Plasmodium falciparum parasites. Broadly reactive antibodies against parasite antigens expressed on the surface of infected erythrocytes  are candidates for anti-malaria therapeutics and vaccines. Among the VSAs, several RIFIN, STEVOR, and SURFIN family members have been demonstrated to be targets of naturally acquired immunity against malaria. For example, RIFIN family members are important ligands for opsonization of P. falciparum infected erythrocytes with specific immunoglobulins (IgG) acquiring broad protective reactivity. However, the global repertoire of human anti-VSAs IgG, its variation in children, and the key protective targets remain poorly understood. Here, we report wheat germ cell-free system-based production and serological profiling of a comprehensive library of A-RIFINs, B-RIFINs, STEVORs, and SURFINs derived from the P. falciparum 3D7 parasite strain. We observed that >98% of assayed proteins (n = 265) were immunogenic in malaria-exposed individuals in Uganda. The overall breadth of immune responses was significantly correlated with age but not with clinical malaria outcome among the study volunteers. However, children with high levels of antibodies to four RIFINs , STEVOR (PF3D7_0732000), and SURFIN 1.2 (PF3D7_0113600) had prospectively reduced the risk of developing febrile malaria, suggesting that the 5 antigens are important targets of protective immunity. Further studies on the significance of repeated exposure to malaria infection and maintenance of such high-level antibodies would contribute to a better understanding of susceptibility and naturally acquired immunity to malaria.Clinical immunity to malaria develops after repeated exposure to  Plasmodium falciparum , surface-associated interspersed gene family (SURFIN), repetitive interspersed family (RIFIN) proteins, and subtelomeric variable open reading frame (STEVOR) proteins   . The SUR300 kDa) , 27. SURic cycle . At leasic cycle , 29.P. falciparum proteins interact with host immunity inducing an array of potentially protective antibodies hence making them important targets of naturally acquired immunity , Uganda National Council for Science and Technology (HS1403) in Uganda; and Ethics Committees of the Research Institute for Microbial Diseases, Osaka University and Ehime University. Written informed consent was obtained from all study participants and/or their parents or guardians before the study. Verbal and written assent was obtained from children aged 6–7 years and 8–17 years, respectively. The assent took precedent over consent from the parents or guardians. The study was conducted in compliance with the International Conference on Harmonization, Good Clinical Practices and the Declaration of Helsinki.rif and 6 stevor genes were designed to be expressed as one truncate  or variable C-terminal regions after removing the central hydrophobic region and C-terminal transmembrane domain ; STEVORs, n = 54 (from 29 unique IDs); and SURFINs, n = 33 (from 10 unique IDs). RIFINs can be sub-grouped as A-RIFINs (n = 120), B1-RIFINs (n = 15), B2-RIFINs (n = 17), and other B-types (n = 26) according to published work  and region-specific primers  and clonhed work  11)..n = 178 P. falciparum proteins were dispensed into a 384-well OptiPlate using a JANUS Automated Workstation dispenser  and mixed with 10 μl of 4000-fold diluted sera in reaction buffer . After 30 min incubation at 26°C, 10 μl of detection mixture containing streptavidin-coated donor-beads (PerkinElmer) and protein G  conjugated acceptor-beads (PerkinElmer) was added, to make a final concentration of 12 μg/ml for both beads. The plate was then incubated in the dark for 1 hr at 26°C to allow optimal binding of the donor and acceptor-beads to the biotinylated protein and human antibody, respectively. Luminescence emitted by acceptor-beads upon excitation of the donor-beads was detected using an EnVision plate reader (PerkinElmer) and captured as raw AlphaScreen Counts (ASCraw). To account for day-to-day and plate-to-plate assay variability, serially diluted biotinylated rabbit IgG (PerkinElmer) was included in each plate, and subsequently used to generate a 5-parameter logistic standardization curve. The assays were randomized to minimize experimental bias.Human serum antibody levels against the RIFIN, STEVOR, and SURFIN protein library were quantified by AlphaScreen as described , 42. BriData analysis was performed using either R software  or Prism 6 .The cut off point for protein seropositivity to human serum was set at half the lowest non-negative ASC value from that of the assayed samples , and a pU-test or Kruskal–Wallis with Bonferroni–Dunn as a post hoc test.Overall survival was defined as the time from first sampling, when all individuals enrolled were blood-smear negative , to the P-value <0.05 was considered significant, and the values presented here were not adjusted for multiple comparisons, rather the interpretation of associations is based on the level of significance and direction of the association between antibody responses and clinical malaria as the primary outcome. Sensitivity survival analysis was conducted using the overall survival outcome of protection  but only with samples obtained from individuals (n = 52) with at least one microscopically confirmed parasitic infection during the follow up period . Analysis while adjusting for different antibody responses as covariates was limited by their highly correlated nature. All tests were two-sided. A p period , 48. ModWGCFS coupled with AlphaScreen is optimal for determining levels of antibodies that interact with linear and conformational epitopes. Here, we assessed antibody responses against a library of all RIFINs, SURFINs, and STEVORs derived from the Pf3D7 strain. We probed the library with 66 sera from Ugandan individuals aged 6–20 years to identify seroreactive domains/regions that are protective against febrile malaria.n = 178), STEVORs (n = 54), and SURFINs (n = 33)  recognized significantly more antigens than sera from children  associate with clinical malaria outcome to any of the VSA family  with at least 1 microscopically confirmed parasite infection during the follow up period , STEVOR (PF3D7_0732000), and SURFIN 1.2 (PF3D7_0113600) had a significantly reduced risk of having febrile malaria (unadjusted Hazard ratio (HR) <1, p period , 48. AllP. falciparum febrile malaria showed reduced risk for the top 13 antigens  with reduced risk to febrile malaria suggesting that they are potential mediators in the development of uncomplicated malaria or are targets of naturally acquired immunity against clinical malaria.Residents in malaria endemic regions acquire immunity to severe malaria early in life before the acquisition of clinical immunity. Thus, it has been proposed that antibodies against VSAs such as RIFINs, SURFINs, and STEVORs that are associated with severe malaria are acquired in early childhood. However, as shown in this study, not all VSAs present in elder groups could be detected in young children  suggestiin vitro studies suggest that the antibodies act by disrupting rosette formation around iRBCs, promoting phagocytosis, and blocking the binding of RIFINs to host B- and NK immune cells immunoglobulin-like receptor B1 (LILRB1)  , 24, 29.proteins , anti-RIRIFINs interact directly with LILRB1 through specific amino acids or conformations within their C-terminal variable region. The binding is important for iRBC interaction with various inhibitory receptors expressed on leucocytes which contribute to the pathogenesis of severe malaria . In thisThe role of B-RIFINs as targets of protective immunity against clinical malaria remains unclear. Indeed, their role in merozoite invasion, sequestration, cytoadherence of infected erythrocytes, and involvement in the development of severe malaria requires further studies. However, with specific B-RIFINs variants such as PF3D7_1300600 showing localization on the surface of merozoites and gametocytes , and onlWe also assessed human acquisition of antibodies to SURFINs and STEVORs. In a recent study, a polyclonal antibody (anti- PF3D7_1254100) with broad specificity to different STEVOR variants localized STEVORs on the surface of non-permeabilized/non-fixed merozoites . Using tsurf1.2 gene is annotated as a pseudogene in the malaria genome database (www.PlasmoDB.org); hence in vivo expression in parasites must be confirmed before further analysis. Other SURFINs such as SURFIN4.2 have been putatively associated with erythrocyte invasion (The function of PF3D7_0113600 (SURFIN1.2) which was associated with reduced risk of febrile malaria selected in this study is unknown, but has been suggested to be involved in cell surface adhesion  and is ainvasion . FurtherFor all RIFINs, SURFINs, and STEVORs, we did not observe any reduced chance of a protein being recognized by sera based on length, and comparisons between truncates of the same protein showed comparable immunoreactivities. The variable C-terminus were relatively more seroprevalent than the conserved N-terminal truncates . In a siAlthough we detected potential correlates of protection in the population, we may have missed weak or rare immune associations or overestimated others due to the limited sample size assayed while analyzing multiple antigens over a wide age range. Another limitation was the absence of infection or clinical malaria historical data prior to conducting the prospective study, which could have reduced heterogeneity in samples and allowed more accurate statistical adjustments. The protein library used may also not accurately reflect the proteins expressed by the local parasite population causing the infection, due to its construction based upon the reference Pf3D7 parasite strain. In addition, some proteins may not have been optimally expressed as native parasite proteins, or may have lacked important epitopes, such as within or associated with surface exposed central hydrophobic regions or variable regions that were excluded during protein truncation to ease recombinant protein expression. Nevertheless, the high immunoreactivities observed in this study suggest that the expressed proteins were similar or shared conformations to native parasite proteins. Further studies are required to clarify the role of the selected antigens in protection from malaria pathogenesis.P. falciparum vaccine antigens or correlates of clinical protection by interrogating human samples obtained from well-founded studies.In conclusion, the extensive global immunoreactivity of RIFINs, SURFINs, and STEVORs presented in this study may reflect a parasite strategy of expressing multiple VSAs in the blood stage, hence enhancing the likelihood of survival and establishment of chronic infections in the host. The identified four B-RIFIN antigens , STEVOR (PF3D7_0732000), and SURFIN 1.2 (PF3D7_0113600) may indicate important targets or correlates of protective immunity against febrile malaria that need further evaluation. The development of this protein library will facilitate high-throughput evaluation of protective immune profiles toward the identification and prioritization of potential The datasets generated for this study are available on request to the corresponding author.The studies involving human participants were reviewed and approved by Institutional Research and Ethics Committee of Lacor Hospital (LHIREC 023/09/13), Uganda National Council for Science and Technology (HS1403) in Uganda, Ethics Committees of the Research Institute for Microbial Diseases, Osaka University and Ehime University. Written informed consent to participate in this study was provided by the participants and/or legal guardians/next of kin.BK, TT, and ET conceived and designed experiments. BK, HN, NP, MW, MM, EN, BB, AY, TE, and TH conducted experiments. BK, MW, TT, and ET analyzed the data. BK, TT, and ET wrote the manuscript. All authors discussed and edited the manuscript.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 malaria is thought to be nonsterile and sustained by persistence of low-level parasitemia. This study assessed the association between baseline microscopic and submicroscopic asymptomatic P. falciparum infections and antimalarial antibody levels and whether these parasitemia modify protective associations between antibody levels and malaria in Ghanaian children. Healthy children  were recruited into a 50-week longitudinal malaria cohort study from January 2016 to January 2017. Baseline asymptomatic parasitemia were determined by microscopy (microscopic parasitemia) and PCR (submicroscopic parasitemia), and antibody levels against crude schizont antigens were measured by enzyme-limited immunosorbent assay (ELISA). Antibody levels, parasite diversity, and risk of malaria in the ensuing transmission season were compared among children who had baseline asymptomatic microscopic or submicroscopic or no P. falciparum infections. Of the 99 asymptomatic baseline infections, 46 (46.5%) were microscopic and 53 (53.5%), submicroscopic. Cox regression analysis adjusting for age group, sex and community found a strong association between both baseline microscopic  and submicroscopic  asymptomatic parasitemia and a reduced risk of febrile malaria compared to those who were uninfected at baseline. Baseline asymptomatic submicroscopic parasitemia had a significant effect on associations between antischizont antibodies and protection against febrile malaria . The study found both baseline P. falciparum asymptomatic microscopic and more strongly submicroscopic infections to be associated with protection against febrile malaria in the ensuing transmission season. This could have important implications for malaria seroepidemiological studies and vaccine trials.Naturally acquired immunity to  Plasmodium falciparum malaria remains a major cause of morbidity and mortality, particularly in pregnant women and children in sub-Saharan Africa, despite significant progress made by current control programs , a phenomenon termed premunition, which is thought to help sustain NAI against malaria  or were older than 13 years (n = 15) or whose parents withdrew consent (n = 7) during screening. Of those enrolled, 848 (87.2%) completed the 50-week longitudinal follow-up. At baseline, a total of 99 (11.7%) children were infected with P. falciparum, of which 46 (46.5%) were detected by microscopy (microscopic) and the remaining 53 (53.5%) by PCR alone (submicroscopic)  compared to those who were younger  (P = 0.0036) lower baseline hemoglobin (Hb) levels  than those with submicroscopic (11.3 [1.6] g/dl) or no P. falciparum infection (11.3 [1.5] g/dl) (P < 0.001) differences in the distributions of children who had either microscopic, submicroscopic, or no P. falciparum infection among the study communities but none for other covariates such as sex, sickle cell status, and bed net use  who met the inclusion criteria were enrolled out of the 997 screened. The excluded children were those who had febrile malaria (oscopic) . The rem = 3.7%) . Childre5] g/dl) . There w net use .2 = 1.42, P = 0.49) different among children with either microscopic, submicroscopic, or no infections at baseline. Nonetheless, the proportion of children who were reported to have taken antimalarial drugs 2 weeks prior to the baseline sampling was slightly higher in the group with submicroscopic infection  compared to those with microscopic  or no infection .Antimalarial drug treatment history up to 30 days prior to testing has been associated with increased prevalence of submicroscopic parasitemia in previous studies ; therefoP. falciparum crude schizont antigens were measured and compared among the different infection statuses at baseline in separate multiple linear regression analyses adjusting for age group, Hb, bed net use, sex, and community. There was no significant difference in baseline IgG levels between children who were uninfected and those with microscopic parasitemia . However, children with submicroscopic parasitemia had significantly higher  IgG levels compared to those of the uninfected group  were infected by P. falciparum at least once (microscopy and/or PCR detection of any parasitemia during monthly finger pricking) but did not develop fever or any malaria symptoms throughout the follow-up period. These participants were classified as protected. The final infection status of the remaining 70.4% (597/848) with respect to P. falciparum was classified as undefined since no parasite was detected in their blood by microscopy during either the monthly finger pricking or at any time point in the study period. This group was excluded from subsequent analysis in order to increase the power to clearly identify the effect of immunity in protection against febrile malaria as has been shown previously  of the study participants experienced at least one febrile malaria episode as defined by fever (measured axillary temperature of >37.5°C or fever reported) and any eviously . P. falcP. falciparum infection status  and febrile malaria episodes in the ensuing transmission season was assessed using a Kaplan-Meier plot and log rank test (P < 0.0001) difference in the probability of children in the different baseline infection status groups to remain free of malaria in the ensuing transmission season. Cox regression analysis adjusting for age group, sex, and community found a strong association between both baseline microscopic  and submicroscopic  asymptomatic parasitemia and a reduced risk of febrile malaria compared to that of those who were uninfected at baseline  higher median IgG levels compared to those of susceptible children; however, IgM levels were similar (P = 0.046) and protection against febrile malaria but not for IgM (P = 0.36) (P = 0.00064) and IgM (P < 0.0001)  . The lik 0.0001) .P. falciparum infections prior to the transmission season  in an area where malaria is endemic in southern Ghana. The overall low prevalence of asymptomatic parasitemia at baseline was expected, since in Ghana, malaria is currently more prevalent in the middle and northern sectors than in the south, where only pockets of areas with high transmission are seen  than in those with microscopic (64%) parasitemia. This may be due to perhaps comparatively more of the baseline asymptomatic microscopic parasitemia later developing into febrile episodes. However, it is not known whether there exists a minimum threshold of parasitemia that is required to sustain functional protective nonsterile immunity against malaria. Naturally acquired protective immunity against malaria is thought to be sustained through persistent exposure to different parasite strains in a population  of the University of Ghana, Accra, Ghana, and by the Ghana Health Service Ethics Committee of the Ministry of Health, Ghana. Written informed consent was given by the parents and guardians of all children before they were enrolled into the study.P. falciparum being the main malaria parasite causing majority of infections. The main health facility serving the study communities is the Danfa Health Centre (DHC), which was also the designated health facility for the study where all study participants were encouraged to attend as their first point of care when ill. Medical cases beyond the capacity of the DHC are referred to bigger hospitals such as the Dodowa, Pentecost, 37 Military, and Ridge hospitals, all within the Greater Accra Region of Ghana.The study was a conducted in six periurban communities, of which four, Danfa, Kweiman, Adoteiman and Otinibi, were in the La-Nkwantanang-Madina Municipal assembly  and two, Ayi-Mensah and Ghana Flag were in the Ga-East Municipal Assembly , all in the Greater Accra Region of Ghana. These communities were selected due to proximity to each other and to a common health facility for easy tracking of malaria cases, as well as proximity to the NMIMR to ensure that samples were processed in time. The study area has different ethnic groups of Ghana—mainly Ga, Akan, Ewes, and Hausa—living together in within the same communities. The area is within the coastal savannah zone of Ghana, and rainfall pattern is bimodal with a major peak around May to July and a minor peak from September to November. Malaria transmission mirrors the rainfall pattern and peaks during or immediately after the rainy seasons, with P. falciparum parasitemia in addition to fever  or whose parent/guardian withdrew consent during the screening process were excluded from enrollment. Those without fever at baseline were not tested with malaria RDT. Children presenting with any chronic medical condition were referred to the DHC for treatment and were also excluded. At enrollment (baseline), each study participant was assigned a unique study identifier and a questionnaire was completed to capture the clinical/medical history and demographic data of participants. Axillary body temperature was measured, and about 3 ml of venous blood (children ≥ 2 years) or 0.5 to 1 ml of finger-prick blood (children < 2 years) samples were collected into EDTA anticoagulant tubes for immunological assays. Blood hemoglobin level measurement by Hemocue-Hb 201  and sickle cell status (sodium metabisulphite test) were determined. In addition, blood slides for malaria parasitemia estimation by light microscopy and filter paper  dried blood spots (DBS) for parasite genetic diversity studies were obtained. There was weekly active follow-up in which trained field assistants visited the home of each child to measure axillary body temperature and completed a morbidity questionnaire with the help of the parent/guardian on the child’s wellbeing. Children found to be unwell or with measured or reported fever were referred to the DHC, where DBS and blood smears for malaria parasite studies were collected and treatment administered by the medical staff in the facility. Passive surveillance was throughout the study period when the parent/guardian visited the DHC outside the scheduled field assistant’s weekly visits when the child was unwell, which they were encouraged to do. DBS and blood film samples were also obtained during such visits. All malaria cases were treated with artemisinin-based combination therapy, which was the standard treatment according to the Ghana Health Service guidelines at the time of the study. Once every month, DBS and blood smears for parasite detection and genetic diversity studies were obtained from each study participant by finger pricking. Participants who missed 3 consecutive monthly finger pricks were excluded from the final analysis.A total of 997 children whose parents or guardians consented for their inclusion in the study were screened, and 973 between the ages of 0.5 to 12 years were enrolled into a 50-week longitudinal cohort study prior to the malaria season in January and February 2016. Children aged <0.5 or >13 years or with a positive malaria rapid diagnostic test (RDT)  and/or Plasmodium parasite examination by light microscopy as described previously  at 2,000 rpm for 10 min, and the plasma and buffy coat were separated and stored at −40°C until use. The DBS, packed individually in resealable zipper lock bags, were kept at −20°C until use. Blood smears were stained with 10% Giemsa for eviously .P. falciparum NF54 schizont extract was obtained as previously described , 0.25 mM mixture of each deoxynucleoside triphosphate (dNTP), 0.25 mM each primer , and 0.0375 U of OneTaq Hot Start DNA polymerase . All reactions had a genomic DNA from a culture of P. falciparum laboratory strain 3D7 as a positive control. The PCR cycling  conditions were an initial activation step of 94°C for 15 min, followed by 35 cycles of denaturation for 30 s at 94°C, annealing for 45 s at 54°C, and extension for 45 s at 68°C. The final extension was carried out for 5 min, also at 68°C. The PCR products were visualized  by electrophoresis on 2% agarose with ethidium bromide staining.All of the samples from baseline already screened by microscopy were also assessed for parasitemia using PCR. This allowed for identification of individuals with submicroscopic parasitemia alone who were defined as being microscopy negative but PCR positive for escribed  with modP. falciparum 18S rRNA gene. Proportions of study participants distributed among age groups, sex, sickle cell status, bed net use, and communities were compared for each variable between the three categories by the chi-square test. Hb was normally distributed and was compared among the groups by analysis of variance (ANOVA). The Mann-Whitney test, which considers ties among values, was used in calculating exact P values to compare the median antibody levels between protected and susceptible children. Antibody data were log (base e) transformed to approximate normality and differences compared among the groups with the uninfected group as the reference in a multiple linear regression adjusting for age group, Hb, bed net use, sex, and community. The association between antischizont antibody levels and protection against malaria was assessed by logistic regression analysis, adjusting for covariates. A likelihood ratio test was used to estimate the impact of baseline parasitemia status on associations between antibody levels and protection against malaria. The associations between baseline infection status and time to first malaria episode in the 50-week follow-up was assessed by Kaplan-Meier plots, and log rank statistics and hazard ratios were calculated by Cox regression analysis. All data analyses were performed in R v3.6.0 (P values of <0.05 were considered statistically significant.Study participants were categorized into three groups based on baseline infection status as either uninfected or having either submicroscopic or microscopic infections. Submicroscopic infections were defined as infections that could not be detected by microscopy and were only detected by PCR targeting the


Malaria is a major travel medicine issue. Retrospective confirmation of a malaria episode diagnosed in an endemic area can have relevant implications in transfusional medicine in Europe, where blood donors are excluded from donation on the basis of positive malaria serology. However, there is scarce evidence on the dynamics of anti-malarial antibodies after a first malaria episode in non-immune individuals. The first aim of this study was to describe the dynamics of anti-malarial antibodies in a first malaria episode in non-immune travellers. Secondary objectives were to assess the sensitivity of serology for a retrospective diagnosis in non-immune travellers diagnosed while abroad and to discuss the implications in transfusional medicine.Plasmodium falciparum and admitted at the IRCCS Sacro Cuore Don Calabria hospital in a 14-year period. The antibody titres were collected at baseline and during further follow up visits. Epidemiological, demographic and laboratory test results  were anonymously recorded in a study specific electronic Case Report Form created with OpenClinica software. Statistical analysis was performed with SAS software version 9.4.Retrospective analysis of the results of an indirect fluorescence antibody test (IFAT) for malaria available for patients with a first malaria episode by Thirty-six patients were included. Among them, all but two were Europeans (one African and one American). Median length of fever before diagnosis was 2 days (IQR 1–3). Thirty-five patients had seroconversion between day 1 and day 4 from admission, and the titre showed a sharply rising titre, often to a very high level in a few days. Only a single patient remained negative in the first 5 days from admission, after which he was no more tested. Six patients were followed up for at least 2 months, and they all showed a decline in IFAT titre, tending to seroreversion .Serology demonstrated reliable for retrospective diagnosis in non-immune travellers. The decline in the anti-malarial titre might be included in the screening algorithms of blood donors, but further studies are needed. Plasmodium falciparum ALL the records responding to the inclusion criteria in the period considered were included. Expected number was of about 40 subjects.Plasmodium-specific  total antibodies (no distinction is made between IgM and IgG) and samples are considered negative when IFAT titre = 0. In strict terms, titres express serial dilutions, where 20 (strictly speaking: 1/20) is the first dilution considered, therefore “0” simply means “negative, or no fluorescence, at the first dilution of 1/20. For clarity in the paper, titres are expressed as whole numbers, where each whole number corresponds to the denominator of the respective dilution. According with the manufacturers’ instructions, weak positivity (titre 20 to 40) is considered to be due to either a) a past infection by P. falciparum; b) a past or active infection by another plasmodial species . Titres ≥ 80 indicate a (past or active) P. falciparum malaria infection. A positive (titre 160) and a negative control are included in each session. In order to estimate its accuracy, the test was carried out on 36 blood donors with no relevant travel history and the result was negative (0 titre) for all. The test was also performed on 84 African subjects recently (up to one year before testing) arrived in Italy from endemic areas, with no acute nor recent malaria symptoms and all tested positive with an antibody titre varying between 160 and 10,120, save for two subjects with a titre of 80.IFAT assay  was performed according to the manufacturer’s instructions. Briefly, the test enables the detection of www.OpenClinica.com.) In the database the patients’ data were anonymized and identified by a study univocal code. Statistical analysis was performed with SAS software version 9.4. All collected data were summarized using descriptive statistics. Estimated parameters were reported with their respective 95% CI. Both, statistical methods and plots were used to assess the data.Data were collected in a standardized electronic Case Report Forms (CRF) using OpenClinica software© . In the plot, for each patient, the set of observations over time would give rise to a curve (or profile) allowing to compare the dynamic of anti-malarial antibodies across time and subjects.P. falciparum malaria known diagnosis on presentation. For subjects with a minimal follow-up of one month, the sensitivity  was calculated as the proportion: positive subjects at ≥ 1 month/all subjects.At each follow-up visit, sensitivity of serology was calculated using the patients The study protocol was approved by the Ethical Committee (Comitato Etico per la Sperimentazione Clinica delle province di Verona e Rovigo), Protocol Number 47963.P. falciparum malaria in non-immune subjects. The dynamics of anti-malarial antibody titre during hospitalization is summarized in Fig. Thirty-six subjects were included. The main demographic, clinical characteristics and laboratory findings of the included patients are summarized in Table th, 2013 with fever after a short journey to Cameroon. He had a positive RDT for P. falciparum and a positive Quantitative Buffy Coat (QBC), but the thick and thin films were both negative, indicating an exceedingly low parasite density. Unfortunately, it was not possible to follow up the patient after he was discharged. Nineteen patients had the last antibody titration after discharge  were still negative at the beginning of the acute malaria episode when all had already a detectable parasitaemia    for patients who had a follow-up until at least 1 month Fig. .As further exploratory analyses, correlations were searched between some key variables. A moderate positive correlation was found between the parasite density and the number of febrile days before diagnosis , while a weak positive correlation was found between the parasite density and the maximum antibody titre attained . On the contrary, a strong negative correlation was found between the parasite density and the days to antibody positivization .The full data set of the antibody titre over time for all study subjects is resumed in Additional file P. falciparum anti-trophozoites) antibody titre in non-immune patients suffering from their first P. falciparum malaria episode, which was confirmed by a negative antibody titre in almost all of them. While there is a paucity of papers describing the natural evolution of anti-malarial antibodies after a natural infection, relevant research has been done on experimental infection, often in the context of studies aimed at malaria vaccine development [This study analysed the evolution of the anti-malarial  role in returning travellers on one side, and at discussing the role of serology as a screening tool in transfusional medicine on the other. The antibodies detected by the test are total antibodies with no distinction between classes, and there are no clues in this study to discuss their protective role, that was not among the study objectives. In particular, it is not possible to state that finding a positive IFAT titre, say, at 1 year or two after the first (and unique) malaria episode would confer a protection in case of re-exposure, although it is logical to suppose that this may be partially so, in comparison with a subject with a negative titre.The scope of this work was of course different. This was a non-experimental, observational study, using a commercially available anti-P. falciparum malaria in a returning traveller with a recent (i.e. 1 month or less) diagnosis while travelling, while a positive test would retrospectively confirm malaria. Five patients had already a detectable antibody titre at their first observation, four at the lowest titre of 20, and one at titre 40. One of them was a (VFR) patient from Africa (Nigeria) who reported no previous malaria history, although a past exposure might have escaped recollection. Unfortunately, the last available antibody titre for this patient  was recorded during hospitalization and no follow-up was available. Although it has been shown that immigrants, even if with a malaria history, tend to lose their semi-immunity after a long stay abroad [To the authors’ knowledge this is the first such study published. Virtually all patients became positive during the acute malaria episode, and the positivity, after a sharp increase in titre, persisted for at least 1 month in the 19 patients who had a follow-up after discharge. Thus, a negative test result would virtually rule out a previous y abroad , 12, theOne patient remained negative until the last observation before discharge (5 days after admission). This was a patient with an exceedingly low parasite density, and given the strong negative correlation between the parasite density and the days to positivization, had the patient had a sufficient observation time a positive antibody titre would probably eventually have appeared. This cannot be stated with certainty, though, as he was unfortunately lost to follow-up after discharge. As for the five patients who had already a positive  antibody titre at their first determination, a possible explanation is a previous, unreported malaria episode, or alternatively this may represent an early positivization (at least for two of them who had both a very high parasite density on admission).Plasmodium parasites, antigen or DNA [In all patients with a sufficiently long follow-up, the antibody titre reached a peak in about a couple of months and then started declining, with no case of rebound, tending to negativization. This could open interesting perspectives in transfusional medicine. Current rules tend to exclude a donor who has a positive serology, irrespective of the titre. However, a declining titre most probably indicates that malaria infection is no longer present and might be taken as sufficient to re-admit a subject to donation, best if in combination with a sensitive molecular test, and without waiting for a complete negativization that would take a comparatively long time. It is reasonable to assume that chronic carriers of a very low or undetectable parasitaemia  would maintain a high antibody titre. This is typically observed in chronic malaria infection . Subjectn or DNA . Of courThe sample size, that is a convenience sample based on data availability and is constrained by the retrospective design of the study, might not be sufficient to adequately describe the antibody dynamic over time. However, this study provides a first indication of the trend of anti-malarial antibodies after a first, successfully treated malaria episode in non-immune subjects. The antibody tests were executed with a method (IFAT–BioMérieux) that is no longer available (unfortunately the manufacturer has ceased production). It would be interesting to validate the results with other, commercially available (ELISA) tests with the serum specimens from the same patients that are still available in the hospital biobank.P. falciparum blood stages invariably appear during the acute attack and rapidly increase in titre. The test is reliable for the retrospective diagnosis or exclusion of malaria in non-immune travellers. In transfusional medicine, specifically targeted, ad-hoc studies are needed to inform a possible new approach to malaria screening in donors, where, in combination with molecular diagnostics, a declining antibody titre may be considered for inclusion in the screening algorithms.Anti-malarial antibodies against Additional file 1: Table S1. Main characteristics of the study population: country of origin, length of fever, laboratory findings.


Few recent descriptions of severe childhood malaria have been published from high-transmission regions. In the current study, the clinical epidemiology of severe malaria in Mbale, Eastern Uganda, is described, where the entomological inoculation rate exceeds 100 infective bites per year.Plasmodium falciparum malaria at Mbale Regional Referral Hospital in Eastern Uganda. All children aged 2 months–12 years who presented on Mondays to Fridays between 8.00 am and 5.00 pm from 5th May 2011 until 30th April 2012 were screened for parasitaemia. Clinical and laboratory data were then collected from all P. falciparum positive children with features of WHO-defined severe malaria by use of a standardized proforma.A prospective descriptive study was conducted to determine the prevalence, clinical spectrum and outcome of severe P = 0.0002], hyperlactataemia , hypoxaemia (OR) 3.64 , and hepatomegaly . No independent association was found between mortality and either coma or hyperparasitaemia.A total of 10 208 children were screened of which 6582 (64%) had a positive blood film. Of these children, 662 (10%) had clinical features of severe malaria and were consented for the current study. Respiratory distress was the most common severity feature , while 365/585 (62.4%) had hyperparasitaemia, 177/662 (26.7%) had clinical jaundice, 169 (25.5%) had severe anaemia, 134/660 (20.2%) had hyperlactataemia (lactate ≥ 5 mmol/L), 93 (14.0%) had passed dark red or black urine, 52 (7.9%) had impaired consciousness and 49/662 (7.4%) had hypoxaemia (oxygen saturations < 90%). In-hospital mortality was 63/662 (9.5%) overall but was higher in children with either cerebral malaria (33.3%) or severe anaemia (19.5%). Factors that were independently associated with mortality on multivariate analysis included severe anaemia [odds ratio (OR) 5.36; 2.16–1.32; Severe childhood malaria remains common in Eastern Uganda where it continues to be associated with high mortality. An unusually high proportion of children with severe malaria had jaundice or gave a history of having recently passed dark red or black urine, an issue worthy of further investigation. Plasmodium falciparum malaria remains a major cause of morbidity and mortality in tropical countries, especially within the WHO Africa Region which accounts for the majority of cases worldwide [Despite the recent control strategies of national and international communities, orldwide . Accordiorldwide .In Uganda, the most recent descriptive studies of severe malaria, conducted in the central, south-western and south-eastern parts of the country, were published more than 15 years ago , 3. Wherth May 2011 and 30th April 2012. MRRH is a 470-bed hospital with 95 paediatric beds that is situated in a region of Eastern Uganda with hyper-endemic malaria transmission [P. falciparum blood film and one or more clinical or laboratory features of WHO-defined severe malaria were recruited [We conducted a prospective descriptive study within the Paediatric Acute Care Unit (PACU) of Mbale Regional Referral Hospital (MRRH) between 5smission . All chiecruited , 9–12  or coma  ≤ 2) where no other cause other than malaria could be identified. Multiple convulsions, defined as > 2 episodes in the 24 h period prior to admission. Respiratory distress was defined as increased work of breathing manifesting as deep, fast or very slow breathing, including retractions and the use of accessory muscles. Spontaneous bleeding was defined as physically un-induced and irrepressible bleeding from at least two non-traumatized sites in a patient with severe malaria without a previous history of abnormal bleeding. Haemoglobinuria was defined when the parent reported a history of, and the clinician macroscopically observed, dark red or black urine according to a score of 5 or more on the Hillman urine colour scale [2) of < 90%, as measured using a standard pulse oximeter. Definitions of laboratory criteria included: hypoglycaemia (blood glucose < 2.2 mmol/L), metabolic acidosis (plasma bicarbonate < 15 mmol/L), severe anaemia (Hb < 5 g/dL), hyperparasitaemia (> 5% or 250 000/μL) [Standard case report forms (CRFs) were used to collect data on clinical, laboratory and socio-demographic factors, along with subsequent treatment and outcome. Patients displaying any of the following clinical features were recruited if their peripheral blood smears were positive for asexual forms of ur scale , a descrur scale , 15, Nigur scale  and Buruur scale . Clinica 000/μL) , and hypEligible patients with any of the above features of severity were invited to participate after informed consent was sought from the patient’s parent or guardian. Data were captured on a case report form (CRF), which was logically sequenced to capture data on: socio-demographic features, clinical features (symptoms and signs), laboratory data, treatment and outcome. Blood samples for the study were collected for parasitological microscopy as well as estimation of lactate using , and random blood sugar levels using On Call Plus . In addition, a small volume of blood was collected for additional tests including 2 mls of whole blood for a complete blood count (CBC) and 0.1 mls for a quality-controlled blood slide. At the hospital, HIV testing was routinely done as part of the Ministry of Health policy.Plasmodium parasites. Blood smears were stained with 10% Giemsa, a stable methanol based Romanowsky stain, at pH 7.2. The stained blood smears were examined microscopically under  x400 and x1000 objectives. The presence of Plasmodium parasites was noted based on their staining features. Malaria parasites were further quantified by examining microscopically under the x100 objective. The number of Plasmodium parasites in the positive smears was counted against 500 WBCs and the value that was obtained was multiplied by 16 in order to obtain the Plasmodium parasitaemia/μl of whole blood.Initial thick and thin blood smears were made from finger prick blood samples for identification and typing of ®. When available, blood transfusion dosed at 20 mls of whole blood/kg was administered to children with severe anaemia. In addition, oxygen therapy was administered to patients with hypoxaemia. Hypoglycaemic patients were administered 25% dextrose at a dose of 2 mls/kg. Dehydration was treated with fluids according to the WHO protocols for moderate (Plan B) and severe (Plan C) dehydration. Referrals for specific care were made where appropriate, including to the Paediatric Infectious Diseases service for HIV infected children, the TB service for children with tuberculosis, the nutrition unit for malnourished children and physiotherapy for children requiring rehabilitation services. Temperature control with paracetamol and intermittent tepid sponging was administered to patients with temperatures of > 38.5 °C.All patient management at the PACU followed two protocols. First, the Emergency Triage and Treatment (ETAT) guidelines  were fol2 tests and continuous variables using Student’s t-tests or ANOVA, as appropriate. Non-normally distributed variables were normalized by log10-transformation before analysis. P-values of < 0.05 were considered statistically significant. Variables found to be significantly associated with mortality on univariate analysis were included in logistic regression models investigating independent predictors of mortality. All analyses were conducted using R statistical software (http://www.R-project.org/).Categorical variables were compared by use of χWritten informed consent was provided by the parents or guardians of all study participants. Ethical permission for the study was granted by both the MRRH Research and Ethics Committee in Mbale and the Uganda National Council of Science and Technology (UNCST) in Kampala, both in Uganda.P. falciparum malaria, of which 662 (10%) had one or more clinical features of WHO-defined severe malaria and were included in the current study. Their median age was 18 (IQR 10-33) months while the majority  were < 5 years old  had a positive blood film for The clinical and laboratory characteristics of recruited children, stratified by survival to hospital discharge, are summarized in Table The laboratory characteristics of recruited children are summarised in Table P = 0.004), hyperlactataemia was strongly associated with mortality (P < 0.0001). Hypoglycaemia was also associated with mortality (P < 0.001), but no correlation was found between admission parasite density and outcome. In general, CFRs increased with the number of severity features reported , hypoxaemia (P = 0.008), hyperlactataemia (P = 0.001) and hepatomegaly (P = 0.004) , often associated with an increased rate of breathing . Deep brP < 0.0001) [In keeping with the local epidemiology of heavy perennial malaria transmission , severe  0.0001) . The cas 0.0001) , 27, 28. 0.0001) , even in 0.0001) , potenti 0.0001) .In contrast to the recent mortality rates reported from the TRACT trial , 32, whiP. falciparum malaria among children in the current case-series were respiratory distress, clinical jaundice and severe anaemia. Clinical jaundice and the recent passage of dark red or black urine, neither of which have been commonly reported in similar studies from other parts of Africa, were frequent. Overall mortality was high, with severe anaemia, hyperlactataemia, hypoxaemia and hepatomegaly being independently associated with death. Observations from this study justify further work aimed at investigating the causes and best treatment for blackwater fever in children in Eastern Uganda.In conclusion the common clinical features of severe


Malaria during pregnancy may result in unfavourable outcomes in both mothers and their foetuses. This study sought to document the current burden and factors associated with malaria and anaemia among pregnant women attending their first antenatal clinic visit in an area of Ghana with perennial malaria transmission.A total of 1655 pregnant women aged 18 years and above with a gestational age of 13–22 weeks, who attended an antenatal care (ANC) clinic for the first time, were consented and enrolled into the study. A structured questionnaire was used to collect socio-demographic and obstetric data and information on use of malaria preventive measures. Venous blood (2 mL) was collected before sulfadoxine-pyrimethamine administration. Malaria parasitaemia and haemoglobin concentration were determined using microscopy and an automated haematology analyser, respectively. Data analysis was carried out using Stata 14.Mean age (SD) and gestational age (SD) of women at enrolment were 27.4 (6.2) years and 16.7 (4.3) weeks, respectively. Overall malaria parasite prevalence was 20.4% (95% CI 18.5–22.4%). Geometric mean parasite density was 442 parasites/µL (95% CI 380–515). Among women with parasitaemia, the proportion of very low (1–199 parasites/µL), low (200–999 parasites/µL), medium (1000–9999 parasites/µL) and high  parasite density were 31.1, 47.0, 18.9, and 3.0%, respectively. Age ≥ 25 years , multigravid , educated to high school level or above  and in household with higher socio-economic status  were associated with a lower risk of malaria parasitaemia. The prevalence of anaemia (< 11.0 g/dL) was 56.0%, and the mean haemoglobin concentration in women with or without parasitaemia was 9.9 g/dL or 10.9 g/dL, respectively.Plasmodium falciparum infection. Majority of the infections were below 1000 parasites/µL and with associated anaemia. There is a need to strengthen existing malaria prevention strategies to prevent unfavourable maternal and fetal birth outcomes in this population.One out of five pregnant women attending their first ANC clinic visit in an area of perennial malaria transmission in the middle belt of Ghana had  Plasmodium falciparum accounting for 99.7% of the cases [P. falciparum infection in pregnancy is associated with unfavourable pregnancy outcomes such as stillbirth, low birth weight (LBW), pre-term delivery, abortion and maternal anaemia [Despite efforts aimed at controlling and eliminating malaria, the disease still remains a major public health problem. It was estimated that 228 million cases of malaria occurred worldwide in 2018 (decreased from 231 million cases in 2017), and that there were 405,000 deaths in 2018 compared to 416,000 in 2017. Most malaria cases 93%) and deaths (94%) occurred in the World Health Organization (WHO) Africa Region, with 3% and de anaemia .As the burden of malaria varies in different geographical locations and population groups, surveillance of malaria cases has been recommended by WHO to identify areas or population groups most affected by malaria so that the necessary resources and interventions can be targeted at these groups , and theThis study aimed at describing the prevalence and risk factors for malaria and anaemia among pregnant women living in an area of high malaria transmission in the middle belt of Ghana, prior to administration of intermittent preventive treatment with sulfadoxine-pyrimethamine (IPTp-SP).Anopheles gambiae and Anopheles funestus and the transmission is perennial, but peaks between April and October. The annual entomological inoculation rate was 269 infective bites per person per year in 2005 [This study was conducted in four adjoining administrative areas, the Kintampo North Municipality, Kintampo South District, Nkoranza South Municipality, and Nkoranza North District, located in the Bono East Region, within the forest-savannah, transitional ecological zone in the middle belt of Ghana Fig. . The Kin in 2005 , 15, butThis was an analysis of baseline data collected from pregnant women enrolled into a cohort study evaluating the effectiveness and safety of four or more doses of IPTp-SP. Eligibility criteria included pregnant women of any gravidity who attended the antenatal care (ANC) clinic for the first time, who had a gestational age 13–22 weeks, were aged 18 years or above, were known negative for human immunodeficiency virus (HIV) infection, and were willing to participate in the study (demonstrated by signing or thumb-printing an informed consent form).Enrolment of study participants was done at ANC clinics within the study area from July 2017 to March 2019. A general introduction on the nature of the study was given to the pregnant women by the nursing staff at the ANC during their health talks. An initial screening was done to determine eligibility. For those meeting the inclusion criteria, a member of the study team then explained the study further including what an informed consent is, why the study was being done, what it involved in terms of benefits, risks, compensation, confidentiality, right to refuse to be part of the study, and the right to still receive all routine ANC services. Following collection of informed consent, eligible study participants had a questionnaire completed which included information on demographics, obstetric history, socio-economic status (SES), malaria symptoms, anti-malarial drug use, insecticide-treated bed net (ITN) use, and use of other malaria vector preventive measures.2-EDTA tube  for a full blood count. Samples were kept in a transport box containing cold packs and transported to the laboratory in Kintampo for analysis.Prior to commencement of IPTp-SP, a blood sample (2 mL) was collected by a trained member of the study team using sterile venepuncture and transferred into a K2-EDTA anticoagulated blood on a validated and calibrated Horiba-ABX Micros 60 or Pentra 60 C+ haematology analyser  within 8 h of blood draw in the Haematology Unit of the Kintampo Health Research Centre (KHRC) Clinical Laboratory. In addition to regular user maintenance, routine servicing of the analysers was performed quarterly. Three levels of quality control samples  were tested daily prior to analysis of study samples to ensure the reliability of results. The laboratory also participated in monthly external quality assessment schemes organized by the UK National External Quality Assessment Scheme .Haemoglobin estimations were performed using KPreparation and reading of malaria blood slides was done in the Parasitology Unit of KHRC Clinical Laboratory, as described by Swysen et al.  and WHO A total of 1655 pregnant women who were enrolled in the main study designed to evaluate the impact of four or more doses of SP on placental malaria, LBW and anaemia were included in this cross-sectional study. With a malaria prevalence of 17.6% reported among pregnant women at outpatient departments (OPDs) in Ghana by the National Malaria Control Programme , the stuClinical malaria was defined as parasitaemia and an axillary temperature ≥ 37.5 °C or a history of fever within the past 48 h, while asymptomatic malaria was defined as any level of parasitaemia without fever. The level of parasitaemia was classified as very low (1–199 parasites/µL), low (200–999 parasites/μL), medium , and high  after modification of published cut-offs to create the very low category . ITN useEnrolment, haematology and parasitology data were entered into pre-coded questionnaires and checked for completeness. Completed questionnaires were double-entered and verified using a database designed with CSharp application as front end and SQL Server as back end. Data processing and statistical analysis were carried out using Stata 14 . Principal components analysis (PCA) was used to derive a wealth index variable (SES) based on each woman’s household assets and characteristics. Socio-demographic, obstetric and malaria vector prevention characteristics of all women enrolled in the study were summarized.Wilcoxon rank-sum test was used to assess the association between parasite density and age (< 25 or ≥ 25) and gestational age (< 18 weeks or ≥ 18 weeks). The association of gravidity  with parasite density was explored using Kruskal–Wallis test. The prevalence of malaria parasitaemia was computed together with its 95% confidence interval. Univariate and multivariate logistic regression was used to study the association between socio-demographic, obstetric and malaria vector prevention characteristics and the risk of malaria parasitaemia. Variables in the univariate analysis with a p-value < 0.15 were included in a multivariate logistic regression model. The interaction of age and gravidity on the risk of malaria parasitaemia was also explored.Haemoglobin concentration in participants who had malaria parasitaemia was compared to those who did not have malaria parasitaemia using Wilcoxon rank-sum test. The prevalence of anaemia was further classified into mild, moderate and severe, and reported with 95% confidence intervals. Univariate and multivariate logistic regression was used to assess the association of socio-demographic, obstetric and malaria vector prevention characteristics of study participants with the risk of anaemia. Similar to the approach used in the analysis of malaria parasitaemia, variables from the univariate analysis with a p-value < 0.15 were included in a multivariate logistic regression model. The interaction of age and gravidity on the risk of anaemia was also explored. In all multivariate models, significance was established at a 5% level.A total of 1655 participants were eligible for inclusion in the study. Malaria microscopy results were available for 1647 (99.5%) participants, while haemoglobin results were available for 1465 participants (88.5%); samples from 182 were not adequate for haemoglobin estimation. Mean age (± SD) of the participants was 27.4 (± 6.2) years, with nearly half (49.6%) of them aged 25–34 years and the mean gestational age at enrolment (± SD) was 16.7 (± 4.3) weeks, with about two-thirds (67.4%) of women having a gestational age ≤ 18 weeks. Primigravidae and secundigravidae formed 21.0% (n = 355) and 23.7% (n = 392) of the participants, respectively. About one-fifth, 20.9% (n = 346) had completed high school or higher education.Almost 90.0% 1488/1655) of participants reported possessing an ITN, of which 12.1% (180/1488) were reported torn. Reported ITN use among all women and those possessing an ITN was 66.2% (1095/1655) and 73.6% (1095/1488), respectively. About one-fifth of the participants reported using a mosquito coil within the last 7 days  was 442 parasites per µL (95% CI 380–515). Density was also higher in women aged < 25 years  compared to those ≥ 25 years , and in primigravidae  compared with multigravidae   and the prevalence of clinical malaria was 1.5% (95% CI 1.0–2.2%). The prevalence of parasitaemia was higher in women aged < 25 years  compared to those ≥ 25 years , and also higher in primigravidae  compared to multigravidae . In the unadjusted analyses, age, education, household SES, marital status, profession, gravidity, and ITN use were associated with risk of malaria parasitaemia Table . In the Figure Mean (± SE) haemoglobin concentration for the pregnant women at first ANC visit was 10.7 g/dL ± 0.04). Among women with malaria parasitaemia, mean haemoglobin concentration was significantly lower than in those without malaria parasitaemia  Fig. . Overall. Among wIn the univariate analysis, age, gestational age, education, household SES, marital status, profession, gravidity, and malaria parasitaemia status were associated with risk of anaemia Table . In the The effect of the interaction between age and gravidity on prevalence of anaemia is shown in Fig. This study observed a high prevalence of malaria parasitaemia among pregnant women during their first ANC visit in the middle belt of Ghana where malaria transmission is high and perennial. The overall malaria parasitaemia prevalence in this study population 20.4%) is similar to that reported from Hohoe municipality in Ghana (20.3%) but is much higher than that reported from other sites in Ghana: Madina, Accra (5.0%)  and Ejis0.4% is sP. falciparum was detected using microscopy in this study, this is not surprising. The use of the PCR technique may have resulted in detection of other species, as a study of non-falciparum malaria in northern Ghana showed a prevalence of 0.96% [The overall GMPD reported in this study (442 parasites/µL) is similar to that among pregnant women in Mount Cameroon, Cameroon , but mucof 0.96% . The finof 0.96% , Ghana [of 0.96% , 28 and Surprisingly, this study found reported ITN use to be associated with higher risk of malaria parasitaemia in multivariate analysis, which is contrary to what has been reported in many studies , 34 wherThe findings on the overall prevalence of anaemia among pregnant women in this study is higher than that reported from the Sunyani Municipality of Ghana , similarThe strength of this study is that it is adequately powered to provide accurate information on the high prevalence of malaria in pregnant women in the study area and the need for effective measures of control and for monitoring their progress. The study, however, has some limitations. Firstly, malaria microscopy has been shown to underestimate malaria parasite prevalence compared to molecular methods due to the presence of sub-microscopic levels of parasitaemia. However, quality-assured malaria microscopy remains the gold standard for diagnosis and surveillance. Strict adherence to standard operating procedures was ensured and internationally certified microscopists used for the reading of malaria slides. Secondly, although helminth infections haemoglobinopathies have been identified as important risk factors of anaemia, stool examination and haemoglobin genotyping could not be performed during this study.P. falciparum infection during a first ANC visit in the middle belt of Ghana is high, with one out of five women infected and one out of three first-time pregnant women aged < 25 years infected. More than three-quarters of these infections were below 999 parasites/µL of blood. There is the need to strengthen existing malaria prevention strategies and also to employ targeted interventions to control malaria in the study area. This study provides indicators for regular monitoring of progress made towards malaria control or elimination.The prevalence of


Falciparum malaria is clinically heterogeneous and the relative contribution of parasite and host in shaping disease severity remains unclear. We explored the interaction between inflammation and parasite variant surface antigen (VSA) expression, asking whether this relationship underpins the variation observed in controlled human malaria infection (CHMI). We uncovered marked heterogeneity in the host response to blood challenge; some volunteers remained quiescent, others triggered interferon-stimulated inflammation and some showed transcriptional evidence of myeloid cell suppression. Significantly, only inflammatory volunteers experienced hallmark symptoms of malaria. When we tracked temporal changes in parasite VSA expression to ask whether variants associated with severe disease rapidly expand in naive hosts, we found no transcriptional evidence to support this hypothesis. These data indicate that parasite variants that dominate severe malaria do not have an intrinsic growth or survival advantage; instead, they presumably rely upon infection-induced changes in their within-host environment for selection. Plasmodium falciparum is well-recognised  and can bind the endothelium with high affinity in sensitive sites, such as brain  known as group A and DC8 d adults . These vas brain . It is uvar genes in naive hosts, with predominant expression of group B variants and no marked differences between volunteers  by direct intravenous inoculation . To reveal the diversity of responses within our cohort, each volunteer's time-course was analysed independently by tracking their dynamic changes in gene expression as the infection progressed. Importantly, this approach does not assume shared features between individuals, nor does it bias against uncommon (or rare) responses.To explore host-intrinsic variation in the immune response to culation . This enculation . SystemiAs a first step, we measured the variance of every gene through time and visualised the 100 protein-coding genes with highest variance in each volunteer . To contIn contrast, variance  and distCsf3r) and recruitment and activation  based on their apparent downregulation of clusters 1 and 3 at diagnosis . SignifiCollectively, these data reveal marked variation in the human immune response to falciparum malaria with at least three possible outcomes of blood-stage infection – immune quiescence, interferon-stimulated inflammation, and suppression of myeloid-associated gene expression.We next explored the influence of the pre-erythrocytic-stages of infection on the immune response to blood-stage parasites. Whilst blood challenge uses a recently mosquito-transmitted parasite line (< 3 blood cycles from liver egress ) this mo−1, range 9133–273,247) than the rest of the cohort  . Parasite burden may therefore promote an inflammatory response in this challenge model but cannot fully explain it; for example, 2/4 unresponsive volunteers (v018 and v020) had parasite densities that were comparable to 4/8 inflammatory volunteers at diagnosis  varied We next set out to ask whether immune variation had clinical consequences. Symptomatology was scored throughout infection with adverse events graded as absent (0), mild (1), moderate (2), or severe (3). Volunteers in the unresponsive and suppressor groups were essentially asymptomatic at diagnosis, whereas inflammatory volunteers exhibited more frequent and/or severe adverse events, including fever . When weP. falciparum, and their response is indistinguishable from inflammatory hosts.In light of these findings, we examined the potential impact of inflammation on host metabolism to better resolve the consequences of immune variation. Here, we used an LC-MS-based metabolomics platform to measure changes in circulating metabolites during infection, and applied range-scaling to ensure all metabolites  were equally weighted . When weAll together, these data reveal that immune decision-making, systemic metabolism and parasite replication all operate independently in the early stages of the blood cycle. And they further reveal a clear relationship between the intensity of inflammation and symptomatology in falciparum malaria.var gene expression in the inoculum used for blood challenge; this derived from a single malaria-infected volunteer infected by mosquito bite and was cryopreserved ex vivo (without culture) after just three cycles of asexual replication  transcription of most virulence-associated group A or DC8 var genes  and PF3D7_0800300 (aka PF08_0140)) ) . Our datpulation .var genes was essentially unchanged from that observed in the inoculum . Evidently, there was no preferential expansion of group A or DC8 variants within the time-frame of this study.Current theory predicts that group A and DC8 variants should rapidly and preferentially expand in naive hosts because of an intrinsic growth and/or survival advantage favouring their selection . Blood cinoculum . Indeed,iagnosis . Surprisiagnosis . We coulrifin genes we still could find no evidence of gene switching or selection. Indeed, the only rifin that was differentially expressed at diagnosis  was already dominantly transcribed at the start of infection  is less suited to rifins, which are predominantly transcribed at later stages of the parasite life cycle (var or rifin genes (adj p<0.01). In fact, only one differentially expressed gene could be identified (Pfck1) - this gene encodes casein kinase one and was more highly transcribed in the inflammatory group . The diverse clinical outcomes observed in this trial cannot therefore be easily explained by variation in parasite gene expression; instead, they must largely originate from host-intrinsic immune variation.When we expanded our analysis to include nfection . It shoufe cycle . We thervar genes, which are associated with severe malaria in children and adults, come to dominate infections when parasites egress from the liver expressing mainly group B and C var genes. One leading hypothesis is that group A/DC8 variants have an intrinsic growth and/or survival advantage leading to their rapid expansion in naive hosts. To directly test this hypothesis, we developed methodology that allowed us to measure var gene expression across 10 days of infection in a human blood challenge model. Surprisingly, we found no transcriptional evidence for the preferential expansion of group A or DC8 variants. These data indicate that other factors  must be required to promote the selection of these variants in hosts that develop severe disease. We also found that the diverse outcomes of controlled human malaria infection must largely originate from host-intrinsic variation as just one differentially expressed parasite gene could be identified between volunteers that trigger systemic inflammation  and those that remain asymptomatic (by preventing or delaying the acute phase response). Taken together, these data emphasise the importance of the human host in determining their own fate.In this study, we set out to map immune variation in falciparum malaria and explore the interplay between parasite and host factors in shaping outcome of infection. Of particular interest was how group A and DC8 This conclusion is well supported by epidemiological studies showing that the clinical manifestations of severe malaria depend upon host age  and chil+ T cells – the key orchestrators of innate and adaptive immunity – would provide a downstream readout of these critical tissue-specific interactions in human volunteers. Unfortunately, we cannot begin to examine these outcomes in our current datasets because at our final time-point (diagnosis) CD4+ T cells are still sequestered in the tissue. Nevertheless, future studies could extend sampling beyond drug treatment to analyse resolution of the acute phase response and examine the heterogeneity between volunteers in T cell profiles as they are released back into the circulation. Due to the inherent adaptive plasticity of human T cells  had parasite densities comparable to half of the inflammatory group (4/8). And furthermore, v017 triggered a measurable inflammatory response at just 157 parasites mlagnitude . Immune agnitude . This isagnitude  or it maagnitude . This mevar genes, which encode PfEMP1 variants associated with uncomplicated malaria, are dominantly expressed in human volunteers infected by mosquito bite  used the 3D7 ine NF54 . The 3D7ine NF54  and couline NF54 . This co culture . Further in vivo .var gene switching would have been observed if a larger number of volunteers were examined, but this cannot be excluded. An alternative explanation is that a parasite density threshold needs to be crossed to promote var gene switching in vivo, and this threshold is not reached in controlled human malaria infection. In support of this argument, parasite density is known to be an important factor in severe malaria  and the small number of volunteers in this study would be unlikely to capture a rare event. Nevertheless, it is clear that the incidence of severe malaria is much higher in naive hosts . And mor malaria  and this malaria . Howeverentified .var genes become predominantly transcribed in patients with severe malaria? The most plausible explanation for our data is that the short duration of CHMI does not allow enough time for the selection of these variants, and that this would have been observed if drug treatment was withheld. However, the preferential expansion of group A/DC8 variants would need to occur rapidly because at the mean parasite multiplication rate of 10 seen in this study only 2–3 more cycles of replication would have been required for some volunteers to reach parasite burdens at which severe disease can occur (5000 parasites μl−1 or 0.1% parasitaemia  Although this is well written manuscript describing a study that represents a large body of carefully collected and interpreted data on a topic that is of importance to the malaria community, the conclusion regarding the absence of marked changes in transcripts of var and rif genes is at variance with the current thinking about the pathogenesis of P. falciparum malaria.var and rifin genes was unexpected, and in our view it is the counter-intuitive nature of this result that makes it so exciting. We agree that these data do not challenge the paradigm that a subset of var genes are associated with severe malaria; instead we believe these data offer new insights into how group A/DC8 variants may come to dominate severe infections. Specifically, they indicate that group A/DC8 variants do not have an intrinsic growth or survival advantage (a leading hypothesis) but instead may rely upon changes in the host environment to promote their selection. We therefore anticipate that this study will stimulate much discussion and further research on the selection of pathogenic parasite variants in vivo. We also agree that CHMI undoubtedly has limitations, and we had intended to describe these fully in our original manuscript. However, we obviously failed to communicate this clearly, so we have substantially re-written parts of the manuscript in order to more clearly and carefully state our conclusions and explore the caveats relating to our data. These changes are described in detail below.The absence of marked changes in transcription of Revisions for this paper:As concerns the current studies, the reviewers are requesting extensive revisions of the manuscript. These revisions are directed at the major aspects of the observations/findings and the authors' data interpretations.There was a general agreement that the results concerning immune transcripts recapitulated to some extent published data. The data showing Plasmodium gene expression and specifically the absence of any changes in the expression of the var and rif genes generated quite a bit of discussion amongst the reviewers because these results seem at odds what has been reported for host with little malaria experience. In the absence of more definite evidence than what is presented, it is imperative that the authors reduced the rhetoric and entertain a hypothesis that is much better supported by current scientific evidence . In general, the reviewers agreed that the authors over-interpret their findings on var and rif gene transcription, and not least their significance and therefore, please tone down your claims.var genes in severe disease – this was not our intention. To qualify our conclusions and describe them in more accurate and measured tones we have made extensive changes to the text. The specific changes made are as follows:We are sorry that our wording gave the impression that we were questioning the importance of antigenic variation in malaria or the role of var gene switching in naïve hosts infected with Plasmodium falciparum”. We hope that this title emphasises the unique nature of this study  without erroneously giving the impression that we are challenging the role of var genes in malaria pathogenesis.Title: we have changed the title to “Mapping immune variation and Abstract: We have altered the wording of the Abstract to more carefully communicate our conclusions and to explicitly acknowledge the dominant association of specific parasite variants with severe malaria. We have also made it clear that our data are based exclusively on transcriptional analysis:“When we tracked temporal changes in parasite VSA expression to ask whether variants associated with severe disease rapidly expand in naïve hosts we found no transcriptional evidence to support this hypothesis. These data indicate that parasite variants that dominate severe malaria do not have an intrinsic growth or survival advantage; instead, they presumably rely upon infection-induced changes in their within-host environment for selection.”Results: we have modified the text to clearly set out the hypothesis that we are testing: “Current theory predicts that group A and DC8 variants should rapidly and preferentially expand in naive hosts because of an intrinsic growth and/or survival advantage”.And we have qualified our major conclusions:“Evidently, there was no preferential expansion of group A or DC8 variants within the time-frame of this study” and “The diverse clinical outcomes observed in this trial cannot therefore be easily explained by variation in parasite gene expression; instead, they must largely originate from host-intrinsic immune variation”.Discussion: we have added three new paragraphs to discuss the limitations of CHMI and to examine the possible reasons why preferential expansion of group A/DC8 variants was not observed within the time-frame of this study. Importantly, of the myriad possibilities we state our preferred hypothesis: that group A/DC8 variants would expand if CHMI were prolonged beyond 10-days and this would result from enhanced selection (due to changes in the host environment) rather than increased switching rates. We make it clear that further experiments are required.In the Discussion we:– summarise the limitations of CHMI;– discuss the limitations imposed by the use of a single parasite genotype, and the possibility that different parasite isolates may vary in their capacity to switch towards group A or DC8 variants;– discuss the limitations of small sample size, and the possibility that preferential expansion of group A/DC8 variants might be observed if more volunteers are studied;var gene switching/selection;– discuss the possibility that parasite density is not high enough in CHMI to trigger – discuss the relatively short duration of CHMI and ask whether the expansion of group A/DC8 variants would be observed in longer infections. We then explore in detail why a longer timeframe is likely to be required and the changes that may trigger expansion of pathogenic variants.And finally, the summary paragraph at the end of the Discussion has been modified to qualify our conclusions and again emphasise the dominant association of specific parasite variants with severe malaria.It would have been reassuring, if the authors could demonstrate that the parasites used in the CHMI were indeed capable of changing transcription pattern in response to selection for specific adhesion specificities, in particular adhesion specificities that have been associated with development of severe complications . Without such a capacity, the analysis of parasite gene transcription loses its significance. While the data in Peters et al., 2002, support the authors' assumption that the parasites used can indeed switch, direct evidence would be preferable. Because the experiments that would be most informative are not really feasible, the authors must acknowledge this in the Discussion.var genes and that this should be assessed in future trials. In the meantime, we have expanded our Discussion to explore this idea further and to state that additional direct experimental evidence are still required:We agree that one possible explanation for our data is that the 3D7 parasites used for blood challenge could have lost their ability to switch towards group A/DC8 var gene switching.”“The 3D7 clone was cultured for many years before preparation of the inoculum used for blood challenge  and could have acquired mutations that affect its behaviour in vivo . […] Overall, we consider it possible, but unlikely, that our data can be explained by a mutation in 3D7 that affects The short duration of the experiment and the relatively small number of participants may simply be insufficient to yield the transcriptomic changes one might expect on the basis of the existing literature. While the authors acknowledge these caveats, they need to consider them in their conclusions, as well as the Abstract. Another point to consider is the parasite density. If the density is the trigger to switch var gene type, then densities may not be high enough in any host. Parasite density is not adequately explored and neither is the contradiction about parasite density and immune status where the inflammatory group had longer infections.var gene switching/selection.As outlined above, we have carefully re-worded our Abstract and conclusions (subsection “Parasite variants associated with severe disease do not rapidly expand in naïve hosts”) to take account of the limitations of the study. Furthermore, we now discuss in detail the possibility that the short duration of infection, the small number of volunteers or the parasite density observed in CHMI (Discussion) may explain the absence of Because the CHMI model necessitated a strict requirement for drug treatment in early infection, it did not essentially recapitulate the natural P. falciparum infection. It is possible, therefore, that in this model there was a critical absence of an important positive feedback loops that would have been activated once inflammation started and which created an environment that may be critical in selection of more virulent parasite types by altering endothelial receptor expression. Again all these are crucial issues to consider, as the present story challenges the existing paradigm of the origins of severe malaria.var genes in malaria pathogenesis.We would like to re-iterate that it was not our intention to challenge the paradigm that group A/DC8 variants are associated with severe malaria, but instead to raise the question of how these variants come to dominate severe infections. Our data do not support the hypothesis that group A/DC8 variants have an intrinsic growth or survival advantage in naïve hosts; instead, our data indicate that their selection will likely require infection-induced changes in the host environment. We agree that infections were probably terminated too early to observe the effects of these changes on parasite selection and this is discussed in detail in the revised manuscript (Discussion). Furthermore, all of our conclusions have been carefully re-worded so that we don’t erroneously give the impression that we are challenging the role of 1) Please comment on the implications of the shortest infection in immunologically unresponsive participants.We think it likely that the unresponsive volunteers would have mounted an immune response if their infections had continued for a longer time period:“Indeed, it remains a possibility that all naive hosts  will eventually converge on interferon-stimulated inflammation as the infection progresses. […] This metabolic switch may therefore represent a hardwired mechanism of self-regulation because systemic inflammation is an unavoidable fate in naive hosts.”2) Subsection “Systemic inflammation coincides with the onset of clinical symptoms”: Why were only three of eight "inflammatory" volunteers included in the analysis discussed? What was the result when all eight were included?Unfortunately, we were only able to carry out metabolomic analysis on a total of 21 samples. We therefore chose 3 time-points from 7 volunteers, and selected 3 from the inflammatory group (those with the highest CXCL10/IFNγ levels), 2 from the unresponsive group and the 2 suppressor volunteers. This has been clarified in the Materials and methods.3) Please include discussion on the role of innate immunity to malaria and the impact of inflammation on the induction of adaptive immunity also should be mentioned.The role of innate immunity and inflammation (and the activation of adaptive immunity) is discussed:“Notably, only inflammatory volunteers developed hallmark signs and symptoms of malaria (e.g. pyrexia and lymphopenia) providing further support for the idea that inflammation causes the earliest manifestations of disease. […] It therefore appears that we might need to re-assess the role of innate immunity in the first hours and days of the pathogenic blood cycle – rather than serving as an early brake on parasite replication it may be that systemic inflammation primarily functions to switch bone marrow production in favour of myelopoiesis and mobilise the required effector cells to the spleen.”+ T cells – the key orchestrators of innate and adaptive immunity – would provide a downstream readout of these critical tissue-specific interactions in human volunteers.”“The innate response to malaria therefore quickly re-structures and co-localises key strands of the immune system; whether this leads to an effective response that can limit parasite burden and minimise collateral damage will depend upon the outcome of cell-cell circuits in the spleen. Measuring the activation and differentiation of CD44) A more expanded information and discussion on parasitological aspects are needed to strengthen the conclusion. Please include the following points in your Discussion/conclusion: parasite multiplication which was the same in the various groups, while Infections lasted longer and parasite densities were higher in the inflammatory group. There appeared to be highly variable density thresholds to induce inflammation, and therefore a possibility exists that there was a hidden or sequestered biomass in some individuals as a consequence of an inflammatory response. Please also comment on the synchronicity of infections in individuals as this will impact the density result.We have expanded our discussion of the parasitological aspects in the revised manuscript (Discussion) and clearly highlighted the limitations of the model. Furthermore, we have qualified all of our conclusions to take account of these limitations.The apparent contradiction that parasite multiplication rates are comparable between groups but the course of infection (and parasite density) is increased in inflammatory volunteers almost certainly arises from using microscopy for diagnosis. A positive thick blood smear was the primary end-point for drug treatment and inevitably when circulating parasite densities are around the limit of detection  diagnosis becomes fairly stochastic. As one example, inflammatory volunteer 016 was diagnosed with a parasite density of 273,247 parasites per ml but if they had been diagnosed just 12-hours earlier their final parasite density would have been just 3,636 parasites per ml. At both time-points this volunteer was inflammatory and symptomatic. Because of the stochastic nature of microscopy we no longer use this as an endpoint in our clinical trials. Length of infection and parasite density are discussed in the Discussion.With regards to a sequestered parasite biomass: we tried to measure PfHRP2 levels in plasma . However, PfHRP2 levels were below the limit of detection in all volunteers. It therefore remains possible that a hidden parasite biomass in some volunteers (but not others) explains the different outcomes of infection. However, in our view a more simple explanation is that there is between-host variation in the parasite density threshold required to trigger inflammation. This is well supported by a large body of evidence that comes from the in vitro study of human immune variation . Furthermore, whilst we are unable to comment in detail on the synchronicity of these sub-patent infections, we do note that the characteristic saw-tooth pattern of parasite density (by qPCR) was observed in every volunteer in our blood challenge model . This would suggest that sequestration is occurring in all volunteers.5) One of the earliest reports, Lavstsen et al., 2005, on var gene transcription in experimental P. falciparum infections, reached a conclusions that transcription of group A and B var genes did increase over early days following CHM. Since these are different conclusions re NF54 parasites from which 3D7 was derived, this point requires a discussion. Bachmann et al., 2019, does not actually study naïve hosts but suggests hosts with existing immunity exert more effect on var gene profiles than non-immune hosts.The Lavstsen et al., 2005, is now discussed:var gene expression changes within the first few days of blood-stage infection . However, in that study parasites were isolated from volunteers and then placed into culture for one month before var gene profiling – switching may therefore have occurred in vitro confounding interpretation of these data. Our study, which measured var gene expression ex vivo, avoids any bias introduced by culturing parasites but nevertheless has limitations that require careful consideration.”“This is in contrast to a previous study that suggested Regarding reference to Bachmann et al., 2019, we apologise for the mistake – the work using parasite strain 7G8 in CHMI by Bachmann et al. has been presented at meetings, but is not published yet. This has been corrected in the revised manuscript.6) Subsection “Immune decision-making in falciparum malaria”, second paragraph: are these the same 4 volunteers identified earlier in the same paragraph?Yes, this is shown in Supplementary file 2.7) Discussion, third paragraph: It is hard to envisage a scenario in which CHMI would be allowed to proceed further due to the inherent risks to the volunteer of rapidly-increasing parasite density.We have modified this sentence to make it clear that it would be beneficial to prolong CHMI trials if safe to do so:“CHMI trials that follow volunteers for one or two more blood cycles would be required to explore the consequences of increased Pfck1 transcription and of prolonged systemic inflammation, and consideration should be given as to whether this can be done safely.”8) Discussion, fifth paragraph: it seems that inflammation was only present at the time of treatment and one life cycle (48 h) before, based on Figure 1 – so it may be that there was not enough time for inflammation-mediated alterations in host receptor expression to feed through into the var gene profiles by providing a selective advantage to certain adhesive types.We completely agree and discuss this possibility in depth in the Discussion.9) "as part of a custom-design multiplex assay from BioLegend" – does that mean other markers were also measured and were not informative? This would be useful to know given the emphasis on an "inflammatory" response when there is no evidence of classic inflammatory cytokines .TNF and IL-1b have short half-lives in vivo and would not be expected to increase in plasma within 1-2 days of the start of an inflammatory response. These cytokines are not the triggers for inflammation  but rather the downstream effector molecules released by activated myeloid cells. We can detect increased transcription of TNF and IL-1b in whole blood but no increase in circulating protein within the timeframe of this study. For clarity, we have now included reference to TNF in the Discussion:“However, circulating levels of plasma TNF had not yet increased at diagnosis and so more time is clearly required for interferon signaling to drive the cytokine cascades necessary to activate endothelial cells.”10) Subsection “Analysis of parasite var and rifin expression”: were the var reads measured on each of these three days ? The Materials and methods don't seem to state which day the samples from the blood challenge were collected for var analysis, although it seems clear from results.var reads were merged.We apologise that we did not make this clear. We have now revised the Materials and methods to state exactly when parasites were collected during each study and the circumstances under which In the blood challenge study, parasites were collected only at diagnosis:“Blood challenge: 50ml whole blood was drawn into lithium heparin vacutainers at diagnosis (immediately before drug treatment) and white cells were removed by passing the blood through a leucoflex LXT filter (Macopharma).”In the mosquito challenge study, parasites were collected on days 9, 10 and 11:“Mosquito challenge: parasites were isolated from 20ml whole blood drawn on days 9, 10 and 11 post-infection according to the blood challenge protocol, with the following modifications.”var gene expression at the start of the blood cycle in every volunteer: “For every volunteer infected by mosquito bite read counts were pooled from days 9, 10 & 11 post-infection to generate a comprehensive parasite transcriptome 2-3 cycles after liver egress.”Parasite reads were pooled for each volunteer infected by mosquito bite from the data collected on days 9, 10 and 11. This was to ensure sufficient coverage to map 11) Please comment on the relevance of these finding to clinical vaccine and drug trials.A major aim of this study was to map the variation between hosts in their response to infection. Between-host variation has been well documented in vaccine trials  and our data therefore support the need to account for individual variation when calculating sample sizes for vaccine (and drug) trials. Having said that, the sample size in our study is likely not sufficient to reveal the full breadth of immune variation in falciparum malaria and we would therefore be reluctant to include a statement in the manuscript about sample sizes in vaccine or drug trials, as we would not be able to substantiate any claims.12) Please revise the title of your manuscript to reflect the changes in your revised manuscript.var gene switching in naïve hosts infected with Plasmodium falciparum”. We hope that this title emphasises the unique nature of this study – the transcriptional analysis of both parasite and host through time.The title has been changed to “Mapping immune variation and Revisions expected in follow-up work:Given how challenging this work has been, no additional experiments are being suggested for this study, but repeating similar studies with different parasite lines and eventually in malaria endemic areas would be important to consider for future experiments.We agree, and have emphasised the need for further research (including different parasite lines and in endemic settings) in our revised Discussion.