Declining antibody affinity over time after human vaccination with a Plasmodium falciparum merozoite vaccine candidate.

Maintaining high affinity antibodies after vaccination may be important for long-lasting immunity to malaria, but data on induction and kinetics of affinity is lacking. In a Phase 1 malaria vaccine trial, antibody affinity increased following a second vaccination but declined substantially over 12-months, suggesting poor maintenance of high affinity antibodies.

How to Detect Antibodies Against Babesia divergens in Human Blood Samples.

Background: Today only indirect fluorescent antibody assays (IFAs) are commercially available to detect antibodies against Babesia divergens in humans. IFA is subjective and requires highly experienced staff. We have therefore developed an enzyme-linked immunosorbent assay (ELISA)-based method for measuring anti-B. divergens immunoglobulin G antibodies in human blood samples. Methods: Crude merozoite extract from in vitro cultures of a new B. divergens isolate was used in ELISA to detect antibodies in different sets of samples: Borrelia burgdorferi-positive samples, healthy individuals, tick-bitten individuals including follow-up samples 3 months later, positive control samples from patients with an active Babesia infection, and samples from malaria-endemic regions. As a reference, IFA was used to detect antibodies in the tick-bitten samples. Western blot was used to evaluate reactions against specific bands in extracts with/without parasites. Results: Using IFA as the reference method, the sensitivity and specificity of the ELISA were 86% (12/14) and 100% (52/52). There was a very high correlation (r = -0.84; P = .0004) between IFA dilution factors and ELISA absorbances among the samples classified as positive. Five percent of the B. burgdorferi-positive samples were judged as weakly positive and 5% as strongly positive in our ELISA. Western blot showed that the immunodominant antigens (∼120 kDa) were from merozoites and not from erythrocytes. Conclusions: This ELISA can detect antibodies directed against B. divergens, and it can be a useful and easy assay to handle compared with IFA. The ELISA can also measure high and low levels of antibodies, which could give insight into the recency of a B. divergens infection.

Acquisition of complement fixing antibodies targeting Plasmodium falciparum merozoites in infants and their mothers in Uganda.

Background: Antibody-mediated complement fixation has previously been associated with protection against malaria in naturally acquired immunity. However, the process of early-life development of complement-fixing antibodies in infants, both in comparison to their respective mothers and to other immune parameters, remains less clear. Results: We measured complement-fixing antibodies in newborns and their mothers in a malaria endemic area over 5 years follow-up and found that infants' complement-fixing antibody levels were highest at birth, decreased until six months, then increased progressively until they were similar to birth at five years. Infants with high levels at birth experienced a faster decay of complement-fixing antibodies but showed similar levels to the low response group of newborns thereafter. No difference was observed in antibody levels between infant cord blood and mothers at delivery. The same result was found when categorized into high and low response groups, indicating placental transfer of antibodies. Complement-fixing antibodies were positively correlated with total schizont-specific IgG and IgM levels in mothers and infants at several time points. At nine months, complement-fixing antibodies were negatively correlated with total B cell frequency and osteopontin concentrations in the infants, while positively correlated with atypical memory B cells and P. falciparum-positive atypical memory B cells. Conclusion: This study indicates that complement-fixing antibodies against P. falciparum merozoites are produced in the mothers and placentally-transferred, and they are acquired in infants over time during the first years of life. Understanding early life immune responses is crucial for developing a functional, long lasting malaria vaccine.

Head-to-head comparison of two loop-mediated isothermal amplification (LAMP) kits for diagnosis of malaria in a non-endemic setting.

BACKGROUND: Light microscopy and rapid diagnostic tests (RDT) have long been the recommended diagnostic methods for malaria. However, in recent years, loop-mediated isothermal amplification (LAMP) techniques have been shown to offer superior performance, in particular concerning low-grade parasitaemia, by delivering higher sensitivity and specificity with low laboratory capacity requirements in little more than an hour. In this study, the diagnostic performance of two LAMP kits were assessed head-to-head, compared to highly sensitive quantitative real time PCR (qPCR), in a non-endemic setting. METHODS: In this retrospective validation study two LAMP kits; Alethia® Illumigene Malaria kit and HumaTurb Loopamp™ Malaria Pan Detection (PDT) kit, were evaluated head-to-head for detection of Plasmodium-DNA in 133 biobanked blood samples from suspected malaria cases at the Clinical Microbiology Laboratory of Region Skåne, Sweden to determine their diagnostic performance compared to qPCR. RESULTS: Of the 133 samples tested, qPCR detected Plasmodium DNA in 41 samples (defined as true positives), and the two LAMP methods detected 41 and 37 of those, respectively. The results from the HumaTurb Loopamp™ Malaria PDT kit were in complete congruence with the qPCR, with a sensitivity of 100% (95% CI 91.40-100%) and specificity of 100% (95% CI 96.07-100%). The Alethia® Illumigene Malaria kit had a sensitivity of 90.24% (95% CI 76.87-97.28) and a specificity of 95.65% (95% CI 89.24-98.80) as compared to qPCR. CONCLUSIONS: This head-to-head comparison showed higher performance indicators of the HumaTurb Loopamp™ Malaria PDT kit compared to the Alethia® illumigene Malaria kit for detection of malaria.

Babesia divergens Shows Equal Predilection for Human ABO Blood Types in an In Vitro Erythrocyte Preference Assay.

Babesia is spread to humans via ticks or blood transfusions. Severity of Plasmodium falciparum malaria is strongly correlated to the ABO blood group of the patient. Babesia divergens is an intraerythrocytic parasite with many similarities to malaria, but the impact of ABO on the susceptibility to and progression of the infection in humans is unknown. We have now cultured B. divergens in human group A, B and O erythrocytes in vitro and measured rates of multiplication. The predilection for the different erythrocyte types was also determined using an in vitro erythrocyte preference assay when the parasites were grown in group A, B or O erythrocytes over time and then offered to invade differently stained erythrocytes of all the blood types at the same time. The results showed no difference in multiplication rates for the different blood types, and the parasite exhibited no obvious morphological differences in the different blood types. When cultured first in one blood type and then offered to grow in the others, the preference assay showed that there was no difference between the A, B or O blood groups. In conclusion, this indicates that individuals of the different ABO blood types are likely to be equally susceptible to B. divergens infections.

Anti-phosphatidylserine antibody levels are low in multigravid pregnant women in a malaria-endemic area in Nigeria, and do not correlate with anti-VAR2CSA antibodies.

Anemia is a common malaria-associated complication in pregnant women in endemic regions. Phosphatidylserine (PS) is exposed to the immune system during the massive destruction of red blood cells (RBCs) that accompany malaria, and antibodies against PS have been linked to anemia through destruction of uninfected RBCs. We determined levels of anti-PS IgG antibodies in pregnant women in Ibadan, Nigeria and correlated them to parameters of importance in development of anemia and immunity. Anti-PS correlated inversely with Packed Cell Volume (PCV), indicating that the antibodies could contribute to anemia. There was no correlation with anti-VAR2CSA IgG, haptoglobin or parasitemia, indicating that the modulation of anti-PS response is multifactorial in nature. Anti-PS levels were lowest in multigravidae compared to both primigravidae and secundigravidae and correlated inversely with age. In conclusion, lower levels of anti-PS in multigravidae could be beneficial in avoiding anemia.

Autoantibodies against red blood cell antigens are common in a malaria endemic area.

Plasmodium falciparum malaria can cause severe anemia. Even after treatment, hematocrit can decrease. The role of autoantibodies against erythrocytes is not clearly elucidated and how common they are, or what they are directed against, is still largely unknown. We have investigated antibodies against erythrocytes in healthy adult men living in a highly malaria endemic area in Uganda. We found antibodies in more than half of the individuals, which is significantly more than in a non-endemic area (Sweden). Some of the Ugandan samples had a broad reactivity where it was not possible to determine the exact target of the autoantibodies, but we also found specific antibodies directed against erythrocyte surface antigens known to be of importance for merozoite invasion such as glycophorin A (anti-Ena, anti-M) and glycophorin B (anti-U, anti-S). In addition, several autoantibodies had partial specificities against glycophorin C and the blood group systems Rh, Diego (located on Band 3), Duffy (located on ACKR1), and Cromer (located on CD55), all of which have been described to be important for malaria and therefore of interest for understanding how autoantibodies could potentially stop parasites from entering the erythrocyte. In conclusion, specific autoantibodies against erythrocytes are common in a malaria endemic area.

Prevalence and risk factors for transactional sex among Swedish-born and foreign-born MSM in Sweden.

BACKGROUND: Little is known about transactional sex (TS) (selling and buying sex) among men who have sex with men (MSM) in Sweden, especially among foreign-born MSM. This study aims to assess the prevalence and risk factors of TS (ever and in the previous five years) among MSM living in Sweden and to determine if there is a difference between Swedish-born MSM and foreign-born MSM. METHODS: Swedish data from a multicountry online banner survey (EMIS-2017) was used (n = 4443). Multivariable regression analysis was applied to analyse the data. RESULTS: The prevalence of ever-selling sex among all MSM participants was 13.2% and 5.9% in the previous five years. Selling sex ever and in the previous five years was higher among foreign-born MSM (16% and 8.4%, respectively) than Swedish-born MSM (12.7% and 5.4%, respectively). Among all participants, younger age (aOR:3.19, 95% CI:1.57-6.45) and really struggling to live on current income (aOR:3.37, 95% CI:2.29-4.96) increased the odds of selling sex. Being foreign-born MSM (aOR:1.33, 95% CI:1.02-1.73) and having had sex with a woman in the previous 12 months increased the odds of selling sex (aOR:1.44, 95% CI:1.00-2.07). The prevalence of ever buying sex among MSM participants in Sweden was 10.8% and 6.7% in the previous five years, with the same trend among foreign-born MSM (11.6% and 6.9%, respectively) and Swedish-born MSM (10.7% and 6.6%, respectively). Higher education and not having a current partner increased the odds of buying sex. Younger age was protective for buying sex (aOR:0.05, 95% CI:0.02-0.14). Among the foreign-born MSM, the length of stay in Sweden decreased the odds of buying sex (aOR: 0.98, 95% CI: 0.96-0.99). CONCLUSIONS: The comparatively high prevalence of TS among MSM participants in Sweden, where buying sex is illegal, with a higher prevalence among foreign-born MSM participants, calls for sexual and reproductive health and rights interventions in this population. Increased attention, including HIV prevention programming and education, should be aimed at younger MSM, MSM struggling with their current income, and foreign-born MSM, as they are more likely to report selling sex.

Flow Cytometry Assay of Plasmodium Falciparum-Specific B-Cell Proportions.

Detection of P. falciparum-specific subpopulations of B-cells is important for studies of immunity in malaria. This protocol relies on the photostability and protein loading capacity of carboxylated quantum dots to detect a broad range of different P. falciparum-specific B-cells. Infected red blood cell ghosts, obtained by permeabilization of infected cells with Streptolysin O, are coupled with carboxyl quantum dots using N-ethyl-N-dimethylaminopropyl-carbodiimide condensation. Immunophenotyping of P. falciparum-specific B-cells is performed by flow cytometry using Fc-receptor block, quantum dot-infected red blood cell ghost conjugates, and fluorochrome-conjugated anti-human CD19 mouse monoclonal antibodies.

Conceptualizing safer sex in a new era: Risk perception and decision-making process among highly sexually active men who have sex with men.

BACKGROUND: Men who have sex with men (MSM) are at the epicenter of the HIV epidemic. Efforts to prevent sexually transmitted infections (STIs) and HIV transmission have traditionally focused on condoms and abstinence from high risk sexual practices. Recently, additional methods such as pre-exposure prophylaxis (PrEP) and viral load sorting have been introduced. The aim of this study was to gain understanding about risk management and risk perception strategies for HIV among highly sexually active Swedish MSM with men in Berlin. METHODS: Eighteen sexually active Swedish MSM who travelled to or lived in Berlin were recruited and interviewed in this study. The data were analyzed using content analysis. RESULTS AND DISCUSSION: These men represent a group of knowledgeable MSM in terms of HIV. They acknowledged that having sex with men in Berlin was linked to high sexual risk taking due to the higher prevalence of HIV/STIs than in Sweden, but reported that they nevertheless did not alter their risk management strategies. The analysis resulted in a conceptual model of risk assessment that allows for a deeper understanding of the complexity of the risk reduction decision-making process. Three ontological perceptions of risk were identified: accepting, minimizing and rejecting risk. Seven practiced risk reduction methods were described. Some informants applied their preferred method or set of methods to all settings and partners, while others faced complex decision-making processes. CONCLUSION: HIV is integrated into the core of MSM's sexuality, independently of how they ontologically related to the idea of risk. A constant navigation between pleasure, risk and safety, alongside having to relate to risk created a complex process. Efforts were made to remove HIV from their lives by rejecting the idea of risk, and thereby reject the idea of the homosexual body being a possible vessel for a virus and an epidemic.

Platelets inhibit erythrocyte invasion by Plasmodium falciparum at physiological platelet:erythrocyte ratios.

OBJECTIVE: To evaluate the effect of platelet:erythrocyte (P:E) ratios on Plasmodium falciparum erythrocyte invasion. BACKGROUND: Recent reports have shown that platelets are directly involved in the immune response towards P. falciparum during erythrocyte invasion. However, the literature both supports and conflicts with a role for platelets in limiting invasion. Also, the effect of platelet numbers on invasion (parasitemia) has not been thoroughly investigated. METHODS/MATERIALS: The P. falciparum strains FCR3S1.2 and W2mef were cultured with group O erythrocytes. The cultures were synchronised and supplemented with pooled platelets at P:E ratios ranging from 1:100 to 1:2. Parasitemia was measured at 40 h by flow cytometry and by microscopy of blood smears. RESULTS: A linear relationship was observed between reduced invasion and increased platelet numbers at P:E ratios ranging from 1:100 to 1:20. However, this effect was reversed at lower ratios (1:10-1:2). Microscopic evaluation revealed aggregation and attachment of platelets to erythrocytes, but not specifically to parasitised erythrocytes. CONCLUSION: We have shown that under physiological P:E ratios (approx. 1:10-1:40), platelets inhibited P. falciparum invasion in a dose-dependent manner. At ratios of 1:10 and below, platelets did not further increase the inhibitory effect and, although the trend was reversed, inhibition was still maintained.

Osteopontin and malaria: no direct effect on parasite growth, but correlation with P. falciparum-specific B cells and BAFF in a malaria endemic area.

BACKGROUND: The dysregulation of B cell activation is prevalent during naturally acquired immunity against malaria. Osteopontin (OPN), a protein produced by various cells including B cells, is a phosphorylated glycoprotein that participates in immune regulation and has been suggested to be involved in the immune response against malaria. Here we studied the longitudinal concentrations of OPN in infants and their mothers living in Uganda, and how OPN concentrations correlated with B cell subsets specific for P. falciparum and B cell activating factor (BAFF). We also investigated the direct effect of OPN on P. falciparum in vitro. RESULTS: The OPN concentration was higher in the infants compared to the mothers, and OPN concentration in infants decreased from birth until 9 months. OPN concentration in infants during 9 months were independent of OPN concentrations in corresponding mothers. OPN concentrations in infants were inversely correlated with total atypical memory B cells (MBCs) as well as P. falciparum-specific atypical MBCs. There was a positive correlation between OPN and BAFF concentrations in both mothers and infants. When OPN was added to P. falciparum cultured in vitro, parasitemia was unaffected regardless of OPN concentration. CONCLUSIONS: The concentrations of OPN in infants were higher and independent of the OPN concentrations in corresponding mothers. In vitro, OPN does not have a direct effect on P. falciparum growth. Our correlation analysis results suggest that OPN could have a role in the B cell immune response and acquisition of natural immunity against malaria.

Naturally Acquired Antibodies against Plasmodium falciparum: Friend or Foe?

Antibodies are central to acquired immunity against malaria. Plasmodium falciparum elicits antibody responses against many of its protein components, but there is also formation of antibodies against different parts of the red blood cells, in which the parasites spend most of their time. In the absence of a decisive intervention such as a vaccine, people living in malaria endemic regions largely depend on naturally acquired antibodies for protection. However, these antibodies do not confer sterile immunity and the mechanisms of action are still unclear. Most studies have focused on the inhibitory effect of antibodies, but here, we review both the beneficial as well as the potentially harmful roles of naturally acquired antibodies, as well as autoantibodies formed in malaria. We discuss different studies that have sought to understand acquired antibody responses against P. falciparum antigens, and potential problems when different antibodies are combined, such as in naturally acquired immunity.

Direct contact between Plasmodium falciparum and human B-cells in a novel co-culture increases parasite growth and affects B-cell growth.

BACKGROUND: Plasmodium falciparum parasites cause malaria and co-exist in humans together with B-cells for long periods of time. Immunity is only achieved after repeated exposure. There has been a lack of methods to mimic the in vivo co-occurrence, where cells and parasites can be grown together for many days, and it has been difficult with long time in vitro studies. METHODS AND RESULTS: A new method for growing P. falciparum in 5% CO2 with a specially formulated culture medium is described. This knowledge was used to establish the co-culture of live P. falciparum together with human B-cells in vitro for 10 days. The presence of B-cells clearly enhanced parasite growth, but less so when Transwell inserts were used (not allowing passage of cells or merozoites), showing that direct contact is advantageous. B-cells also proliferated more in presence of parasites. Symbiotic parasitic growth was verified using CESS cell-line and it showed similar results, indicating that B-cells are indeed the cells responsible for the effect. In malaria endemic areas, people often have increased levels of atypical memory B-cells in the blood, and in this assay it was demonstrated that when parasites were present there was an increase in the proportion of CD19 + CD20 + CD27 - FCRL4 + B-cells, and a contraction of classical memory B-cells. This effect was most clearly seen when direct contact between B-cells and parasites was allowed. CONCLUSIONS: These results demonstrate that P. falciparum and B-cells undoubtedly can affect each other when allowed to multiply together, which is valuable information for future vaccine studies.

Sex, drugs and techno - a qualitative study on finding the balance between risk, safety and pleasure among men who have sex with men engaging in recreational and sexualised drug use.

BACKGROUND: Recreational and sexual drug use among men who have sex with men may result in increased risk of poor health. The aim of this study was to better understand drug use and harm reduction techniques among Swedish men who have sex with men traveling to Berlin in order to improve the health of this population and inform public health strategies. METHODS: A qualitative study based on semi-structured interviews with 15 Swedish men aged 23-44 with experience of drug use were recruited through network sampling. Interviews were conducted in Stockholm and Berlin and analysed using content analysis. The interview guide included questions on drug use, context, health and safety. RESULTS: The participants engaged in drug use in both settings and in various contexts. Participants saw themselves as capable of finding a balance between pleasure, safety and risk with the aim to maximize positive effects while minimizing negative ones. The different risks of drug use were known, and participants relied on knowledge, harm reduction strategies and self-defined rules of intake to stay safe and healthy in a broad sense, both short term (i.e. during each session) and long term. Choice of drug and, frequency of intake, multi-use, risk of overdose, risk of HIV, purpose and context of use, how often, etc. were all part of the overall strategy. Knowledge of these methods was spread within the community and on-line rather than from counsellors or other health care providers. However, it did not always translate perfectly into practice and some had experienced overdoses and problematic use. CONCLUSIONS: The findings of this study point to the need for increased adoption of harm reduction techniques in this population focusing on mitigating harm and prevention of risk of problematic use or starting injection drugs. Existing traditional services require adaptations to become more accessible and acceptable to sub-groups of drug users, including low-threshold services providing non-judgemental, evidence-based information. This will require funding of alternative providers such as STI/HIV clinics, among others, and health care providers to increase adoption of prevention strategies, specifically pre-exposure prophylaxis for HIV.

A Serosurvey of Tick-Borne Encephalitis Virus in Sweden: Different Populations and Geographical Locations.

Background: New risk areas for tick-borne encephalitis (TBE) are emerging and the spread of disease and vaccine coverage is unclear in Sweden. We wanted to study the prevalence and levels of TBE-virus (TBEV) antibodies in southern Sweden, and to investigate whether there were individuals with undiagnosed TBE. Materials and Methods: Two cohorts of sera were collected: One group of anonymous individuals in rural areas (AIRA) in Skåne and one group of volunteers who often got tick-bites (tick-bitten individuals [TBI]). An enzyme-linked immunosorbent assay for TBEV IgM and IgG was performed, as well as a TBEV neutralization test (NT) in selected individuals. Results: In the AIRA group, there was an IgG seropositivity of 5.3%. There were individuals with high antibody levels both in areas previously considered as risk areas (Bromölla and Knislinge), as well as in another area (Tyringe). In the TBI group, 45% of the individuals were vaccinated according to the questionnaires and IgG seropositivity was 28%. A lower seroprevalence and levels of antibodies were seen in the middle-aged group (50-69 years) compared with younger or elderly study participants. A positive NT revealed several individuals with suspected undiagnosed episodes of TBE. Conclusion: Subclinical or misdiagnosed cases have probably occurred in Skåne. Middle-aged individuals had lower levels of IgG, which could indicate either less tick exposure or a lower vaccine response. Less than half of the TBI were vaccinated, an indication that more information about the disease and vaccine might be needed. We conclude that the study motivates an increased awareness of TBEV in the region.

A longitudinal study of plasma BAFF levels in mothers and their infants in Uganda, and correlations with subsets of B cells.

Malaria is a potentially life-threatening disease with approximately half of the world's population at risk. Young children and pregnant women are hit hardest by the disease. B cells and antibodies are part of an adaptive immune response protecting individuals continuously exposed to the parasite. An infection with Plasmodium falciparum can cause dysregulation of B cell homeostasis, while antibodies are known to be key in controlling symptoms and parasitemia. BAFF is an instrumental cytokine for the development and maintenance of B cells. Pregnancy alters the immune status and renders previously clinically immune women at risk of severe malaria, potentially due to altered B cell responses associated with changes in BAFF levels. In this prospective study, we investigated the levels of BAFF in a malaria-endemic area in mothers and their infants from birth up to 9 months. We found that BAFF-levels are significantly higher in infants than in mothers. BAFF is highest in cord blood and then drops rapidly, but remains significantly higher in infants compared to mothers even at 9 months of age. We further correlated BAFF levels to P. falciparum-specific antibody levels and B cell frequencies and found a negative correlation between BAFF and both P. falciparum-specific and total proportions of IgG+ memory B cells, as well as CD27- memory B cells, indicating that exposure to both malaria and other diseases affect the development of B-cell memory and that BAFF plays a part in this. In conclusion, we have provided new information on how natural immunity against malaria is formed.

Structured Observation of Motor Performance in Infants: Level and quality associated with later motor development.

AIM: The aim of this study was to investigate the level of motor development and the quality of motor performance during the first 10 months in relation to the Bayley Scales of Infant Development-third edition (Bayley-III) motor index at 2.5 years. METHODS: Children born very preterm from a population-based study (n = 113) were assessed with the Structured Observation of Motor Performance in Infants (SOMP-I) at 2, 4, 6 and 10 months corrected age and the Bayley-III motor index at 2.5 years corrected age (n = 98). Logistic regressions were performed to investigate the independent association of each SOMP-I domain to Bayley-III motor index. RESULTS: There were significant associations between the SOMP-I-scores and Bayley-III motor index per every assessment age. At 4 months, both level and quality were independently associated with a later motor outcome, OR for level was 1.26 (95% CI = 1.08-1.50, P = .002) and for quality, 0.75 (95% CI = 0.63-0.90, P = .002). Quality was independently associated with the Bayley-III motor index at 6 and 10 months: OR 0.080 (95% CI = 0.67-0.95 P = .010) and 0.79 (95% CI = 0.64-0.97, P = .026). CONCLUSION: Both SOMP-I domains, level and quality, are markers to identify motor problems early. Quality became more important with age.

[Babesiosis could be more common in Sweden than previously thought]. / Babesia-infektion kan vara vanligare i Sverige än vi tidigare trott - Parasiten kan spridas via fästingar och blodtransfusion.

Babesia is a malaria-like, intraerythrocytic parasite with more than 100 different species. It is a zoonosis and some of the species are transmitted to humans by ticks and also as a possible transfusion-transmitted infection. In Sweden the disease has been well known in veterinary medicine for a long time, but only a few but severe cases have been published in humans during the last decades. Common symptoms from human Babesia infections (babesiosis) are fever, chills and myalgia and they vary from subclinical to potentially fatal among those with risk factors such as immunosuppression and splenectomy. In the U.S. more than 2,000 cases of babesiosis are found yearly and it is one of the most frequent fatal infections following blood transfusion. A study from southern Sweden has recently revealed a seroprevalence of 16% of Babesia antibodies among Borrelia-infected persons. These results indicate that there is a need to broaden awareness of Babesia in Sweden.