The ability of Interleukin-10 to negate haemozoin-related pro-inflammatory effects has the potential to restore impaired macrophage function associated with malaria infection.

BACKGROUND: Although pro-inflammatory cytokines are involved in the clearance of Plasmodium falciparum during the early stages of the infection, increased levels of these cytokines have been implicated in the pathogenesis of severe malaria. Amongst various parasite-derived inducers of inflammation, the malarial pigment haemozoin (Hz), which accumulates in monocytes, macrophages and other immune cells during infection, has been shown to significantly contribute to dysregulation of the normal inflammatory cascades. METHODS: The direct effect of Hz-loading on cytokine production by monocytes and the indirect effect of Hz on cytokine production by myeloid cells was investigated during acute malaria and convalescence using archived plasma samples from studies investigating P. falciparum malaria pathogenesis in Malawian subjects. Further, the possible inhibitory effect of IL-10 on Hz-loaded cells was examined, and the proportion of cytokine-producing T-cells and monocytes during acute malaria and in convalescence was characterized. RESULTS: Hz contributed towards an increase in the production of inflammatory cytokines, such as Interferon Gamma (IFN-γ), Tumor Necrosis Factor (TNF) and Interleukin 2 (IL-2) by various cells. In contrast, the cytokine IL-10 was observed to have a dose-dependent suppressive effect on the production of TNF among other cytokines. Cerebral malaria (CM) was characterized by impaired monocyte functions, which normalized in convalescence. CM was also characterized by reduced levels of IFN-γ-producing T cell subsets, and reduced expression of immune recognition receptors HLA-DR and CD 86, which also normalized in convalescence. However, CM and other clinical malaria groups were characterized by significantly higher plasma levels of pro-inflammatory cytokines than healthy controls, implicating anti-inflammatory cytokines in balancing the immune response. CONCLUSIONS: Acute CM was characterized by elevated plasma levels of pro-inflammatory cytokines and chemokines but lower proportions of cytokine-producing T-cells and monocytes that normalize during convalescence. IL-10 is also shown to have the potential to indirectly prevent excessive inflammation. Cytokine production dysregulated by the accumulation of Hz appears to impair the balance of the immune response to malaria and exacerbates pathology.

Variation in competent and respectful delivery care in Kenya and Malawi: a retrospective analysis of national facility surveys.

OBJECTIVE: Although substantial progress has been made in increasing access to care during childbirth, reductions in maternal and neonatal mortality have been slower. Poor-quality care may be to blame. In this study, we measure the quality of labour and delivery services in Kenya and Malawi using data from observations of deliveries and explore factors associated with levels of competent and respectful care. METHODS: We used data from nationally representative health facility assessment surveys. A total of 1100 deliveries in 392 facilities across Kenya and Malawi were observed and quality was assessed using two indices: the quality of the process of intrapartum and immediate postpartum care (QoPIIPC) index and a previously validated index of respectful maternity care. Data from standardised observations of care were analysed using descriptive statistics and multivariable random-intercept regression models to examine factors associated with variation in quality of care. We also quantified the variance in quality explained by each domain of covariates (patient-, provider- and facility-level and subnational divisions). RESULTS: Only 61-66% of basic elements of competent and respectful care were performed. In adjusted models, better-staffed facilities, private hospitals and morning deliveries were associated with higher levels of competent and respectful care. In Malawi, younger, primipara and HIV-positive women received higher-quality care. Quality also differed substantially across regions in Kenya, with a 25 percentage-point gap between Nairobi and the Coast region. Quality was also higher in higher-volume facilities and those with caesarean section capacity. Most of the explained variance in quality was due to regions in Kenya and to facility, and patient-level characteristics in Malawi. CONCLUSIONS: Our findings suggest considerable scope for improvement in quality. Increasing staffing and shifting births to higher-volume facilities - along with promotion of respectful care in these facilities - should be considered in sub-Saharan Africa to improve outcomes for mothers and newborns.

OBJECTIF: Bien que des progrès substantiels aient été accomplis dans l'amélioration de l'accès aux soins pendant l'accouchement, les réductions de la mortalité maternelle et néonatale ont été plus lentes. Des soins de mauvaise qualité peuvent être à blâmer. Dans cette étude, nous mesurons la qualité de la main-d'œuvre et des services d'accouchement au Kenya et au Malawi en utilisant les données des observations des accouchements et explorons les facteurs associés aux niveaux de la compétence et du respect dans les soins. MÉTHODES: Nous avons utilisé les données d'enquêtes d'évaluation des établissements de santé représentatives au niveau national. 1100 accouchements dans 392 établissements au Kenya et au Malawi ont été observés et la qualité a été évaluée à l'aide de deux indices: l'indice de qualité du processus de soins intra-partum et postpartum immédiat (QoPIIPC) et un indice précédemment validé de soins maternels respectueux. Les données des observations normalisées des soins ont été analysées à l'aide de statistiques descriptives et de modèles de régression à interceptions aléatoires multivariables pour examiner les facteurs associés à la variation de la qualité des soins. Nous avons également quantifié la variance de la qualité expliquée par chaque domaine de covariables (divisions au niveau des patients, des prestataires et des établissements, et infranationales). RÉSULTATS: Seuls 61% à 66% des éléments de base de soins compétents et respectueux ont été réalisés. Dans les modèles ajustés, des établissements mieux dotés en personnel, des hôpitaux privés et des accouchements le matin étaient associés à des niveaux plus élevés de soins compétents et respectueux. Au Malawi, les femmes plus jeunes, primipares et VIH positives ont reçu des soins de meilleure qualité. La qualité différait également considérablement d'une région à l'autre au Kenya, avec un écart de 25 points de pourcentage entre Nairobi et la région côtière. La qualité était également plus élevée dans les établissements avec un volume plus élevé et ceux ayant une capacité de césarienne. La majeure partie des raisons de la variance dans la qualité était liée aux régions du Kenya et à l'établissement et aux caractéristiques des patients au Malawi. CONCLUSIONS: Nos résultats suggèrent une marge considérable pour l'amélioration de la qualité. L'augmentation du personnel et le déplacement des naissances vers des établissements de plus grand volume - ainsi que la promotion de soins respectueux dans ces établissements - devraient être envisagés en Afrique subsaharienne pour améliorer les résultats pour les mères et les nouveau-nés.

Brain swelling is independent of peripheral plasma cytokine levels in Malawian children with cerebral malaria.

BACKGROUND: Cerebral malaria (CM) is often fatal, and severe brain swelling is a predictor of CM-related mortality. CM is characterized by elevated circulating pro-inflammatory cytokines TNF and IFN-γ and anti-inflammatory cytokine IL-10, however whether cytokine levels correlate with brain swelling severity is unknown. This study therefore was conducted to investigate the relationship between cytokine levels and brain swelling severity in children presenting with CM. METHODS: A total of 195 Malawian children presenting with CM were recruited and had the concentrations of plasma cytokines determined and compared to brain swelling severity, determined by MRI examination, and graded as severe, moderate, mild or none. RESULTS: Levels of IL-1ß, IL-6, IL-8 and IL-10 did not differ between CM patients with and without severe brain swelling. Compared to children without brain swelling, IL-12 levels were higher in children with severe swelling (p < 0.01, no swelling 1 pg/mL, IQR [1] vs. severe swelling 18.7 pg/mL, IQR [1-27]), whereas TNF concentrations were higher in children with moderate brain swelling compared to children with no swelling (p < 0.01, no swelling 3 pg/mL, IQR [1-20] vs. moderate swelling 24 pg/mL, IQR [8-58]. Multivariate analysis showed that no single cytokine independently predicted brain swelling. CONCLUSION: Severe brain swelling in paediatric CM was independent of tested blood pro-inflammatory and anti-inflammatory cytokines which are markers of systemic inflammation.

Convalescent Plasmodium falciparum-specific seroreactivity does not correlate with paediatric malaria severity or Plasmodium antigen exposure.

BACKGROUND: Antibody immunity is thought to be essential to prevent severe Plasmodium falciparum infection, but the exact correlates of protection are unknown. Over time, children in endemic areas acquire non-sterile immunity to malaria that correlates with development of antibodies to merozoite invasion proteins and parasite proteins expressed on the surface of infected erythrocytes. RESULTS: A 1000 feature P. falciparum 3D7 protein microarray was used to compare P. falciparum-specific seroreactivity during acute infection and 30 days after infection in 23 children with uncomplicated malaria (UM) and 25 children with retinopathy-positive cerebral malaria (CM). All children had broad P. falciparum antibody reactivity during acute disease. IgM reactivity decreased and IgG reactivity increased in convalescence. Antibody reactivity to CIDR domains of "virulent" PfEMP1 proteins was low with robust reactivity to the highly conserved, intracellular ATS domain of PfEMP1 in both groups. Although children with UM and CM differed markedly in parasite burden and PfEMP1 exposure during acute disease, neither acute nor convalescent PfEMP1 seroreactivity differed between groups. Greater seroprevalence to a conserved Group A-associated ICAM binding extracellular domain was observed relative to linked extracellular CIDRα1 domains in both case groups. Pooled immune IgG from Malawian adults revealed greater reactivity to PfEMP1 than observed in children. CONCLUSIONS: Children with uncomplicated and cerebral malaria have similar breadth and magnitude of P. falciparum antibody reactivity. The utility of protein microarrays to measure serological recognition of polymorphic PfEMP1 antigens needs to be studied further, but the study findings support the hypothesis that conserved domains of PfEMP1 are more prominent targets of cross reactive antibodies than variable domains in children with symptomatic malaria. Protein microarrays represent an additional tool to identify cross-reactive Plasmodium antigens including PfEMP1 domains that can be investigated as strain-transcendent vaccine candidates.

Differential PfEMP1 expression is associated with cerebral malaria pathology.

Plasmodium falciparum is unique among human malarias in its ability to sequester in post-capillary venules of host organs. The main variant antigens implicated are the P. falciparum erythrocyte membrane protein 1 (PfEMP1), which can be divided into three major groups (A-C). Our study was a unique examination of sequestered populations of parasites for genetic background and expression of PfEMP1 groups. We collected post-mortem tissue from twenty paediatric hosts with pathologically different forms of cerebral malaria (CM1 and CM2) and parasitaemic controls (PC) to directly examine sequestered populations of parasites in the brain, heart and gut. Use of two different techniques to investigate this question produced divergent results. By quantitative PCR, group A var genes were upregulated in all three organs of CM2 and PC cases. In contrast, in CM1 infections displaying high levels of sequestration but negligible vascular pathology, there was high expression of group B var. Cloning and sequencing of var transcript tags from the same samples indicated a uniformly low expression of group A-like var. Generally, within an organ sample, 1-2 sequences were expressed at dominant levels. 23% of var tags were detected in multiple patients despite the P. falciparum infections being genetically distinct, and two tags were observed in up to seven hosts each with high expression in the brains of 3-4 patients. This study is a novel examination of the sequestered parasites responsible for fatal cerebral malaria and describes expression patterns of the major cytoadherence ligand in three organ-derived populations and three pathological states.

Delayed acquisition of Plasmodium falciparum antigen-specific CD4(+) T cell responses in HIV-exposed uninfected Malawian children receiving daily cotrimoxazole prophylaxis.

BACKGROUND: Cotrimoxazole (CTX) prophylaxis, recommended in HIV-exposed uninfected (HEU) children primarily against HIV-related opportunistic infections, has been shown to have some efficacy against Plasmodium falciparum malaria. The effects of CTX prophylaxis on the acquisition of P. falciparum antigen specific CD4(+) T cells-mediated immunity in HEU children is still not fully understood. METHODS: Peripheral blood was collected from HEU and HIV-unexposed uninfected (HUU) children at 6, 12 and 18 months of age. Proportion of CD4(+) T cells subsets were determined by immunophenotyping. P. falciparum antigen-specific CD4(+) T cells responses were measured by intracellular cytokine staining assay. RESULTS: There were no differences in the proportions of naïve, effector and memory CD4(+) T cell subsets between HEU and HUU children at all ages. There was a trend showing acquisition of P. falciparum-specific IFN-γ and TNF-producing CD4(+) T cells with age in both HUU and HEU children. There was, however, lower frequency of P. falciparum-specific IFN-γ-producing CD4(+) T cells in HEU compared to HUU at 6 and 12 months, which normalized 6 months after stopping CTX prophylaxis. CONCLUSION: The results demonstrate that there is delayed acquisition of P. falciparum-specific IFN-γ-producing CD4(+) T cells in HEU children on daily cotrimoxazole prophylaxis, which is evident at 6 and 12 months of age in comparison to HUU age-matched controls. However, whether this delayed acquisition of P. falciparum-specific IFN-γ-producing CD4(+) T cells leads to higher risk to malaria disease remains unknown and warrants further investigation.

Proportions of CD4+, CD8+ and B cell subsets are not affected by exposure to HIV or to Cotrimoxazole prophylaxis in Malawian HIV-uninfected but exposed children.

BACKGROUND: As a result of successful PMTCT programs, children born from HIV-infected mothers are now effectively protected from contracting the infection. However, it is not well known whether in utero exposure to the virus and the subsequent exposure to Cotrimoxazole (CTX) prophylaxis affect the cell mediated immune system of the children. This observational prospective study was aimed at determining how CD4(+) T, CD8(+) T and B cell subsets varied in HIV-exposed but uninfected (HEU) children at different ages. METHODS: We recruited HEU and HIV-unexposed and uninfected (HUU) children from 6 months of age and followed them up until they were 18 months old. HEU children received daily CTX prophylaxis beginning at 6 weeks of age until when 12 months of age. Venous blood samples were collected 6 monthly and analysed for different subsets of CD8(+) T, B cells and totalCD4(+) T cells. RESULTS: At 6 months of age, HEU children had a lower percentage of total CD4(+) T cells compared to HUU children and a lower proportion of naïve CD8(+) T cells but higher percentage of effector memory CD8(+) T cells compared to HUU children. HEU and HUU children had similar proportions of all B cell subsets at all ages. CONCLUSIONS: The study showed that the subtle variations in CD4(+) and CD8(+) T cell subsets observed at 6 months do not last beyond 12 months of age, suggesting that HEU children have a robust cell-mediated immune system during first year of life. TRIAL REGISTRATION: This article report is not based on results of a controlled health-care intervention.

Sequential acquisition of T cells and antibodies to nontyphoidal Salmonella in Malawian children.

BACKGROUND: Salmonella Typhimurium (STm) remain a prominent cause of bacteremia in sub-Saharan Africa. Complement-fixing antibodies to STm develop by 2 years of age. We hypothesized that STm-specific CD4⁺ T cells develop alongside this process. METHODS: Eighty healthy Malawian children aged 0-60 months were recruited. STm-specific CD4⁺ T cells producing interferon γ, tumor necrosis factor α, and interleukin 2 were quantified using intracellular cytokine staining. Antibodies to STm were measured by serum bactericidal activity (SBA) assay, and anti-STm immunoglobulin G antibodies by enzyme-linked immunosorbent assay. RESULTS: Between 2006 and 2011, STm bacteremias were detected in 449 children <5 years old. STm-specific CD4⁺ T cells were acquired in infancy, peaked at 14 months, and then declined. STm-specific SBA was detectable in newborns, declined in the first 8 months, and then increased to a peak at age 35 months. Acquisition of SBA correlated with acquisition of anti-STm-lipopolysaccharide (LPS) immunoglobulin G (r = 0.329 [95% confidence interval, .552-.062]; P = .01) but not anti-STm-outer membrane protein or anti-STm-flagellar protein (FliC). CONCLUSIONS: Acquisition of STm-specific CD4⁺ T cells in early childhood is consistent with early exposure to STm or cross-reactive protein antigens priming this T-cell development. STm-specific CD4⁺ T cells seem insufficient to protect against invasive nontyphoidal Salmonella disease, but sequential acquisition of SBA to STm LPS is associated with a decline in its incidence.

Prothrombotic autoantibodies targeting platelet factor 4/polyanion are associated with pediatric cerebral malaria.

BACKGROUNDFeatures of consumptive coagulopathy and thromboinflammation are prominent in cerebral malaria (CM). We hypothesized that thrombogenic autoantibodies contribute to a procoagulant state in CM.METHODSPlasma from children with uncomplicated malaria (UM) (n = 124) and CM (n = 136) was analyzed by ELISA for a panel of 8 autoantibodies including anti-platelet factor 4/polyanion (anti-PF4/P), anti-phospholipid, anti-phosphatidylserine, anti-myeloperoxidase, anti-proteinase 3, anti-dsDNA, anti-ß-2-glycoprotein I, and anti-cardiolipin. Plasma samples from individuals with nonmalarial coma (NMC) (n = 49) and healthy controls (HCs) (n = 56) were assayed for comparison. Associations with clinical and immune biomarkers were determined using univariate and logistic regression analyses.RESULTSMedian anti-PF4/P and anti-PS IgG levels were elevated in individuals with malaria infection relative to levels in HCs (P < 0.001) and patients with NMC (PF4/P: P < 0.001). Anti-PF4/P IgG levels were elevated in children with CM (median = 0.27, IQR: 0.19-0.41) compared with those with UM (median = 0.19, IQR: 0.14-0.22, P < 0.0001). Anti-PS IgG levels did not differ between patients with UM and those with CM (P = 0.39). When patients with CM were stratified by malaria retinopathy (Ret) status, the levels of anti-PF4/P IgG correlated negatively with the peripheral platelet count in patients with Ret+ CM (Spearman's rho [Rs] = 0.201, P = 0.04) and associated positively with mortality (OR = 15.2, 95% CI: 1.02-275, P = 0.048). Plasma from patients with CM induced greater platelet activation in an ex vivo assay relative to plasma from patients with UM (P = 0.02), and the observed platelet activation was associated with anti-PF4/P IgG levels (Rs= 0.293, P = 0.035).CONCLUSIONSThrombosis mediated by elevated anti-PF4/P autoantibodies may be one mechanism contributing to the clinical complications of CM.

Expression of CD11a, CD11b, CD11c, and CD18 on Neutrophils from Different Clinical Types of Malaria in Malawian Children.

BACKGROUND: Malaria in individuals who have never had an infection before is usually characterized by an inflammatory response that is linked to the expression of specific activation markers on cells of the innate immune system. METHODS: This study investigated absolute white blood cell (WBC) and neutrophil counts and expression of several adhesion markers on neutrophils from HIV-uninfected children who were suffering from cerebral malaria (n=35), severe malarial anemia (SMA, n=39), and uncomplicated malaria (n=49) and healthy aparasitemic children (n=33) in Blantyre, Malawi. RESULTS: All clinical malaria groups had higher WBC and neutrophil counts compared to healthy controls, with the acute SMA group having significantly (p<0.0001) higher WBC counts than the controls. These elevated counts normalized during recovery. Surprisingly, in all clinical malaria groups, the surface expression of CD11b, CD11c, and CD18 on neutrophils was lower than in healthy controls, again normalizing during convalescence. CONCLUSION: In areas where Plasmodium falciparum malaria is hyperendemic, such as where this study was conducted, neutrophils have reduced expression of adhesion molecules and activation markers during acute stages of the infection, regardless of the clinical type of malaria. This reduced expression could be due to an individual's past exposure to P. falciparum or other parasite-related factors that manifest during active malaria that still need to be investigated.

Characterization of Lymphocyte Subsets in Lymph Node and Spleen Sections in Fatal Pediatric Malaria.

Secondary lymphoid tissues play a major role in the human immune response to P. falciparum infection. Previous studies have shown that acute falciparum malaria is associated with marked perturbations of the cellular immune system characterized by lowered frequency and absolute number of circulating T cell subsets. A temporary relocation of T cells, possibly by infiltration to secondary lymphoid tissue, or their permanent loss through apoptosis, are two proposed explanations for this observation. We conducted the present study to determine the phenotype of lymphocyte subsets that accumulate in the lymph node and spleen during acute stages of falciparum malaria infection in Malawian children, and to test the hypothesis that lymphocytes are relocated to lymphoid tissues during acute infection. We stained tissue sections from children who had died of the two common clinical forms of severe malaria in Malawi, namely severe malarial anemia (SMA, n = 1) and cerebral malaria (CM, n = 3), and used tissue sections from pediatric patients who had died of non-malaria sepsis (n = 2) as controls. Both lymph node and spleen tissue (red pulp) sections from CM patients had higher percentages of T cells (CD4+ and CD8+) compared to the SMA patient. In the latter, we observed a higher percentage of CD20+ B cells in the lymph nodes compared to CM patients, whereas the opposite was observed in the spleen. Both lymph node and spleen sections from CM patients had increased percentages of CD69+ and CD45RO+ cells compared to tissue sections from the SMA patient. These results support the hypothesis that the relocation of lymphocytes to spleen and lymph node may contribute to the pan-lymphopenia observed in acute CM.

Acute Malaria in Malawian Children and Adults is Characterized by Thrombocytopenia That Normalizes in Convalescence.

Background: Plasmodium falciparum malaria has been linked with significant perturbations of the peripheral cell-mediated immune system during acute phase. Some of these changes include lower than normal platelet counts. Although the exact mechanisms that drive thrombocytopenia in P. falciparum malaria are not fully known, a number of hypotheses have been proposed. We conducted two sets of studies with one aimed at determining platelet counts in Malawian children, and the other in adults during acute P. falciparum malaria and a month post treatment. Materials and Methods: We recruited a total of 113 HIV-uninfected children with acute malaria [n=54 with uncomplicated malaria (UCM), n=30 with severe malarial anemia (SMA), n=29 presenting with cerebral malaria (CM)]. We also recruited 42 HIV-uninfected healthy controls. Out of the 113 participants with malaria, 73 (65%) [n=34 (63%) UCM, n=21 (70%) SMA and n=18 (62%) CM] were successfully followed-up one month after treatment. A 5mL peripheral blood sample was collected for platelet count using HMX Haematological Analyzer analysis both at baseline (acute malaria) and at follow-up a month later. Platelet counts were also determined in blood samples of 106 HIV-uninfected adults, 47 of whom presented with UCM and 29 with severe malaria (SM) and these counts were compared to those of 30 healthy controls. Of the malaria cases, platelet counts for 44 UCM and 21 SM were determined again during follow-up a month after treatment. Results: In both children and adults, platelet counts were significantly lower during acute disease compared to the levels in the healthy controls with the lowest levels observed in CM (children) or SM (adults). These lower than normal levels increased close to normal levels a month post treatment. Conclusion: P. falciparum malaria in Malawian children and adults was characterized by profound thrombocytopenia which recovered during convalescence.

Lymphocyte subsets in healthy Malawians: implications for immunologic assessment of HIV infection in Africa.

BACKGROUND: CD4(+)T lymphocyte measurements are the most important indicator of mortality in HIV-infected individuals in resource-limited settings. There is currently a lack of comprehensive immunophenotyping data from African populations to guide the immunologic assessment of HIV infection. OBJECTIVE: To quantify variation in absolute and relative lymphocyte subsets with age in healthy Malawians. METHODS: Lymphocyte subsets in peripheral blood of 539 healthy HIV-uninfected Malawians stratified by age were enumerated by flow cytometry. RESULTS: B and T-lymphocyte and T-lymphocyte subset absolute concentrations peaked in early childhood then decreased to adult levels, whereas lymphocyte subset proportions demonstrated much less variation with age. Adult lymphocyte subsets were similar to those in developed countries. In contrast, high B-lymphocyte and CD8(+)T-lymphocyte levels among children under 2 years, relative to those in developed countries, resulted in low CD4(+)T-lymphocyte percentages that varied little between 0 and 5 years (35% to 39%). The CD4(+)T-lymphocyte percentages in 35% of healthy children under 1 year and 18% of children age 1 to 3 years were below the World Health Organization threshold defining immunodeficiency in HIV-infected children in resource-limited settings. Thirteen percent of healthy children under 18 months old had a CD4:CD8T-lymphocyte ratio <1.0, which is commonly associated with HIV infection. All immunologic parameters except absolute natural killer lymphocyte concentration varied significantly with age, and percentage and overall absolute CD4(+)T-lymphocyte counts were higher in females than males. CONCLUSION: Although lymphocyte subsets in Malawian adults are similar to those from developed countries, CD4(+)T-lymphocyte percentages in young children are comparatively low. These findings need to be considered when assessing the severity of HIV-related immunodeficiency in African children under 3 years.

Variation of B cell subsets with age in healthy Malawians.

Although a number of previous studies have shown that different lymphocyte subsets, including B cells, vary with age, how different B cell subsets vary with age in Malawian population has not been shown before. We recruited Malawian participants of different ages and analyzed their venous blood samples for different B cell subsets. We found that both percentage and absolute counts of B cells varied with age peaking in the 7 to 12 months age group. Proportion of naïve B cells was highest in neonates and decreased with age whereas the percentage of memory B cells was lowest in neonates and increased with age. When we zeroed in on the age band within which the proportion of B cells was highest, both classical and activated memory B cells increased with age and the naïve followed the opposite trend. These results provide additional knowledge in our understanding of the dynamics of B cell subsets in individuals of a specific ethnicity as they age.

SARS-CoV-2 and HIV-1: Should HIV-1-Infected Individuals in Sub-Saharan Africa Be Considered a Priority Group for the COVID-19 Vaccines?

Since its emergence in 2019 SARS-CoV-2 has proven to have a higher level of morbidity and mortality compared to the other prevailing coronaviruses. Although initially most African countries were spared from the devastating effect of SARS-CoV-2, at present almost every country has been affected. Although no association has been established between being HIV-1-infected and being more vulnerable to contracting COVID-19, HIV-1-infected individuals have a greater risk of developing severe COVID-19 and of COVID-19 related mortality. The rapid development of the various types of COVID-19 vaccines has gone a long way in mitigating the devastating effects of the virus and has controlled its spread. However, global vaccine deployment has been uneven particularly in Africa. The emergence of SARS-CoV-2 variants, such as Beta and Delta, which seem to show some subtle resistance to the existing vaccines, suggests COVID-19 will still be a high-risk infection for years. In this review we report on the current impact of COVID-19 on HIV-1-infected individuals from an immunological perspective and attempt to make a case for prioritising COVID-19 vaccination for those living with HIV-1 in Sub-Saharan Africa (SSA) countries like Malawi as one way of minimising the impact of COVID-19 in these countries.

The role of different components of the immune system against Plasmodium falciparum malaria: Possible contribution towards malaria vaccine development.

Plasmodium falciparum malaria still remains a major global public health challenge with over 220 million new cases and well over 400,000 deaths annually. Most of the deaths occur in sub-Saharan Africa which bears 90 % of the malaria cases. Such high P. falciparum malaria-related morbidity and mortality rates pose a huge burden on the health and economic wellbeing of the countries affected. Lately, substantial gains have been made in reducing malaria morbidity and mortality through intense malaria control initiatives such as use of effective antimalarials, intensive distribution and use of insecticide-treated nets (ITNs), and implementation of massive indoor residual spraying (IRS) campaigns. However, these gains are being threatened by widespread resistance of the parasite to antimalarials, and the vector to insecticides. Over the years the use of vaccines has proven to be the most reliable, cost-effective and efficient method for controlling the burden and spread of many infectious diseases, especially in resource poor settings with limited public health infrastructure. Nonetheless, this had not been the case with malaria until the most promising malaria vaccine candidate, RTS,S/AS01, was approved for pilot implementation programme in three African countries in 2015. This was regarded as the most important breakthrough in the fight against malaria. However, RTS,S/AS01 has been found to have some limitations, the main ones being low efficacy in certain age groups, poor immunogenicity and need for almost three boosters to attain a reasonable efficacy. Thus, the search for a more robust and effective malaria vaccine still continues and a better understanding of naturally acquired immune responses to the various stages, including the transmissible stages of the parasite, could be crucial in rational vaccine design. This review therefore compiles what is currently known about the basic biology of P. falciparum and the natural malaria immune response against malaria and progress made towards vaccine development.

Optimization of stimulation and staining conditions for intracellular cytokine staining (ICS) for determination of cytokine-producing T cells and monocytes.

Cell-mediated responses to immunological stimuli are often localised in inflammatory sites and involve a number of cell types. These responses can be functionally characterised at the single-cell level on the basis of the types of cytokines expressed either in whole blood or PBMCs. The ability to measure antigen-specific cell responses at the single cell level is an important tool with a wide range of potential applications ranging from studies of disease pathogenesis to the evaluation of vaccines. A number of experiments were performed in this study in order to establish the optimal conditions for in vitro stimulation of cytokine production by T cells and monocytes in whole blood samples collected from healthy adult Malawian participants and the optimal staining conditions for various cytokine producing cells. Different stimulation methods and conditions, different culture tubes and incubators and different antibody labelling conditions were assessed in order to establish optimal conditions for detecting cytokine-producing cells in whole blood samples. The use of PMA plus Ionomycin produced highest cytokine-producing T cells whereas LPS was a better stimulant for cytokine producing monocytes. Stimulation of whole blood for 5 h was optimal for cytokine detection in T cells whereas 4 h was optimal for monocytes. BFA was found to be a better Golgi blocker than Monensin and the use of 15 ml Falcon-type polypropylene tubes while stationary resulted in the detection of the highest proportion of cytokine-producing cells. T cells were found to be producers of mainly TNF-α, IFN-γ and IL-2 whereas Monocytes were mainly producing TNF-α and IL-6. Anti-CD3-PerCP (used at a ratio of 1:25), anti-CD14-APC (used at a ratio of 1:50) and anti-cytokine-PE (used at a ratio of 1:12.5) resulted in the best results. The highest cytokine production monocytes were detected when 1 X FACS Lysing solution was used at a volume of 40X that of the whole blood sample compared to the other volumes. These optimal conditions are essential in determination of proportion of cytokine-producing cells using ICS in whole blood.

Acceptability of Vegetable Fortified Ugali in Sub-Saharan Africa.

Corn flour-based porridge like dough, ugali, is the staple food of low-income population in sub-Saharan Africa. Lack of vitamin A, carotenoids, and dietary fibers brings about serious health issues to this population. In this study, vegetables including bok choy, broccoli, cabbage, carrot, Chinese onion stalk (C_onion), mushroom, are added during the cooking of ugali, as nutritional supplements. The freeze-dried powder of each vegetable was used for its long storage, stable nutrients, and similar particle size. Sub-Saharan African assessors were trained and sensory evaluated the six different vegetable fortified ugali with the plain, unfortified as the control on five attributes. The plain ugali was indistinguishable with the C_onion stalk fortified in color, with the carrot and C_onion stalk fortified in odor, with all vegetables (except broccoli and mushroom) fortified ugali in taste, with carrot and C_onion stalk fortified in granularity, and with cabbage, carrot, C_onion stalk fortified in viscosity. Preference ranking analysis showed that the C_onion stalk fortified ugali is even more favorably preferred than the plain, unfortified ugali, probably due to the umami components in C_onion that serve as the taste enhancer. This study indicates that Chinese onion stalk is a potential vegetable supplement to population in the sub-Saharan Africa.

Determinants of brain swelling in pediatric and adult cerebral malaria.

Cerebral malaria (CM) affects children and adults, but brain swelling is more severe in children. To investigate features associated with brain swelling in malaria, we performed blood profiling and brain MRI in a cohort of pediatric and adult patients with CM in Rourkela, India, and compared them with an African pediatric CM cohort in Malawi. We determined that higher plasma Plasmodium falciparum histidine rich protein 2 (PfHRP2) levels and elevated var transcripts that encode for binding to endothelial protein C receptor (EPCR) were linked to CM at both sites. Machine learning models trained on the African pediatric cohort could classify brain swelling in Indian children CM cases but had weaker performance for adult classification, due to overall lower parasite var transcript levels in this age group and more severe thrombocytopenia in Rourkela adults. Subgrouping of patients with CM revealed higher parasite biomass linked to severe thrombocytopenia and higher Group A-EPCR var transcripts in mild thrombocytopenia. Overall, these findings provide evidence that higher parasite biomass and a subset of Group A-EPCR binding variants are common features in children and adult CM cases, despite age differences in brain swelling.