply , 44, 45,e supply . In addie supply .Even though CCM did not detect the reciprocal relationship between FBG and Cp, this observation could be explained by the fact that it was only possible to detect and follow-up malaria parasite load in one participant, thereby increasing the chances for type II error. Nonetheless, increased glucose production and higher FBG in patients with uncomplicated falciparum malaria, possibly driven by increased hepatic gluconeogenesis in the host, have been previously reported –49. In aAnopheles vector, ambient temperature modifies the interaction between gametocyte density and infectivity to the mosquito vector [The relationships between weather parameters and parasite load have not been sufficiently explored. However, in SSA, a statistically significant variation of mean parasite density  has been previously reported to be influenced by different seasons . In the o vector .Also, the observed relationship between physical activity and FBG accords with existing scientific evidence. Physical activity has a glucose-lowering effect  whereforPlasmodium sp. Lastly, diabetes has been indicated to be a key risk factor for severe malaria infection [Pending verification of the hypothesis, individuals with chronically increased blood glucose need to be advised to strongly adhere to malaria protective measures. This may have implications for patients with poor blood glucose control living in SSA. In fact, only 37% of adults with T2DM in rural Ghana receive glucose-lowering medication, and of these, only 63% have good glucose control . In addinfection . TravelePlasmodium sp. density. However, the results of the study need to be interpreted with caution. Even though the number of participants was fair enough for time series analysis, only one individual developed Plasmodium sp. infection, thereby leading to type II error and thus limiting the ability to verify the hypothesis that higher FBG leads to higher parasite load. No examinations were conducted on Sundays, which interrupted the time series assessments. Yet, for the participant with asymptomatic infection, any issues arising from missing data were overcome by leave-one-out cross validation and linear interpolation. Still, independent studies with longer observation periods and larger sample sizes are warranted for a better in-depth explanation of this causal relationship and to verify the direction of causality.This is the first study, in which time series analysis has been applied, to determine the causal relatedness of FBG to In conclusion, CCM has been applied to investigate whether FBG among Ghanaian adults is causally linked with malaria parasite density. Since metabolic processes regulating blood glucose and malaria parasite growth are inherently non-linear, the EDM framework for non-linear dynamics in time series data was utilised. In this healthy study population without DM, the common observation of malaria-related hypoglycemia has been verified. Yet, the confirmation of the original hypothesis is pending until longer and larger time series analysis can provide causal links between chronically increased FBG and malaria parasite growth.Additional file 1: Table S1. FBG parameters for all patients. Note that only patient 2 was infected with Plasmodium falciparum. Comparing variability  of infected and uninfected patients using Shapiro-Wilk-Test with Levene’s Test revealed that variability does not differ significantly (P > 0.05).


Plasmodium life cycle with special emphasis on how complement-fixing antibodies contribute to protective immunity. We briefly touch upon the action of anaphylatoxins, the assembly of membrane attack complex, and the possible reasons underlying the resistance of infected erythrocytes towards antibody-mediated complement lysis, relevant to their prolonged survival in the blood of the human host. We make suggestions for further research on effector functions of antibody-mediated complement activation that would guide future researchers in deploying complement-fixing antibodies in preventive or therapeutic strategies against malaria.Antibody immunity against malaria is effective but non-sterile. In addition to antibody-mediated inhibition, neutralisation or opsonisation of malaria parasites, antibody-mediated complement activation is also important in defense against infection. Antibodies form immune complexes with parasite-derived antigens that can activate the classical complement pathway. The complement system provides efficient surveillance for infection, and its activation leads to parasite lysis or parasite opsonisation for phagocytosis. The induction of complement-fixing antibodies contributes significantly to the development of protective immunity against clinical malaria. These complement-fixing antibodies can form immune complexes that are recognised by complement receptors on innate cells of the immune system. The efficient clearance of immune complexes is accompanied by complement receptor internalisation, abrogating the detrimental consequences of excess complement activation. Here, we review the mechanisms of activation of complement by alternative, classical, and lectin pathways in human malaria at different stages of the  Malaria remains one of the major causes of severe morbidity and mortality globally. In 2019 alone, there were 229 million clinical episodes of malaria causing 0.4 million deaths. The most vulnerable groups include children under five years of age and pregnant women and the heaviest burden of disease is concentrated in sub-Saharan Africa ]. In preIn people living in malaria-endemic areas, immunity to malaria is gradually acquired following repeated exposure so that over time individuals become relatively protected from malaria and its complications [reviewed in ]. This nThe leading malaria vaccine candidate RTS,S induces antibodies to the circumsporozoite protein, and both naturally acquired and vaccine-induced antibodies fix complement and engage Fc receptors –10, discPlasmodium parasites within human erythrocytes is responsible for clinical symptoms of malaria. The parasite has a complex life cycle initiated by a Plasmodium-infected mosquito bite .The asexual replication of Plasmodium life cycle, together with the mechanisms that parasites adopt to evade complement attack to promote their survival. A deeper understanding of antibody-mediated complement activation across the Plasmodium life cycle will provide insights into harnessing complement activation in antibody-mediated protection in malaria.We focus on the importance of understanding the roles of antibody-mediated complement activation in these different stages of the via three pathways, namely the classical, alternative, and mannose-binding lectin (MBL) . They aiewed in .The binding of globular head domains of the complement factor C1q with the antibody constant (Fc) domain regions of IgG hexamers or IgM binding antigen activates the classical complement pathway . The abiP. falciparum within the human host. We discuss the mechanisms of clearance of the parasites via complement-mediated lysis that are brought about by activation of the classical complement pathway in the presence of ICs. We also review the influence of intrinsic and extrinsic properties of both host and parasite that could have a potential impact on complement-dependent lysis of different parasitic stages.In the next section, we provide a brief overview of antibody-mediated complement fixation on different stages of Plasmodium spp. sporozoites enter the blood vessels and move through the circulation and invade hepatocytes, where they divide to produce merozoites which are released to initiate blood-stage infection  and IgG3  such as MSP1iewed in ], as impiewed in . The antBoth naturally acquired  and vaccGametocyte-IEs are the infective stages of the parasite that enable transmission of infection from human to mosquito [reviewed in ]. There When an Anopheles mosquito takes a blood meal, host serum components like complement proteins and antibodies are taken in along with the gametocyte-IEs Figure 1P. falciparum sexual stages revolve around the major gametocyte surface antigen, Pfs230 . The coP. yoelii showed a downregulation of the level of expression of CR1 on monocytes or macrophages, a similar though less pronounced downregulation was reported in patients infected with P. falciparum and P. vivax compared to non-infected controls  that would generate complement-fixing antibodies for protective immunity.Plasmodium antigens.Determine the association between antibody Fc region variations and antibody functionality for optimum complement activation by opsonised Other than erythrophagocytosis, erythrocytes with C3b-containing ICs are taken up by splenic reticuloendothelial cells. This may lead to stripping off of the CR1 from the erythrocyte surface , 79, 80.via the cleavage of C5, by C5 convertase  by innate immune cells are not discussed in detail in this review.Irrespective of the exposure of blood-stage parasites to serum complement over a relatively prolonged asexual blood-stage Figure 1One reason why the IEs are resistant to complement-mediated lysis may be that the IEs may have low number of target sites for antibody-binding and complement activation. If this is below a certain threshold, even an excess of antigen-specific antibodies and serum complement may not activate the classical complement pathway .P. falciparum-IEs also utilise soluble complement factors like factor I (FI) and factor H (FH) (Membrane-bound CRPs that act at different phases of the complement cascade tightly regulate complement activation [reviewed in ]. These r H (FH) –93.ex vivo study of subjects with severe malaria anaemia, the levels of expression of CRPs were lower on uninfected erythrocytes than on IEs  indicate that neutralising antibody alone requires high titres , merozoites , and sexual stages of P. falciparum  can be greatly augmented by the presence of complement. The binding of C1q to IgG-opsonised merozoites and sporozoites has shown to be associated with protective immunity in malaria. We also emphasise that IEs are resistant to complement mediated lysis in comparison to other parasitic stages. This may be due to some intrinsic properties of IEs, including the expression of complement regulatory proteins, an insufficient number of antigenic sites as targets for antibody binding, and the orientation of antigens on the parasite surface for restricted antibody binding for complement deposition. The attempts made to evaluate the underlying mechanisms of antibody-mediated complement activation during malaria will provide a deeper understanding of the processes that mediate complement-mediated protection and/or evasion.In this review, we summarise antibody-dependent complement activation by different stages of parasites during DR gathered papers, organised, and drafted the review. Both EA and SR provided intellectual feedback, critically revised, and approved the final manuscript for submission. All authors contributed to the article and approved the submitted version.DR is supported by the Melbourne Research Scholarship from the University of Melbourne and Miller Foundation Top-up Scholarship. The work of EA and SR is supported by grants from the National Health and Medical Research Council of Australia (GNT1092789 and GNT1143946), and by the Centre for Research Excellence in Malaria Elimination (GNT1134989).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Plasmodium falciparum have spread across the Greater Mekong Subregion compromising antimalarial treatment. The current 3-day artemether-lumefantrine regimen has been associated with high treatment failure rates in pregnant women. Although ACTs are recommended for treating Plasmodium vivax malaria, no clinical trials in pregnancy have been reported.Artemisinin and artemisinin-based combination therapy (ACT) partner drug resistance in +). The primary endpoint for P. falciparum infections was the PCR-corrected cure rate and for P. vivax infections was recurrent parasitaemia, before delivery or day 63, whichever was longer, assessed by Kaplan-Meier estimate.Pregnant women with uncomplicated malaria on the Thailand-Myanmar border participated in an open-label randomized controlled trial comparing dihydroartemisinin-piperaquine (DP), artesunate-mefloquine (ASMQ) and a 4-day artemether-lumefantrine regimen  were randomized to either DP (n=170), ASMQ (n=169) or AL+ (n=172) treatments. Successful malaria elimination efforts in the region resulted in premature termination of the trial. The majority of women had recurrent malaria . Recurrence-free proportions  for vivax malaria were 20.6% (5.1–43.4) for DP (n=125), 46.0% (30.9–60.0) for ASMQ (n=117) and 28.7% (10.0–50.8) for AL+ (n=126). DP and ASMQ provided longer recurrence-free intervals. PCR-corrected cure rates (95% CI) for falciparum malaria were 93.7% (81.6–97.9) for DP (n=49), 79.6% (66.1–88.1) for AMSQ (n=55) and 87.5% (74.3–94.2) for AL+ (n=50). Overall 65% (85/130) of P. falciparum infections had Pfkelch13 propeller mutations which increased over time and recrudescence occurred almost exclusively in them; risk ratio 9.42 (95% CI 1.30–68.29). Among the women with falciparum malaria, 24.0% (95% CI 16.8–33.6) had P. vivax parasitaemia within 4 months. Nausea, vomiting, dizziness and sleep disturbance were more frequent with ASMQ. Miscarriage, small-for-gestational-age and preterm birth did not differ significantly among the treatment groups, including first trimester exposures (n=46).Between February 2010 and August 2016, 511 pregnant women with malaria (353 P. falciparum-infected pregnant woman in this area of widespread artemisinin resistance. Vivax malaria recurrences are very common and warrant chloroquine prophylaxis after antimalarial treatment in this area.DP was well tolerated and safe, and was the only drug providing satisfactory efficacy for ClinicalTrials.gov identifier NCT01054248, registered on 22 January 2010.The online version contains supplementary material available at 10.1186/s12916-021-02002-8. Plasmodium vivax also is prevalent.Malaria contributes substantially to poor pregnancy outcomes in tropical areas including miscarriage, preterm birth, stillbirth, small for gestational age (SGA) at birth, and at worst, maternal mortality , 2. ChemArtemisinin-based combination therapy (ACT) is recommended as the first-line treatment for both falciparum and non-falciparum malaria . In pregPlasmodium falciparum. Multiple drug resistance in P. falciparum is widespread, and in Indonesia and Oceania, P. vivax is often highly resistant to chloroquine  in pregnant women in this area between 2004 and 2006 [Pfkelch13 propeller mutations was very low at that time [The efficacy of the extended ALand 2006 . Howeverhat time . The 5-dhat time  would haP. vivax infections as well as women in the first trimester of pregnancy to be included in an RCT in pregnant women. One earlier small RCT in Columbia included 20 pregnant women in their first trimester and compared efficacy and tolerability, but not pregnancy outcomes, of chloroquine and amodiaquine [P. vivax malaria in pregnancy. As radical cure with primaquine cannot be given in pregnancy, the efficacy reported here is a composite of blood stage cure and relapse suppression. P. vivax recurrence was very common in this study, as in Colombia where 30% of women experienced recurrences within 120 days after chloroquine treatment [P. vivax occurred in approximately one quarter of women within 4 months, and it was more common than P. falciparum recrudescence. In vivax malaria, recurrence of P. vivax occurred in approximately two thirds of women. Subsequent recurrences of P. vivax were suppressed by the slowly eliminated antimalarials, with mefloquine slightly outperforming piperaquine and, as expected, lumefantrine [This study reports the largest number of diaquine . There hreatment . Recurrefantrine . Most, i+ or DP with higher proportions of nausea, vomiting, and sleep disturbance [P. falciparum and P. vivax). This has been implemented as a consequence of this trial.There were no surprises in the comparative tolerability evaluations. ASMQ was less well tolerated than ALturbance , 26, 36.turbance , 8. ACTsturbance . The preturbance , but simturbance . The posturbance , 38. ThiP. vivax in this area [+ had never been tested in this area, DP was not in use in pregnancy, the observed effects such as vomiting are objective and the findings from this trial are consistent with tolerability from pregnancy treatment studies elsewhere [Limitations of this study include termination of the RCT before the sample size was reached and loss-to-follow-up before delivery 8.4%), both of which reduced the statistical power of the study. Chloroquine was not included partly because of concerns over chloroquine-resistance his area  althoughhis area , which i%, both olsewhere .P. vivax recurrence but was less tolerated than DP or AL+. Identification of species is thus important for selecting the first-line drug. The main cause of recurrent malaria for all infections was P. vivax relapse. Prevention of vivax malaria recurrence requires suppressive prophylaxis with chloroquine. Hence treatment on the Thailand-Myanmar border for uncomplicated malaria is currently the same for pregnant and non-pregnant patients: DP for falciparum and chloroquine for vivax malaria. Importantly, because primaquine radical cure cannot be given, chloroquine prophylaxis is now given from the third week after treatment until term. For the fetus, all three ACTs appeared safe as treatment including in the first trimester.In this RCT of three ACTs conducted on the Thailand-Myanmar border during a period of increasing artemisinin resistance, DP was the only ACT providing greater than 90% cure of falciparum malaria. On the other hand, ASMQ provided the longest post-treatment suppression of Additional file 1: S1 Table. Definition of primary efficacy endpoint by initial species. S2 Table. Dose of each compound given to the participants. S3 Table. The baseline characteristics of women with Plasmodium falciparum mono-infection. S4 Table. The baseline characteristics of women with Plasmodium vivax mono-infection. S5 Table. The baseline characteristics of patients with Plasmodium falciparum infection, including both P. falciparum mono-infection and co-infection of P. falciparum and Plasmodium vivax. S6 Table. Cumulative proportion of fever and parasite clearance of falciparum and vivax for each treatment arm among pregnant women with history of fever or documented fever at enrolment. S7 Table. Description of gametocyte carriage, congenital and placental malaria. S8 Table. Univariable and multivariable analyses of the risk of PCR-corrected treatment failure in pregnant women with uncomplicated falciparum malaria using a Cox proportional hazard model. S9 Table. Univariable and multivariable analyses on the risk of PCR-corrected treatment failure in pregnant women with uncomplicated falciparum malaria using a Cox proportional hazard model with Pfkelch13 mutations. S10 Table. Prevalence of symptoms before and after treatment. S11 Table. Description of pregnancy outcomes. S12 Table. Details of pregnant women who had miscarriage or stillbirth. S13 Table. Details of congenital abnormality and ICD-10 coding. S14 Table. Details of neonatal mortality. S15 Table. Univariable and multivariable linear regression analyses of the characteristics associated with gestational week at birth among those who had malaria in pregnancy. S16 Table. Univariable and multivariable linear regression analyses of the characteristics associated with SGA z-score . S17 Table. Birth outcomes in pregnant women who had malaria and were enrolled in the first trimester. S1 Figure. Monthly number of pregnant women with malaria for each malaria species over the study period (from February 2010 to August 2016). S2 Figure. Kaplan-Meier survival curves for antimalarials in pregnancy: Panel a. recurrence of any malaria species, Panel b. Plasmodium falciparum PCR-corrected efficacy censoring intercalated Plasmodium vivax malaria, Panel c. P. falciparum PCR-uncorrected efficacy , and Panel d. P. vivax parasitaemia after P. falciparum mono-infection. S3 Figure. Kaplan-Meier survival curves for parasite clearance of Plasmodium falciparum (Panel a) or Plasmodium vivax (Panel b) for each treatment arm. S4 Figure. Fractional change in haematocrit from baseline during the follow-up for each species (Panel a) or treatment stratified by malaria species (Panel b).


Plasmodium falciparum (Pf) sporozoite (SPZ) malaria vaccines. A large, pivotal trial of the safety and efficacy of the radiation-attenuated Sanaria® PfSPZ Vaccine against P. falciparum is planned for 2022. This study assessed the incidence of malaria at the phase 3 study site and characterized the influence of socio-demographic factors on the burden of malaria to guide trial design.Extensive malaria control measures have been implemented on Bioko Island, Equatorial Guinea over the past 16 years, reducing parasite prevalence and malaria-related morbidity and mortality, but without achieving elimination. Malaria vaccines offer hope for reducing the burden to zero. Three phase 1/2 studies have been conducted successfully on Bioko Island to evaluate the safety and efficacy of whole A cohort of 240 randomly selected individuals aged 6 months to 45 years from selected areas of North Bioko Province, Bioko Island, was followed for 24 weeks after clearance of parasitaemia. Assessment of clinical presentation consistent with malaria and thick blood smears were performed every 2 weeks. Incidence of first and multiple malaria infections per person-time of follow-up was estimated, compared between age groups, and examined for associated socio-demographic risk factors.There were 58 malaria infection episodes observed during the follow up period, including 47 first and 11 repeat infections. The incidence of malaria was 0.25 [95% CI ] and of first malaria was 0.23 [95% CI ] per person per 24 weeks . Incidence of first malaria with symptoms was 0.13 [95% CI ] per person per 24 weeks . Multivariate assessment showed that study area, gender, malaria positivity at screening, and household socioeconomic status independently predicted the observed incidence of malaria.P. falciparum malaria.Despite intensive malaria control efforts on Bioko Island, local transmission remains and is spread evenly throughout age groups. These incidence rates indicate moderate malaria transmission which may be sufficient to support future larger trials of PfSPZ Vaccine. The long-term goal is to conduct mass vaccination programmes to halt transmission and eliminate  Plasmodium falciparum (Pf). Children and pregnant women are the most vulnerable for dying of the disease, and progress with burden reduction has plateaued [The burden of malaria is concentrated in sub-Saharan Africa, where 94% of the estimated 229 million malaria cases occurred in 2019, the large majority caused by lateaued .P. falciparum sporozoite (PfSPZ) malaria vaccine approach, with radiation-attenuated Sanaria® PfSPZ Vaccine and the chemo-attenuated Sanaria® PfSPZ-CVac, the two leading products under development [The Government of Equatorial Guinea (EG) is partnering with Medical Care Development International (MCDI), Ifakara Health Institute (IHI), Swiss Tropical and Public Health Institute (Swiss TPH), and Sanaria Inc. to evaluate the safety and efficacy of the whole elopment , 5. PfSPThree phase 1/2 studies of PfSPZ Vaccines have been conducted in EG. The first study was the first clinical trial in the history of EG and evaluated the safety and tolerability of PfSPZ Vaccine in adults . The secAn efficacious pre-erythrocytic vaccine, like PfSPZ Vaccine, which interrupts malaria transmission since the development of asexual blood stages and associated sexual stages is prevented . TherefoInformation on the expected incidence of malaria and malaria with symptoms on Bioko Island is needed to properly design the upcoming trial, including estimating the appropriate sample size , 10. HenThe study was conducted in communities in an urban/peri-urban area of Malabo District in the Bioko North Province on Bioko Island, EG, where the capital city, Malabo, is located. The population of Malabo District is estimated to be 191,671 individuals living in 68,306 households with an average household size of 2.8 individuals . The population is composed of several ethnic groups, including the Bubi, Fang, Annobones, Ndowe, Bisio, and Fernandino. The study area  . The maievalence –15.Anopheles melas and Anopheles coluzzii [The major vectors maintaining residual transmission on Bioko are coluzzii . Significoluzzii . Since 2coluzzii . Despitecoluzzii , 18.For this study, communities with high malaria prevalence (parasitaemia above 15% by rapid diagnostic test (RDT)) based on a 3-year average (2016–2018) of data from the annual cross-sectional malaria indicator survey were selected. The selected communities were mainly from the western and southwestern regions of the Malabo District; they were categorized based on the administrative boundaries and geolocation into four study areas (area I–IV)  augmented by assessing any acute febrile illnesses (passive surveillance). Information on co-morbidities that occurred was collected from participants during study visits through updates on medical history, physical examination, and laboratory investigations. The study targeted the enrollment of 240 participants split evenly into three age groups: 6–59 months, 5–17 years, and 18–45 years.Permission was obtained from local government officials and community representatives to use the household database from the BIMCP/NMCP to identify household members to be invited to participate in the study . SubsequA multistage sampling was done, first communities were selected primarily due to relatively high parasite prevalence in non-travelling children aged 2–14 years and proximity to the Sampaka Health Centre (no more than 6 kms from the facility) based on the BIMCP/NMCP 2018 health census. Individual demographic information from the 15 selected communities, was aggregated to the household level to determine the number of eligible individuals per age group permanently residing in inhabited households within the study area. Households with at least one eligible individual were included in the sampling frame, representing 20,240 eligible individuals residing in 7901 eligible households.Thereafter, Python 2.7, PANDAS package was used to randomly select and order individuals from unique households for each of the three study age groups. The number of individuals to sample within each of the communities was pre-determined to ensure a representative geographic distribution of sampled individuals proportionate to community sizes. Once an age group was filled, individuals were only invited to participate if contributing to the unfilled age groups.Potential adult participants, or the parents or guardians of potential pediatric participants, present during household visits were asked to come to SHC to provide written consent, 9–17-year-olds were asked to provide written assent, and 6–8-year-olds were asked to provide verbal assent. Screening evaluation included the history of previous illnesses, and physical examination included vital signs and anthropometric measurements. Blood samples were collected for analysis of malaria parasitaemia , the latter performed retrospectively), complete blood count with differential, and biochemistry including alanine aminotransferase (ALT), aspartate aminotransferase (AST), total bilirubin, creatinine, and glucose. A urine sample was collected for human chorionic gonadotropin pregnancy testing in women aged 9–45 years. Socio-demographic data were also collected.Participants were considered eligible for the study if they were permanent residents of the study area, age between 6 months and 45 years, and provided consent/assent. Participants were excluded if they had a history or clinical manifestations of serious or chronic disease requiring frequent medical care including human immunodeficiency virus (HIV) infection, clinical tuberculosis, sickle cell disease, malignancies, diabetes mellitus, hypertension, mental illness, or seizures. Additional exclusion criteria were mid-upper arm circumference (MUAC) < 11.5 cm for children under 5 years of age , grade 3 abnormal laboratory parameters , pregnancy, and history of allergy or serious adverse reaction to artemether/lumefantrine (AL). Participants with issues that increased the risk of non-adherence to study procedures, including the intention to move from the study area during the study period, were excluded.® Cipla Ltd, India) according to manufacturer’s instructions [Eligible participants were given a directly observed curative 3-day, six-dose course of artemether–lumefantrine (AL)  tubes and kept in a cooler box to maintain ambient indoor temperature until reaching the laboratory.Participants were treated based on TBS results. For those TBS positive participants who were at the clinic, treatment with an anti-malarial was initiated before they were allowed to go home. For those who were seen at home they were invited back to the clinic by the community health workers and were then reviewed again at the clinic and an anti-malarial treatment initiated.Compliance with study visits and procedures was encouraged by the provision of a study calendar to participants, by using multiple contact methods , by home visits by community mobilizers, and by provision of transportation to the study center. Participants were compensated for time spent during scheduled study related clinic visits (approximately 12 United States dollars), but not for home visits.Study doctors were available to see study participants 24 h–7 days a week. Participants were encouraged to visit the SHC or call the study doctors whenever they were sick. Medical history, physical examination and laboratory investigations were done as needed for clinical evaluation. All participants with history of fever had their blood checked for malaria by TBS, RDT and later qPCR.Plasmodium genes namely the Pan-Plasmodium 18S RNA sequence (Pspp18S) and the P. falciparum-specific acidic terminal sequence of the var genes (PfvarATS). The human Ribonuclease P gene (HsRNaseP) was used as a DNA extraction and qPCR amplification control. All qPCR assays were run in duplicate and both non-template control (Molecular grade nuclease-free water) and P. falciparum 3D7 DNA were included as negative and positive controls, respectively. The sample was considered positive if the quantitation cycles (Cq) for each of the two replicates was Cq < 40 for Plasmodium spp. gene and Cq < 28 for qPCR amplification control. Haematology parameters were established using ABX Pentra 60 C+  and biochemistry parameters using Cobas Integra 400 Plus . Urine pregnancy tests were done using Hexagon hCG 1-step rapid tests .Thick and thin blood smears and the RDTs for assessment of malaria parasites were prepared from samples collected during home visits or at the study clinic. These were prepared and independently analysed at the SHC laboratory to allow blinding and standardization of the preparation and reading of the results. All remaining study related laboratory analyses were done at the public health laboratory in Baney which is located 24 km from the SHC. TBSs were prepared and read as described previously . In shorR. Data were entered and verified by independent teams.Information was collected on paper-based study case reports forms and these were entered into a customized electronic database, Castor EDCThe design assumed an incidence of new malaria infections (parasitaemia) over 24 weeks of 10% for each age group. In order to detect an incidence rate of 10% per person per 24 weeks with a relative precision of 20% (from 8 to 12%) for each age group at 90% confidence level, and considering a 15% rate of loss to follow up, a sample of 80 participants was targeted for recruitment in each age category.Data were analysed using STATA version 15 . Descriptive statistics were used to summarize the data. Household socioeconomic status (SES) was calculated as a weighted sum of data on household possessions and utilities, using principal components analysis and the scores divided into quintiles. Elements included in the measure: ownership of radio, TV, bicycle, refrigerator, mobile phone, computer, bed net, and source of energy . The incidence rate of first infections was computed for 24 weeks of follow up as the total number of first infections observed after enrolment divided by total person time at risk.P. falciparum infection divided by the total number of participants at the start of follow up for each age category.Person-time was censored for loss to follow up or identification of a first infection. Incidence rate for multiple infections was computed for the 24 weeks of follow-up as the total number of malaria infections developing after enrolment divided by the total person-time at risk. Twenty-eight days were censored off from time at risk after each treatment of malaria infection. Kaplan Meier survival curves were used to compare time to first infection between groups, and Cox regression analysis was used to explore factors associated with first incidence of malaria infection. Lastly, proportional incidence of infection was computed as the number of participants developing first Following the ordered sampling frame of participants identified from the BIMCP/NMCP household database who lived in the selected study areas see “”, 545 weThe median age in years of those enrolled was 9.80 with range (IQR) from 1.02 to 42.51  10.0, 18.8), 6 participants in group 1, 11 participants in group 2, and 16 participants in group 3  per person per 24 weeks follow-up. The overall incidence of first infection was 0.23  per person per 24 weeks. The observed incidence rate for first infections was similar in the three age categories: 0.22 , 0.26  and 0.20  infections per 24 weeks in groups 1, 2 and 3, respectively (Table Incidence of infection appeared to be greater in: (1) males, 0.31 95% CI 0.22, 0.45) versus females, 0.16 , the trend observed in all areas, and (2) those living in Areas III, 0.26  and IV, 0.39  compared to those in Areas I, 0.14  and II, 0.12 . Incidence was lowest in the highest socioeconomic group , 42% (n = 10) and 47% (n = 8) of the total cases of malaria recorded in groups 1, 2 and 3 respectively. Of these, 27 were first cases of malaria with symptoms. The incidence rate for all cases of malaria with symptoms was 0.14  and for first malaria with symptoms was 0.13 . The incidence rates of first cases of malaria with symptoms in groups 1, 2, 3 were 0.16 , 0.10 , 0.11 , respectively. Younger children and those living in Area IV tended to have more clinical episodes, but the numbers were small and the differences between areas were not statistically significant  and 157.48 , respectively. The geometric mean parasite density for all cases of malaria with symptoms was 1898 (95% CI 431–8364), 357 (95% CI 88–1449) and 112 (95% CI 26–493) for age groups 6 to 59 months, 5 to 17 years and 18 to 45 years, respectively.Common illnesses that occurred in the study participants included respiratory tract infections, gastroenteritis, diarrhoea, and skin infections. About 70%  of illnesses required medical attention during the 24 weeks of follow up, and the young age group had more events compared to the older age groups  had significantly higher burden compared to the more urban locations. This heterogeneity indicates that malaria control interventions have variably impacted malaria prevalence and morbidity depending on the setting; importantly, however, nowhere have they interrupted transmission, a situation shared with many other settings in Africa –24. The Furthermore, significant local transmission remains despite the impact of the study itself, which, due to active surveillance and prompt treatment, could be expected to reduce the incidence of malaria infection in the participants beyond the effects of ongoing control measures, as has been observed in several sites in Africa , 26. HenThe relatively similar incidences of both asymptomatic and symptomatic malaria among the age groups suggests that intensive control has shifted the age pattern of the risk of infection away from children compared to before the intervention , as seenSurprisingly, the results of this study indicate there was a significantly higher risk of malaria infection in males than in females, but no higher risk for other illness events including pneumonia and diarrhea. This sex difference in malaria infection, contrasting with similar studies in moderate transmission areas in Africa , extendeTravel has been documented to be an important contributing factor to the maintenance of local transmission in Malabo, and Bioko Island as a whole , 35. HowAs expected, the incidence of malaria with symptoms was lower (13%) compared to asymptomatic infections (20%), likely due, at least in part, to intense follow up and prompt treatment  as the mOverall, there were few illnesses during follow up which is similar to what has been observed in other studies in Africa where good clinical care is provided as part of a study , 26. As This study was conducted to serve as a baseline for the conduct of a large safety and efficacy study of PfSPZ Vaccine that has so far shown promising results in EG and elsewhere , 7, 40. The study provided a good platform for capacity development, building the skills and confidence of the research team; the ability to conduct assessments of malaria incidence will be critical for tracking the success of future efforts to eliminate malaria from Bioko Island.Intensive malaria control efforts in Bioko Island have resulted in a large reduction in the prevalence of malaria; local transmission remains, however, and the risk of infection is now spread relatively evenly through all age groups, a finding which is consistent with areas that have changed from high to lower transmission areas. The remaining burden of malaria offers an opportunity to properly evaluate the efficacy and safety of PfSPZ Vaccine and establish the safety and efficacy data required to receive marketing authorization (licensure).


PIEZO1 was associated with protection from severe malaria. Here, we performed a genetic association study of this gain-of-function variant in a large case-control study including 4149 children from the Ashanti Region in Ghana, West Africa. The statistical analysis did not indicate an association with protection from severe malaria and, thus, providing evidence against a strong protective effect of the PIEZO1 E756del variant on severe malaria susceptibility.Recently, a common genetic variant E756del in the human gene  PIEZO1 variant E756 (rs59446030) in infections with Plasmodium falciparum was provided by Ma et al. [n = 4149) on severe malaria (SM) in the Ashanti Region of Ghana, including 2413 SM cases and 1736 unaffected controls. This study group has proven to be a valuable resource in the past [ATP2B4 influencing the susceptibility to SM, which subsequently has been replicated in several other studies [First evidence for a role of the human E756 rs5946030 in PIEZO1 variant E756 (rs59446030) in the Ghanaian study group was done by allele-specific hybridization with fluorescence resonance energy transfer (FRET) in a LightCycler® 480 device [n = 4149)  and, at a lower frequency, an insertion  (Table n = 10) the three different PIEZO1 alleles identified in our study group were validated by Sanger sequencing  after corrections due to multiple testing.We applied the logistic regression model as described by Nguetse et al., which tests for the effect of the heterozygous E756del genotype (WT/del) on SM compared to the homozygous E756 genotype (WT/WT), and we adjusted for sex, age, ethnicity, and HbAS status. The model yielded an odds ratio (OR) of 0.91 . In addition, in their study Nguetse et al. included mild malaria cases as controls, whereas the control group of the Ghanaian study consisted of apparently healthy children at time of recruitment [Discrepancies between the studies may be the result of inherent geographical differences, i.e., the degree of malaria exposure, or different genetic backgrounds of ethnic groups, or sample size (Gabonese study ruitment . As therruitment , it is iPIEZO1 E756del variant on SM susceptibility and underline the need for further studies in populations exposed to falciparum malaria.In conclusion, our results provide evidence against a strong protective genetic effect of the FRET_primerPiezo1 E756del melting curves


Plasmodium vivax, an efficient national/regional elimination plan must include a comprehensive study of this parasite’s genetic diversity. Here, we retrospectively analyzed P. vivax genetic diversity in autochthonous and imported field isolates collected in different endemic regions in Panama from 2007 to 2020, using highly polymorphic markers . We did the analysis using molecular techniques that are cost-effective for malaria molecular surveillance within Mesoamerica. Thus, we used molecular analyses that are feasible for malaria molecular surveillance within the region, and that can provide useful information for policy and decision making about malaria elimination. We also evaluated if haplotypes established by combining the genotypes found in these genes were associated with relevant epidemiological variables and showed structure across the transmission foci that have been observed in Panama. Ten different haplotypes were identified, some of them strongly associated with geographical origin, age, and collection year. Phylogenetic analysis of csp  revealed that both major variant types (vk210 and vk247) were circulating in Panama. Variant vk247 was restricted to the eastern endemic regions, while vk210 was predominant (77.3%) and widespread, displaying higher diversity  and geographically biased alleles. The regional implications of these molecular findings for the control of P. vivax malaria to achieve elimination across Mesoamerica are discussed.Panama and all nations within the Mesoamerican region have committed to eliminate malaria within this decade. With more than 90% of the malaria cases in this region caused by  Plasmodium vivax parasites [Given the relatively low number of malaria cases and deaths, and its strategic geographical position as a corridor connecting South and North America ; Mesoamearasites . From tharasites ,5. Howevarasites ,3, facinarasites ,7,8. P. falciparum transmission in the eastern region of the country [Plasmodium parasites isolated from these imported cases have shown a higher genetic diversity when compared with the genetic structure displayed by local cases [Panama, located at the southern end of Mesoamerica , has als country . Moreove country ,11. Most country ,11,12,13 country . Recent  country ,15. PlasP. vivax [P. vivax biology considering the species peculiarities that hamper its control. In this sense, knowing the genetic structure of P. vivax circulating in the country over years can elucidate not only the origins and connectivity of the transmission, but also evidence the association of certain genotypes with the pathogenesis and epidemiology of the disease. This information is crucial to design appropriate local and regional control measures, which within the Mesoamerican context also must be cost-effective, given that all nations in the region are either middle- or low-income countries. In Mesoamerica, different molecular strategies have been designed to explore P. vivax diversity, including single locus typing, microsatellite analysis, molecular DNA barcodes, and deep sequencing approaches [csp) and the merozoite surface proteins (msp-1 and msp-3α) of genes have been the most common and widely used approaches to assess and compare the genotypes of P. vivax infecting humans and vectors [The majority (95%) of the malaria cases registered in Panama since 2000 have been caused by P. vivax . This faproaches . Of thes vectors . MoreoveP. vivax parasites circulating in countries from Mesoamerica. Here, we retrospectively analyzed P. vivax genetic diversity in autochthonous and imported field isolates collected in different endemic regions in Panama from 2007 to 2020, using the highly polymorphic markers . We then evaluated if haplotypes established by combining the genotypes found in these genes were associated with demographic, epidemiological, and geographical parameters relevant to malaria infection. The regional implications of these molecular findings for the control of P. vivax malaria to attain the shared elimination goal are discussed.So far, few studies have explored the genetic diversity of P. vivax isolates, confirmed by PCR, and collected between 2007 and 2020, were included in the study. Two mixed infections with P. falciparum were detected but were not further included in the genotyping analysis. The patients came from all the recognized endemic regions in the country located west (Veraguas and Bocas del Toro) and east  of the Panama Canal  for msp-1, and 236 (77.6%) for msp-3α  were successfully amplified and genotyped for r msp-3α . Due to csp gene revealed that two described types of P. vivax repeats (vk210 and vk247) were present in the studied samples, but with different proportions (vk210 81.7% vs. vk247 18.3%) and with distributions that generally correspond to their geographical origin  were identified for the I enzyme . Genotypmsp-3 α-amplified product was digested with the Hha I enzyme (Three different patterns (assigned Genotypes I–III) were observed when the I enzyme . GenotypCombined analysis of the three genetic markers allowed for the identification of 10 different haplotypes (termed Haplotypes 1–10) in the 163 samples evaluated . HaplotyIn general, local samples collected in eastern endemic regions  presented a higher diversity compared to the homogeneity observed in samples from the western areas (Bocas del Toro and Veraguas), with some haplotypes geographically restricted to the Darién endemic region closer to the Colombian border .We performed a multiple correspondence analysis (MCA) to study the association between the haplotypes, demographic data from the patients (including sex and age), and data about the sampling season, year, the geographic origin of the sample, parasitic load, and gametocyte presence. A detailed explanation of this method and its assumptions is presented in the csp products: 20 imported and 65 indigenous isolates . A phylogeny tree was estimated using the maximum likelihood method. The final phylogenetic analysis contained 105 nucleotide sequences, including for comparison 20 GenBank reference sequences collected from different endemic regions around the world  were selected for direct sequence analysis on amplified he world . Completcsp vk210 and vk247 types  and were present in all endemic regions and in imported cases, while the vk247-type variant was only observed in 19 (21%) samples from the eastern provinces  and in two imported isolates: one from China (MW556359) and the other from Perú (MW556360).The central repeat domain sequence of the csp gene were identified in this study: 2 in vk247 and 14 in vk210  Panama with those collected in the western side of the country (Veraguas and Bocas del Toro) and with imported cases, a possible geographical bias of certain the alleles was observed  and population (~4 M), Panama exhibits a high biological diversity with a great variety of landscapes and habitats and diverse indigenous territories (comarcas) inhabited by different ethnic groups that together represent around 12% of the total population [P. vivax [P. vivax cases to provide useful information that can be integrated in the national elimination plan as an indicator of the transmission intensity in the different endemic areas. Furthermore, this information will be valuable to assess the origin of infections, the parasite population complexity, and ultimately as a measure of the potential resilience of P. vivax populations to interventions [Panama is narrow strip of land strategically located at the southeastern end of Mesoamerica joining the continents of North and South America . Despitepulation . These fpulation . In geneP. vivax . DespiteP. vivax , the couventions . P. vivax were evaluated as follows: the repeat region of csp, the F2 fragment of msp-1m and the alpha segment of msp-3. Individually, msp-3 was the less informative in our study, as only three genotypes were detected with this marker. However, this may be an underestimation of the real diversity, as the PCR-RFLP approach, and particularly the visual detection method that we used to distinguish alleles in classical gel electrophoresis, have a low discrimination power that can add a degree of subjectivity to the analysis [P. vivax infections will depend not only on different approaches and detection methods, but also may differ between geographical locations due to variations in local malaria epidemiology, that might render noncomparable results [With this purpose, three polymorphic loci in analysis . This fa results .P. vivax parasites circulating in western Panama, contrasting with the high diversity displayed by the ones collected in eastern Panama, particularly from the Darién region that is closer to the Colombian border . Interestingly, a geographical bias in the distribution of the 14 vk210, alleles detected in local isolates was also observed, with some alleles  being unique to the western side, while alleles 1, 4, 6, and 14 were found exclusively in the eastern side  occur in ethnic minorities that inhabit autonomous regions (Comarcas) of Panama. In general, these indigenous groups are socially marginalized and vulnerable populations. In these populations the use of conventional tools for malaria control have been less effective. The failure to control malaria in these areas is not only due to the social and health inequalities that affect these minorities, but also because of a lack of intercultural understanding, and other factors such as geographical isolation, internal movement across the country, and cross-border movement, particularly between Colombia and the eastern indigenous territories of Panama. There are marked differences between the ethnic groups that occupy the “comarcas” in both sides of the country in terms of cultural practices, languages, and lifestyles that are of relevance to malaria risk. The eastern comarcas are closer to Colombia and are mainly inhabited by the Gunas and Embera-Wounan ethnic groups. There is a frequent movement of the Gunas across their homeland, which comprises certain malaria hotspots in northern Colombia where human mobility has been shown to play a major role in malaria persistence. The Embera-Wounan comarca is next to the border with Colombia, and waves of refugees and economic migrants from malaria-endemic regions regularly cross the border and settle temporarily in the Embera-Wounan comarca. In fact, movement of malaria across the porous eastern border with Colombia poses a major obstacle to achieving malaria elimination in Panama. The higher malaria incidence and parasite diversity observed in the neighboring country can heavily impact malaria transmission and the risk of getting infected, hindering malaria elimination efforts in Panama. This situation highlights the need for setting a common agenda for malaria elimination across the internationally boundary, and the development of an integrated policy between Colombia and Panama. To attain maximum benefit, malaria control and preventive activities need to be synchronized between both countries. Similar strategies have been successfully implemented for the mobile Ngäbe populations that inhabits the Ngäbe-Bugle comarca in the western side of the country, many of whom seasonally migrate across the Panama/Costa Rica border for economic reasonsAn. albimanus and An. punctimacula are the most abundant and widespread vectors in both areas, other species such as An. darlingi have only been found in eastern endemic regions closer to the Colombia border [An. vestitipennis and An. neivai, which are confirmed malaria vectors in the northern Mesoamerican countries of Belize [In this line, some regional peculiarities have also been described in the species richness and geographic distribution of anopheline vectors in both sides of the country. For example, although a border ,32. Furtf Belize , Mexico f Belize , and Honf Belize , have prf Belize . Informaf Belize , which mPlasmodium-imported infections have been detected carrying parasites with a different genetic variant than the local ones [P. vivax infections, the long incubation periods, relapses, and early gametocytemia of this species of parasites can facilitate cross-fertilization and recombination events of different parasite genotypes within Anopheles vectors prevalent in the region, resulting in greater genetic variation [Socio-demographic variables and the geographical location of the areas where the local samples were collected might have also contributed to some extent to the parasite genetic structure observed in the different endemic regions of the country. The proximity of Darién and Guna Yala to the border of Colombia, where vector diversity is higher ,37, can cal ones ,38. Thiscal ones . Particuariation  and incrP. vivax that could be seen in the country nor on the endemic regions evaluated. For this reason, we cannot rule out the presence of other genotypes in regions where they were not identified, although overall patterns of increased diversity in eastern Panama when compared with western Panama are likely robust. Second, the Plasmodium genes to be analyzed and the detection method to be used for genotyping should be carefully chosen by the malaria elimination programs from Mesoamerican countries. Otherwise, results between countries will not be comparable and the information will be useless for the elimination regional goal. As stated, in our study, visual detection of different genotypes in agarose gels was rather subjective. Our study presented a number of important limitations that need to be stated. First, the study sample size was rather small and, therefore, our results do not necessarily represent the genetic diversity of P. vivax genotypes between the western and eastern endemic regions of the country. However, to properly address and elucidate this finding, and to determine the possible contribution of imported genotypes in the local parasite diversity, a larger number of samples representative of all endemic regions must be analyzed. Furthermore, a more robust analytical method such as analysis of molecular variance (AMOVA) is required to specifically address these questions.Our descriptive data suggest a geographical difference in distribution of P. vivax samples from Panama and found a low diversity, with most of the samples belonging to a single highly related lineage that has persisted throughout endemic regions in Panama for a least a decade [P. vivax genes . These three polymorphic genes have been widely used and have proven to be suitable to assess and compare genotypes of P. vivax infecting humans and vectors across endemic regions from different latitudes [A recent study using selective whole genome amplification along with SNPs selection evaluated 59 a decade . Neverthatitudes . DiscrepUnlike methods based on genome-wide sequencing, monitoring genetic diversity using the markers and protocols used in this study can be easily implemented through Mesoamerica and Hispaniola Island. This is because these methods are based on simple PCR reactions that can be implemented with equipment, i.e., thermocyclers, available in all national reference laboratories in the region, and sequencing of samples can be done using Sanger methods that can be processed at a low cost (10 US dollars/sample) if sent abroad, or regional cooperation could further reduce prices, using sequencers in reference laboratories that routinely sequence genetic markers of pathogens, as currently done in the ICGES . Beyond P. falciparum and P. vivax have cohabited in malaria-endemic areas located to the east of the Panama Canal, closer to the Colombian border. Interestingly, to the west of the Panama Canal, closer to the Central American countries, only P. vivax transmission has been registered since the early 1960s [Plasmodium vivax infections account for 90% of the total malaria cases registered in the country in the last 20 years [Panama lies at the southeastern end on Mesoamerica, joining southern North America with South America . Becausely 1960s . Geograply 1960s . Plasmod20 years . Plasmodium species, parasite density, and the presence of gametocytes following the national guidelines for malaria control of the Ministry of Health of Panama [Malaria samples and epidemiological data analyzed in this study were collected by the National Malaria Control Program (NMCP) personnel as part of the routine surveillance performed in all endemic areas. Clinical blood samples were collected by finger-pricks between 2007 and 2020 from malaria patients, including autochthonous and imported cases, the latter being inferred by the case travel history. Blood samples were used to prepare thick blood smears for routine diagnosis and spotted onto filter paper for molecular analysis. Giemsa-stained smears were examined for f Panama . Parasitf Panama . The folP. vivax positive smear samples, we used a nested PCR that amplifies the small subunit ribosomal ribonucleic acid (ssrRNA) genes, following a slightly modified methodology from the protocol by Snounou et al. [P. vivax and P. falciparum are the only malaria species circulating in Panama, in the second PCR reaction, only specific primers for both species were included. All amplification reactions were carried out in a total volume of 50 µL using the premixed solution PCR Nucleotide Mix . Additionally, 0.4% of BSA was added to the reaction along with an extra quantity of MgCl2 to reach a final concentration of 2.0 mM. Genomic DNA was extracted from samples with the commercial kit QIAamp DNA mini kit  following the protocol for dried spot blood recommended by the manufacturer. To confirm u et al. . SpecifiP. vivax samples was assessed by genotyping the conventional polymorphic markers: csp, msp-1, and msp-3α. Standard nested PCR-RFLP strategies were initially applied to evaluate diversity within these three genes using primers and protocols previously described [P. vivax repeats in the csp gene (vk210 and vk247), nested PCR products were separately digested with BstN I  following the manufacturer’s instructions [msp-1 gene, the nested PCR products were digested with Alu I  following the manufacturer’s instructions [msp-3 α allelic diversity assessment, the second PCR products were digested with Hha I  [csp, msp-1, and msp-3α) analyzed in this study.Genetic diversity of confirmed escribed . To distructions . To evalructions . For mspga, USA) . DigestiP. vivax samples, the csp gene was sequenced. For this purpose, the csp gene was amplified as previously described [csp from this study were submitted and registered in GenBank under accession numbers MW556323-MW556428 and MW847285-MW847305.To further evaluate diversity in the escribed . The nescsp, msp-1, and msp-3α alleles was calculated as the proportion of the allele detected for each allelic family out of the total alleles detected. The frequency of polyclonal infection was calculated based on the number of samples with more than one amplified fragment in the nested PCR, or by analyzing the restriction patterns after cutting with the respective enzyme. The frequency of A multiple correspondence analysis (MCA) was performed to analyze the pattern of association between haplotypes, built by combining genetic variants of the loci under study, and malaria epidemiological parameters. The MCA was fitted with the command mca from the library MASS  using thThe retrospective molecular analysis of the samples and the protocols used for molecular surveillance were approved by the Departamento de Control de Vectores of the Ministry of Health (No. 147/DCV/DGSP and No. 374/DCV/ICG) and by the Comité de Bioética de la Investigación del Instituto Conmemorativo Gorgas de Estudios de la Salud (No. 468/CNBI/ICGES/06 and No. 413/CNBI/ICGES/12). The search of suspected cases, diagnostic, treatment, and documentation procedures of each malaria case (detected via active and passive surveillance) were conducted by the Departamento de Control de Vectores as part of the routine surveillance system for malaria control. Epidemiological information was also obtained from the Departamento de Control de Vectores from the Ministry of Health databases. The confidentiality of the study subjects with malaria was protected and individual data were not shared.