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ObjectivesFever is common among individuals seeking healthcare after traveling to tropical regions. Despite the association with potentially severe disease, the etiology is often not determined. Cytokines are soluble mediators dynamically regulated in the response to infection. Measuring cytokines in the blood can therefore be informative to understanding the host-response to infection and can potentially indicate the type of pathogen that causes the disease.MethodIn this study, we measured 49 host-response proteins in the plasma of 124 patients with fever after travel to tropical or subtropical regions. The patients had confirmed diagnosis of eitherP. falciparummalaria, dengue virus, influenza virus, bacterial respiratory tract infection, or culture-confirmed bacterial gastroenteritis, representing the most common disease etiologies. We used multivariate and machine learning methods to assess host-response protein profiles between the different disease groups and healthy control subjects with the aim to identify disease-specific protein signatures.ResultsThe host-response varied between disease groups and combinations of cytokines and other proteins could accurately distinguish infected patients from healthy controls, and from each other. Malaria displayed the most unique protein signature, indicating a strong immunoregulatory response with high levels of IL10, sTNFRI and II, and sCD25 but low levels of sCD40L. In contrast, bacterial gastroenteritis had high levels of sCD40L, APRIL, and IFN-γ, while dengue was the only infection with elevated IFNα2.ConclusionsThese results suggest that characterization of the inflammatory profile of individuals with fever can help to identify disease-specific host-responses, which in turn can be used to guide further diagnostic strategies.

The mechanism of acquisition and maintenance of natural immunity against Plasmodium falciparum malaria remains unclear. Although, clinical immunity develops over time with repeated malaria episodes, disease tolerance is more rapidly acquired compared to protective immunity. It remains unclear, how pre-existing immune responses impacts the mechanism responsible for disease tolerance. Here, we investigated a cohort of returning travelers treated for acute symptomatic P. falciparum malaria, either infected for the first time, or with a previous history of malaria. Through repeated sampling over one year in a malaria free setting, we were able to study the acute and longitudinal effects of the infection. We combined comprehensive immune cell and plasma protein profiling with integrated and data driven analysis, describing the immune landscape from acute disease to one year after infection. We identified a strong association between pro-inflammatory signatures and γδ T cell expansion. The association was significantly impacted by previous exposure to malaria, resulting in a dampened pro-inflammatory response, which translated to reduced Vδ2+ γδ T cell expansion compared to primary infected individuals. The dampened inflammatory signal was associated with early expansion of FcγRIII+ monocytes and parasite-specific antibodies of IgG1 and IgG3 isotypes.Our data suggest that the interplay of FcγRIII+ monocytes and a cytophilic parasite-specific IgG during the early blood stage infection lead to lower parasitemia and a dampened pro-inflammatory response with reduced γδ T cell expansion. This enhanced control and reduced inflammation points to a potential mechanism on how tolerance is established following repeated malaria exposure.One Sentence SummaryA systems immunology analysis on natural malaria sheds light on disease tolerance mechanism associated with gamma delta T cell expansion

Background The effect of primaquine in preventing Plasmodium vivax relapses from dormant stages is well established. For Plasmodium ovale, the relapse characteristics and the use of primaquine is not as well studied. We set to evaluate the relapsing properties of these 2 species, in relation to primaquine use among imported malaria cases in a nonendemic setting. Methods We performed a nationwide retrospective study of malaria diagnosed in Sweden 1995–2019, by reviewing medical records of 3254 cases. All episodes of P. vivax (n = 972) and P. ovale (n = 251) were selected for analysis. Results First time relapses were reported in 80/857 (9.3%) P. vivax and 9/220 (4.1%) P. ovale episodes, respectively (P Conclusions The risk of relapse was considerably lower in P. ovale than in P. vivax infections indicating different relapsing features between the two species. Primaquine was effective in preventing P. vivax relapse. In P. ovale, relapse episodes were few, and the supportive evidence for primaquine remains limited.

Strengthening malaria surveillance is a key intervention needed to reduce the global disease burden. Reliable serological markers of recent malaria exposure could dramatically improve current surveillance methods by allowing for accurate estimates of infection incidence from limited data. We studied the IgG antibody response to 111 Plasmodium falciparum proteins in travellers followed longitudinally after a natural malaria infection in complete absence of re-exposure. We identified a novel combination of five serological markers (GAMA, MSP1, MSPDBL1 C- and N-terminal, and PfSEA1) that detect exposure within the previous 3-months with >80% sensitivity and specificity. Using mathematical modelling, we examined the antibody kinetics and determined that responses informative of recent exposure display several distinct characteristics: rapid initial boosting and decay, less inter-individual variation in response kinetics, and minimal persistence over time. These serological exposure markers can be incorporated into routine malaria surveillance to guide efforts for malaria control and elimination.

Natural immunity to malaria develops over time with repeated malaria episodes, but protection against severe malaria and immune regulation limiting immunopathology, called tolerance, develops more rapidly. Here, we comprehensively profile the blood immune system in patients, with or without prior malaria exposure, over 1 year after acute symptomatic Plasmodium falciparum malaria. Using a data-driven analysis approach to describe the immune landscape over time, we show that a dampened inflammatory response is associated with reduced γδ T cell expansion, early expansion of CD16

Background The malaria parasite Plasmodium falciparum holds an extensive genetic polymorphism. In this pooled analysis, we investigate how the multiplicity in asymptomatic P. falciparum infections—that is, the number of coinfecting clones—affects the subsequent risk of clinical malaria in populations living under different levels of transmission. Methods A systematic search of the literature was performed to identify studies in which P. falciparum infections were genotyped in asymptomatic individuals who were followed up prospectively regarding the incidence of clinical malaria. Individual participant data were pooled from 15 studies (n = 3736 individuals). Results Multiclonal asymptomatic infections were associated with a somewhat increased subsequent risk of clinical malaria in the youngest children, followed by an initial declining risk with age irrespective of transmission intensity. At approximately 5 years of age, the risk continued the gradual decline with age in high-transmission settings. However, in older children in moderate-, low-, and seasonal-transmission settings, multiclonal infections were either not significantly associated with the risk of subsequent febrile malaria or were associated with an increased risk. Conclusions The number of clones in asymptomatic P. falciparum infections is associated with different risks of subsequent clinical malaria depending on age and transmission intensity., Asymptomatic Plasmodium falciparum infections are often composed of multiples clones. This pooled analysis of individual data shows that the number of clones predicts different subsequent risk of developing clinical malaria depending on age and transmission intensity.

Humoral immunity is important in limiting clinical disease in malaria, yet the longitudinal B cell response to infection remains unclear. We performed a 1-year prospective study in patients treated for acute Plasmodium falciparum malaria for the first time or with previous exposure to the disease. Using an unbiased exploratory approach with mass cytometry, followed by targeted flow cytometry, we found that approximately 80% of mature B cells that proliferated in response to acute infection expressed CD11c. Only approximately 40% of CD11c(+) B cells displayed an atypical B cell phenotype, with the remaining cells primarily made up of activated and resting memory B cells. The CD11c(+) B cells expanded rapidly following infection, with previous exposure to malaria resulting in a significantly larger increase compared with individuals with primary infection. This was attributed to an expansion of switched CD11c(+) B cells that was absent in primary infected individuals. The rate of contraction of the CD11c(+) B cell compartment was independent of previous exposure to malaria and displayed a slow decay, with a half-life of approximately 300 days. Collectively, these results identify CD11c as a marker of B cells responding to malaria and further highlight differences in primary and secondary B cell responses during infection.

Background Artemisinin-based combination therapy (ACT) is the first-line treatment of Plasmodium falciparum malaria. Since the introduction of artemether-lumefantrine (AL) for treatment of uncomplicated malaria in Sweden, treatment failures have been reported in adults. Methods A retrospective comparative analysis of treatment regimen for P. falciparum malaria in adults in Stockholm during 2000-2015 was performed to evaluate the effectiveness of AL. Parasite genotyping and drug concentrations were investigated in the AL treatment failures. Results Among the total 397 P. falciparum episodes, 310 were treated with oral regimen only (95 AL, 162 mefloquine, 36 atovaquone-proguanil [AP], and 17 others), and 87 were administered initial intravenous therapy (38 artesunate and 49 quinine) followed by oral treatments. Five late treatment failures were detected after AL and one slow response to AP. The effectiveness of AL alone was 94.7% (95% confidence interval [CI], 88.1%-98.3%), compared with 99.5% for other oral regimens (P = .003). All AL failures occurred in European men and the effectiveness in this group was only 73.7% (95% CI, 48.8%-90.0%). Genotyping confirmed recrudescence of the initial parasite populations and drug resistance markers revealed no clinically significant resistance patterns. Lumefantrine concentrations suggested subtherapeutic concentrations in at least 2 cases. Conclusions Our findings indicate a high rate of symptomatic late treatment failures after 6-dose AL regime in nonimmune adults, especially in men. Our report warrants the need to establish optimal dosing of AL in adults and to alert clinicians about the importance of informing patients regarding the risk of parasites reappearing weeks after AL treatment.

Severe malaria (SM) is a life-threatening complication of infection with Plasmodium falciparum . Epidemiological observations have long indicated that immunity against SM is acquired relatively rapidly, but prospective studies to investigate its immunological basis are logistically challenging and have rarely been undertaken. We investigated the merozoite targets and antibody-mediated mechanisms associated with protection against SM in Kenyan children aged 0 to 2 years. We designed a unique prospective matched case-control study of well-characterized SM clinical phenotypes nested within a longitudinal birth cohort of children ( n = 5,949) monitored over the first 2 years of life. We quantified immunological parameters in sera collected before the SM event in cases and their individually matched controls to evaluate the prospective odds of developing SM in the first 2 years of life. Anti-AMA1 antibodies were associated with a significant reduction in the odds of developing SM (odds ratio [OR] = 0.37; 95% confidence interval [CI] = 0.15 to 0.90; P = 0.029) after adjustment for responses to all other merozoite antigens tested, while those against MSP-2, MSP-3, Plasmodium falciparum Rh2 [ Pf Rh2], MSP-1 19 , and the infected red blood cell surface antigens were not. The combined ability of total IgG to inhibit parasite growth and mediate the release of reactive oxygen species from neutrophils was associated with a marked reduction in the odds of developing SM (OR = 0.07; 95% CI = 0.006 to 0.82; P = 0.03). Assays of these two functional mechanisms were poorly correlated (Spearman rank correlation coefficient [ r s ] = 0.12; P = 0.07). Our data provide epidemiological evidence that multiple antibody-dependent mechanisms contribute to protective immunity via distinct targets whose identification could accelerate the development of vaccines to protect against SM.

Global migration has resulted in a large number of asylum applications in Europe. In 2014, clusters of Plasmodium vivax cases were reported among newly arrived Eritreans. This study aimed to assess malaria among Eritrean migrants in Europe from 2011 to 2016. We reviewed European migration numbers and malaria surveillance data for seven countries (Denmark, Germany, Netherlands, Norway, Sweden, Switzerland and the United Kingdom) which received 44,050 (94.3%) of 46,730 Eritreans seeking asylum in Europe in 2014. The overall number of malaria cases, predominantly P. vivax, increased significantly in 2014 compared to previous years, with the largest increases in Germany (44 P. vivax cases in 2013 vs 294 in 2014, p P. vivax arriving in Europe. Our results demonstrate potential for rapid changes in imported malaria patterns, highlighting the need for improved awareness, surveillance efforts and timely healthcare in migrants.

Analysis of B-cell specificities at the single cell level provides important information on how the B-cell compartment responds when challenged by infection or vaccination. We recently developed a reversed B-cell FluoroSpot assay and showed that it could be used to detect B cells specific for different antigens simultaneously in a mouse model. The aim of this study was to further develop the method to detect and quantify antigen-specific memory B cells (MBCs) in humans where circulating antigen-specific cells are less frequent. We show that MBCs specific for three antigens, tetanus toxoid, hepatitis B surface antigen and cytomegalovirus pp65, could be detected simultaneously in one well. In addition to enumerating antigen-specific MBCs, we also assessed the spot volume to estimate the intensity of the response in individual cells and found this to be a new and sensitive approach to study MBC responses after vaccination. This unique B-cell FluoroSpot approach provides a simple and sensitive multiplex analysis of MBCs and can be adapted to most antigens and host species.

Graphical abstract, Highlights • Severe malaria episodes are rare during the first few months of life. • The rate of decay of cord blood IgG is inversely proportional to the starting concentration. • Antibody dependent respiratory burst mediated by cord IgG protects from severe malaria during the first 6 months of infancy., Young infants are less susceptible to severe episodes of malaria but the targets and mechanisms of protection are not clear. Cord blood antibodies may play an important role in mediating protection but many studies have examined their association with the outcome of infection or non-severe malaria. Here, we investigated whether cord blood IgG to Plasmodium falciparum merozoite antigens and antibody-mediated effector functions were associated with reduced odds of developing severe malaria at different time points during the first year of life. We conducted a case-control study of well-defined severe falciparum malaria nested within a longitudinal birth cohort of Kenyan children. We measured cord blood total IgG levels against five recombinant merozoite antigens and antibody function in the growth inhibition activity and neutrophil antibody-dependent respiratory burst assays. We also assessed the decay of maternal antibodies during the first 6 months of life. The mean antibody half-life range was 2.51 months (95% confidence interval (CI): 2.19–2.92) to 4.91 months (95% CI: 4.47–6.07). The rate of decline of maternal antibodies was inversely proportional to the starting concentration. The functional assay of antibody-dependent respiratory burst activity predicted significantly reduced odds of developing severe malaria during the first 6 months of life (Odds ratio (OR) 0.07, 95% CI: 0.007–0.74, P = 0.007). Identification of the targets of antibodies mediating antibody-dependent respiratory burst activity could contribute to the development of malaria vaccines that protect against severe episodes of malaria in early infancy.

Summary Accelerated cellular aging and reduced lifespan have recently been shown in birds chronically infected with malaria parasites. Whether malaria infection also affects cellular aging in humans has not been reported. Here, we assessed the effect of a single acute Plasmodium falciparum malaria infection on cellular aging dynamics in travelers prospectively followed over one year in Sweden. DNA and RNA were extracted from venous blood collected at the time of admission and repeatedly up to one year. Telomere length was measured using real‐time quantitative PCR, while telomerase activity and CDKN2A expression were measured by reverse transcriptase (RT)–qPCR. Our results show that acute malaria infection affects cellular aging as reflected by elevated levels of CDKN2A expression, lower telomerase activity, and substantial telomere shortening during the first three months postinfection. After that CDKN2A expression declined, telomerase activity increased and telomere length was gradually restored over one year, reflecting that cellular aging was reversed. These findings demonstrate that malaria infection affects cellular aging and the underlying cellular mechanism by which pathogens can affect host cellular aging and longevity need to be elucidated. Our results urge the need to investigate whether repeated malaria infections have more pronounced and long‐lasting effects on cellular aging and lifespan (similarly to what was observed in birds) in populations living in malaria endemic areas.

Since May 2014, an increase in Plasmodium vivax malaria has been observed in Sweden. As of 31 August 2014, 105 malaria cases have been reported in newly arrived Eritrean refugees, 84 of them P. vivax. The patients were mainly young men and reported migration through Ethiopia and/or Sudan. Severe anaemia and long symptom duration reflect inadequate healthcare during migration. Countries currently hosting Eritrean refugees need to consider P. vivax malaria in this group of migrants.