Cytoadherence in paediatric malaria: ABO blood group, CD36, and ICAM1 expression and severe Plasmodium falciparum infection

As a leading cause of childhood mortality worldwide, selection pressure by Plasmodium falciparum continues to shape the human genome. Severe disturbances within the microcirculation result from the adhesion of infected erythrocytes to host receptors on monocytes, platelets, and endothelium. In this prospective study, we compared expression of all major host cytoadhesion receptors among Ugandan children presenting with uncomplicated malaria (n = 1078) versus children with severe malaria (n = 855), including cerebral malaria (n = 174), severe anaemia (n = 522), and lactic acidosis (n = 154). We report a significant survival advantage attributed to blood group O and increased monocyte expression of CD36 and ICAM1 (CD54). The high case fatality rate syndromes of cerebral malaria and lactic acidosis were associated with high platelet CD36 expression and thrombocytopenia, and severe malaria anaemia was characterized by low ICAM1 expression. In a logistic regression model of disease severity, odds ratios for the mitigating effects of blood group O, CD36, and ICAM1 phenotypes were greater than that of sickle haemoglobin. Host genetic adaptations to Plasmodium falciparum suggest new potential malaria treatment strategies

Inter-Relationships of Cardinal Features and Outcomes of Symptomatic Pediatric Plasmodium falciparum Malaria in 1,933 Children in Kampala, Uganda

Malaria remains a challenging diagnosis with variable clinical presentation and a wide spectrum of disease severity. Using a structured case report form, we prospectively assessed 1,933 children at Mulago Hospital in Kampala, Uganda with acute Plasmodium falciparum malaria. Children with uncomplicated malaria significantly differed from those with severe disease for 17 features. Among 855 children with severe disease, the case-fatality rate increased as the number of severity features increased. Logistic regression identified five factors independently associated with death: cerebral malaria, hypoxia, severe thrombocytopenia, leukocytosis, and lactic acidosis. Cluster analysis identified two groups: one combining anemia, splenomegaly, and leukocytosis; and a second group centered on death, severe thrombocytopenia, and lactic acidosis, which included cerebral malaria, hypoxia, hypoglycemia, and hyper-parasitemia. Our report updates previous clinical descriptions of severe malaria, quantifies significant clinical and laboratory inter-relationships, and will assist clinicians treating malaria and those planning or assessing future research (NCT00707200) (www.clinicaltrials.gov)

Emergence and spread of a SARS-CoV-2 lineage A variant (A.23.1) with altered spike protein in Uganda

Here, we report SARS-CoV-2 genomic surveillance from March 2020 until January 2021 in Uganda, a landlocked East African country with a population of approximately 40 million people. We report 322 full SARS-CoV-2 genomes from 39,424 reported SARS-CoV-2 infections, thus representing 0.8% of the reported cases. Phylogenetic analyses of these sequences revealed the emergence of lineage A.23.1 from lineage A.23. Lineage A.23.1 represented 88% of the genomes observed in December 2020, then 100% of the genomes observed in January 2021. The A.23.1 lineage was also reported in 26 other countries. Although the precise changes in A.23.1 differ from those reported in the first three SARS-CoV-2 variants of concern (VOCs), the A.23.1 spike-protein-coding region has changes similar to VOCs including a change at position 613, a change in the furin cleavage site that extends the basic amino acid motif and multiple changes in the immunogenic N-terminal domain. In addition, the A.23.1 lineage has changes in non-spike proteins including nsp6, ORF8 and ORF9 that are also altered in other VOCs. The clinical impact of the A.23.1 variant is not yet clear and it has not been designated as a VOC. However, our findings of emergence and spread of this variant indicate that careful monitoring of this variant, together with assessment of the consequences of the spike protein changes for COVID-19 vaccine performance, are advisable

Burden of sickle cell trait and disease in the Uganda Sickle Surveillance Study (US3): a cross-sectional study

Background Sickle cell disease contributes substantially to mortality in children younger than 5 years in sub-Saharan Africa. In Uganda, 20 000 babies per year are thought to be born with sickle cell disease, but accurate data are not available. We did the cross-sectional Uganda Sickle Surveillance Study to assess the burden of disease. Methods The primary objective of the study was to calculate prevalence of sickle cell trait and disease. We obtained punch samples from dried blood spots routinely collected from HIV-exposed infants in ten regions and 112 districts across Uganda for the national Early Infant Diagnosis programme. Haemoglobin electrophoresis by isoelectric focusing was done on all samples to identify those from babies with sickle trait or disease. Findings Between February, 2014, and March, 2015, 99 243 dried blood spots were analysed and results were available for 97 631. The overall number of children with sickle cell trait was 12 979 (13·3%) and with disease was 716 (0·7%). Sickle cell numbers ranged from 631 (4·6%) for trait and 23 (0·2%) for disease of 13 649 in the South Western region to 1306 (19·8%) for trait and 96 (1·5%) for disease of 6581 in the East Central region. Sickle cell trait was seen in all districts. The lowest prevalence was less than 3·0% in two districts. Eight districts had prevalence greater than 20·0%, with the highest being 23·9%. Sickle cell disease was less common in children older than 12 months or who were HIV positive, which is consistent with comorbidity and early mortality. Interpretation Prevalence of sickle cell trait and disease were high in Uganda, with notable variation between regions and districts. The data will help to inform national strategies for sickle cell disease, including neonatal screening

Impaired T-cell proliferation among HAART-treated adults with suboptimal CD4 recovery in an African cohort.

BACKGROUND: Most HIV-infected subjects exhibit a progressive rise in CD4 T-cell counts after initiation of highly active antiretroviral therapy (HAART). However, a subset of individuals exhibit very poor CD4 T-cell recovery despite effective control of HIV-RNA viraemia. We evaluated CD4 T-cell proliferation among suboptimal responders and its correlation with CD4 T-cell activation. METHODS: The magnitude of CD4 increase (difference between absolute CD4 counts at baseline and absolute CD4 counts at 4 years of ART) was grouped into 4 quartiles for the 211 patients with sustained HIV-RNA viral suppression. Cases of 'Suboptimal immune responders' included patients within the lowest quartile [Median CD4 increase 165 (Range -43-298) cells/μl; n=52] and a comparison group of 'Optimal immune responders' was defined as patients within the highest quartile of CD4 increase [Median CD4 increase 528 (Range 417-878) cells/μl; n=52]. Frozen PBMC were thawed and analysed from a convenient sample of 39 suboptimal responders and 48 optimal responders after 4 years of suppressive antiretroviral therapy. T-cell activation was measured by proportions of T-cells expressing surface marker CD38 and HLADR (CD4+CD38+HLA-DR+ and CD8+CD38+HLA-DR+ cells). T-cell proliferation was determined by the extent of carboxyfluorescein diacetate succinimidyl ester (CFSE) dye dilution on culture day 5 of PBMCs in the presence of antigen (SEB, PPD, CMVpp65, GagA and GagD). Samples were analyzed on a FACS Calibur flow cytometer and flow data was analyzed using FlowJo and GraphPad. RESULTS: Overall, CD4 T-cell proliferation on stimulation with SEB, PPD, CMVpp65, Gag A and Gag D.antigens, was lower among suboptimal than optimal responders; this was significant for SEB (CD4+ p=0.003; CD8+ p=0.048) and PPD antigens (CD8+ p=0.038). Among suboptimal responders, T-cell proliferation decreased with increasing immune activation (Negative correlation; slope = -0.13±-0.11) but not among optimal responders. CONCLUSION: T-cell immune activation and exhaustion were associated with poor proliferation among suboptimal responders to HAART despite sustained viral suppression. We recommend studies to further understand the mechanisms leading to impaired T-cell function among suboptimal responders as well as the potential role of immune modulation in optimizing CD4 count and functional recovery after HAART

Avidity of anti-malarial antibodies inversely related to transmission intensity at three sites in Uganda.

BACKGROUND: People living in malaria endemic areas acquire protection from severe malaria quickly, but protection from clinical disease and control of parasitaemia is acquired only after many years of repeated infections. Antibodies play a central role in protection from clinical disease; however, protective antibodies are slow to develop. This study sought to investigate the influence of Plasmodium falciparum exposure on the acquisition of high-avidity antibodies to P. falciparum antigens, which may be associated with protection. METHODS: Cross-sectional surveys were performed in children and adults at three sites in Uganda with varied P. falciparum transmission intensity (entomological inoculation rates; 3.8, 26.6, and 125 infectious bites per person per year). Sandwich ELISA was used to measure antibody responses to two P. falciparum merozoite surface antigens: merozoite surface protein 1-19 (MSP1-19) and apical membrane antigen 1 (AMA1). In individuals with detectable antibody levels, guanidine hydrochloride (GuHCl) was added to measure the relative avidity of antibody responses by ELISA. RESULTS: Within a site, there were no significant differences in median antibody levels between the three age groups. Between sites, median antibody levels were generally higher in the higher transmission sites, with differences more apparent for AMA-1 and in ≥5 year group. Similarly, median avidity index (proportion of high avidity antibodies) showed no significant increase with increasing age but was significantly lower at sites of higher transmission amongst participants ≥5 years of age. Using 5 M GuHCl, the median avidity indices in the ≥5 year group at the highest and lowest transmission sites were 19.9 and 26.8, respectively (p = 0.0002) for MSP1-19 and 12.2 and 17.2 (p = 0.0007) for AMA1. CONCLUSION: Avidity to two different P. falciparum antigens was lower in areas of high transmission intensity compared to areas with lower transmission. Appreciation of the mechanisms behind these findings as well as their clinical consequences will require additional investigation, ideally utilizing longitudinal data and investigation of a broader array of responses

FCRL5 Delineates Functionally Impaired Memory B Cells Associated with Plasmodium falciparum Exposure.

Exposure to Plasmodium falciparum is associated with circulating "atypical" memory B cells (atMBCs), which appear similar to dysfunctional B cells found in HIV-infected individuals. Functional analysis of atMBCs has been limited, with one report suggesting these cells are not dysfunctional but produce protective antibodies. To better understand the function of malaria-associated atMBCs, we performed global transcriptome analysis of these cells, obtained from individuals living in an area of high malaria endemicity in Uganda. Comparison of gene expression data suggested down-modulation of B cell receptor signaling and apoptosis in atMBCs compared to classical MBCs. Additionally, in contrast to previous reports, we found upregulation of Fc receptor-like 5 (FCRL5), but not FCRL4, on atMBCs. Atypical MBCs were poor spontaneous producers of antibody ex vivo, and higher surface expression of FCRL5 defined a distinct subset of atMBCs compromised in its ability to produce antibody upon stimulation. Moreover, higher levels of P. falciparum exposure were associated with increased frequencies of FCRL5+ atMBCs. Together, our findings suggest that FCLR5+ identifies a functionally distinct, and perhaps dysfunctional, subset of MBCs in individuals exposed to P. falciparum

Optimisation and standardisation of a multiplex immunoassay of diverse Plasmodium falciparum antigens to assess changes in malaria transmission using sero-epidemiology.

Background: Antibody responses have been used to characterise transmission and exposure history in malaria-endemic settings for over a decade. Such studies have typically been conducted on well-standardised enzyme-linked immunosorbent assays (ELISAs). However, recently developed quantitative suspension array technologies (qSAT) are now capable of high-throughput and multiplexed screening of up to hundreds of analytes at a time. This study presents a customised protocol for the Luminex MAGPIX © qSAT using a diverse set of malaria antigens. The aim is to develop a standardised assay for routine serological surveillance that is implementable across laboratories and epidemiological settings. Methods: A panel of eight Plasmodium falciparum recombinant antigens, associated with long- and short-lived antibody responses, was designed for the Luminex MAGPIX © platform. The assay was optimised for key steps in the protocol: antigen-bead coupling concentration, buffer composition, serum sample dilution, and bead storage conditions. Quality control procedures and data normalisation methods were developed to address high-throughput assay processing.  Antigen-specific limits of quantification (LOQs) were also estimated using both in-house and WHO reference serum as positive controls. Results: Antigen-specific bead coupling was optimised across five serum dilutions and two positive controls, resulting in concentrations operational within stable analytical ranges. Coupled beads were stable after storage at room temperature (22?C) for up to eight weeks. High sensitivity and specificity for distinguishing positive and negative controls at serum sample dilutions of 1:500 (AUC 0.94 95%CI 0.91-0.96) and 1:1000 (AUC 0.96 95%CI 0.94-0.98) were observed. LOQs were also successfully estimated for all analytes but varied by antigen and positive control. Conclusions: This study demonstrates that developing a standardised malaria-specific qSAT protocol for a diverse set of antigens is achievable, though further optimisations may be required. Quality control and data standardisation methods may also be useful for future analysis of large sero-epidemiological surveys

Elevated plasma abscisic acid is associated with asymptomatic falciparum malaria and with IgG-/caspase-1-dependent immunity in Plasmodium yoelii-infected mice.

Abscisic acid (ABA) is an ancient stress hormone and is detectable in a wide variety of organisms where it regulates innate immunity and inflammation. Previously, we showed that oral supplementation with ABA decreased parasitemia in a mouse model of malaria, decreased liver and spleen pathology and reduced parasite transmission to mosquitoes. Here, we report that higher circulating ABA levels were associated with a reduced risk of symptomatic malaria in a cohort of Plasmodium falciparum-infected Ugandan children. To understand possible mechanisms of ABA protection in malaria, we returned to our mouse model to show that ABA effects on Plasmodium yoelii 17XNL infection were accompanied by minimal effects on complete blood count and blood chemistry analytes, suggesting a benefit to host health. In addition, orally delivered ABA induced patterns of gene expression in mouse liver and spleen that suggested enhancement of host anti-parasite defenses. To test these inferences, we utilized passive immunization and knockout mice to demonstrate that ABA supplementation increases circulating levels of protective, parasite-specific IgG and requires caspase-1 to reduce parasitemia. Collectively, ABA induces host responses that ameliorate infection and disease in an animal model and suggest that further studies of ABA in the context of human malaria are warranted