Impact of COVID-19 on mortality in coastal Kenya: a longitudinal open cohort study.

The mortality impact of COVID-19 in Africa remains controversial because most countries lack vital registration. We analysed excess mortality in Kilifi Health and Demographic Surveillance System, Kenya, using 9 years of baseline data. SARS-CoV-2 seroprevalence studies suggest most adults here were infected before May 2022. During 5 waves of COVID-19 (April 2020-May 2022) an overall excess mortality of 4.8% (95% PI 1.2%, 9.4%) concealed a significant excess (11.6%, 95% PI 5.9%, 18.9%) among older adults ( ≥ 65 years) and a deficit among children aged 1-14 years (-7.7%, 95% PI -20.9%, 6.9%). The excess mortality rate for January 2020-December 2021, age-standardised to the Kenyan population, was 27.4/100,000 person-years (95% CI 23.2-31.6). In Coastal Kenya, excess mortality during the pandemic was substantially lower than in most high-income countries but the significant excess mortality in older adults emphasizes the value of achieving high vaccine coverage in this risk group.

The impact of malaria-protective red blood cell polymorphisms on parasite biomass in children with severe Plasmodium falciparum malaria.

Severe falciparum malaria is a major cause of preventable child mortality in sub-Saharan Africa. Plasma concentrations of P. falciparum Histidine-Rich Protein 2 (PfHRP2) have diagnostic and prognostic value in severe malaria. We investigate the potential use of plasma PfHRP2 and the sequestration index (the ratio of PfHRP2 to parasite density) as quantitative traits for case-only genetic association studies of severe malaria. Data from 2198 Kenyan children diagnosed with severe malaria, genotyped for 14 major candidate genes, show that polymorphisms in four major red cell genes that lead to hemoglobin S, O blood group, α-thalassemia, and the Dantu blood group, are associated with substantially lower admission plasma PfHRP2 concentrations, consistent with protective effects against extensive parasitized erythrocyte sequestration. In contrast the known protective ATP2B4 polymorphism is associated with higher plasma PfHRP2 concentrations, lower parasite densities and a higher sequestration index. We provide testable hypotheses for the mechanism of protection of ATP2B4.

Randomised controlled trial of oxygen therapy and high-flow nasal therapy in African children with pneumonia.

PURPOSE: The life-saving role of oxygen therapy in African children with severe pneumonia is not yet established. METHODS: The open-label fractional-factorial COAST trial randomised eligible Ugandan and Kenyan children aged > 28 days with severe pneumonia and severe hypoxaemia stratum (SpO2 < 80%) to high-flow nasal therapy (HFNT) or low-flow oxygen (LFO: standard care) and hypoxaemia stratum (SpO2 80-91%) to HFNT or LFO (liberal strategies) or permissive hypoxaemia (ratio 1:1:2). Children with cyanotic heart disease, chronic lung disease or > 3 h receipt of oxygen were excluded. The primary endpoint was 48 h mortality; secondary endpoints included mortality or neurocognitive sequelae at 28 days. RESULTS: The trial was stopped early after enrolling 1852/4200 children, including 388 in the severe hypoxaemia stratum (median 7 months; median SpO2 75%) randomised to HFNT (n = 194) or LFO (n = 194) and 1454 in the hypoxaemia stratum (median 9 months; median SpO2 88%) randomised to HFNT (n = 363) vs LFO (n = 364) vs permissive hypoxaemia (n = 727). Per-protocol 15% of patients in the permissive hypoxaemia group received oxygen (when SpO2 < 80%). In the severe hypoxaemia stratum, 48-h mortality was 9.3% for HFNT vs. 13.4% for LFO groups. In the hypoxaemia stratum, 48-h mortality was 1.1% for HFNT vs. 2.5% LFO and 1.4% for permissive hypoxaemia. In the hypoxaemia stratum, adjusted odds ratio for 48-h mortality in liberal vs permissive comparison was 1.16 (0.49-2.74; p = 0.73); HFNT vs LFO comparison was 0.60 (0.33-1.06; p = 0.08). Strata-specific 28 day mortality rates were, respectively: 18.6, 23.4 and 3.3, 4.1, 3.9%. Neurocognitive sequelae were rare. CONCLUSIONS: Respiratory support with HFNT showing potential benefit should prompt further trials.

IgM in human immunity to Plasmodium falciparum malaria.

Most studies on human immunity to malaria have focused on the roles of immunoglobulin G (IgG), whereas the roles of IgM remain undefined. Analyzing multiple human cohorts to assess the dynamics of malaria-specific IgM during experimentally induced and naturally acquired malaria, we identified IgM activity against blood-stage parasites. We found that merozoite-specific IgM appears rapidly in Plasmodium falciparum infection and is prominent during malaria in children and adults with lifetime exposure, together with IgG. Unexpectedly, IgM persisted for extended periods of time; we found no difference in decay of merozoite-specific IgM over time compared to that of IgG. IgM blocked merozoite invasion of red blood cells in a complement-dependent manner. IgM was also associated with significantly reduced risk of clinical malaria in a longitudinal cohort of children. These findings suggest that merozoite-specific IgM is an important functional and long-lived antibody response targeting blood-stage malaria parasites that contributes to malaria immunity.

No Evidence that Knops Blood Group Polymorphisms Affect Complement Receptor 1 Clustering on Erythrocytes.

Clustering of Complement Receptor 1 (CR1) in the erythrocyte membrane is important for immune-complex transfer and clearance. CR1 contains the Knops blood group antigens, including the antithetical pairs Swain-Langley 1 and 2 (Sl1 and Sl2) and McCoy a and b (McCa and McCb), whose functional effects are unknown. We tested the hypothesis that the Sl and McC polymorphisms might influence CR1 clustering on erythrocyte membranes. Blood samples from 125 healthy Kenyan children were analysed by immunofluorescence and confocal microscopy to determine CR1 cluster number and volume. In agreement with previous reports, CR1 cluster number and volume were positively associated with CR1 copy number (mean number of CR1 molecules per erythrocyte). Individuals with the McC b /McC b genotype had more clusters per cell than McC a /McC a individuals. However, this association was lost when the strong effect of CR1 copy number was included in the model. No association was observed between Sl genotype, sickle cell genotype, α+thalassaemia genotype, gender or age and CR1 cluster number or volume. Therefore, after correction for CR1 copy number, the Sl and McCoy polymorphisms did not influence erythrocyte CR1 clustering, and the effects of the Knops polymorphisms on CR1 function remains unknown.

Red blood cell complement receptor one level varies with Knops blood group, α(+)thalassaemia and age among Kenyan children.

Both the invasion of red blood cells (RBCs) by Plasmodium falciparum parasites and the sequestration of parasite-infected RBCs in the microvasculature are mediated in part by complement receptor one (CR1). RBC surface CR1 level can vary between individuals by more than 20-fold and may be associated with the risk of severe malaria. The factors that influence RBC CR1 level variation are poorly understood, particularly in African populations. We studied 3535 child residents of a malaria-endemic region of coastal Kenya and report, for the first time, that the CR1 Knops blood group alleles Sl2 and McC(b), and homozygous HbSS are positively associated with RBC CR1 level. Sickle cell trait and ABO blood group did not influence RBC CR1 level. We also confirm the previous observation that α(+)thalassaemia is associated with reduced RBC CR1 level, possibly due to small RBC volume, and that age-related changes in RBC CR1 expression occur throughout childhood. RBC CR1 level in malaria-endemic African populations is a complex phenotype influenced by multiple factors that should be taken into account in the design and interpretation of future studies on CR1 and malaria susceptibility.

The population genetics and dynamics of the thalassemias.

The inherited disorders of hemoglobin, including the thalassemias, are by far the commonest monogenic diseases. Although several factors are responsible for their very high frequency, the major mechanism seems to be natural selection mediated by heterozygote protection against severe forms of malaria. Recent work has highlighted the complexity of the interplay among the different hemoglobin variants themselves and among different levels of malaria resistance, and is helping to explain the extraordinary heterogeneity in the distribution of the hemoglobin disorders even within short geographical distances. Some progress has also been made toward understanding the cellular and immune mechanisms that may underlie heterozygote protection against malaria in these conditions. In addition to providing valuable information about human evolutionary biology, work in this field has an increasingly important influence on the development of programs for the better management of the hemoglobin disorders, particularly in the poorer countries of the tropical world.

NFKBIZ polymorphisms and susceptibility to pneumococcal disease in European and African populations.

The proinflammatory transcription factor nuclear factor-kappaB (NF-kappaB) has a central role in host defence against pneumococcal disease. Both rare mutations and common polymorphisms in the NFKBIA gene encoding the NF-kappaB inhibitor, IkappaB-alpha, associate with susceptibility to bacterial disease, but the possible role of polymorphisms within the related IkappaB-zeta gene NFKBIZ in the development of invasive pneumococcal disease (IPD) has not been reported previously. To investigate this further, we examined the frequencies of 22 single-nucleotide polymorphisms spanning NFKBIZ in two case-control studies, comprising UK Caucasian (n=1008) and Kenyan (n=723) individuals. Nine polymorphisms within a single UK linkage disequilibrium (LD) block and all four polymorphisms within the equivalent, shorter Kenyan LD block displayed either a significant association with IPD or a trend towards association. For each polymorphism, heterozygosity was associated with protection from IPD when compared with the combined homozygous states (for example, for rs600718, Mantel-Haenszel 2 x 2 chi(2)=7.576, P=0.006, odds ratio (OR)=0.67, 95% confidence interval (95% CI) for OR: 0.51-0.88; for rs616597, Mantel-Haenszel 2 x 2 chi(2)=8.715, P=0.003, OR=0.65, 95% CI: 0.49-0.86). We conclude that multiple NFKBIZ polymorphisms associate with susceptibility to IPD in humans. The study of multiple populations may aid in fine mapping of associations within extensive regions of strong LD ('transethnic mapping').

Variation in the ICAM1 gene is not associated with severe malaria phenotypes.

Evidence from autopsy and in vitro binding studies suggests that adhesion of erythrocytes infected with Plasmodium falciparum to the human host intercellular adhesion molecule (ICAM)-1 receptor is important in the pathogenesis of severe malaria. Previous association studies between polymorphisms in the ICAM1 gene and susceptibility to severe malarial phenotypes have been inconclusive and often contradictory. We performed genetic association studies with 15 single nucleotide polymorphisms (SNPs) around the ICAM1 locus. All SNPs were screened in a family study of 1071 trios from The Gambia, Malawi and Kenya. Two key non-synonymous SNPs with previously reported associations, rs5491 (K56M or 'ICAM-1(Kilifi)') and rs5498 (K469E), were tested in an additional 708 Gambian trios and a case-control study of 4058 individuals. None of the polymorphisms were associated with severe malaria phenotypes. Pooled results across our studies for ICAM-1(Kilifi) were, in severe malaria, odds ratio (OR) 1.02, 95% confidence interval (CI) 0.96-1.09, P=0.54, and cerebral malaria OR 1.07, CI 0.97-1.17, P=0.17. We assess the available epidemiological, population genetic and functional evidence that links ICAM-1(Kilifi) to severe malaria susceptibility.

Failure to respond to the surface of Plasmodium falciparum infected erythrocytes predicts susceptibility to clinical malaria amongst African children.

Following infection with Plasmodium falciparum malaria, children in endemic areas develop antibodies specific to antigens on the parasite-infected red cell surface of the infecting isolate, antibodies associated with protection against subsequent infection with that isolate. In some circumstances induction of antibodies to heterologous parasite isolates also occurs and this has been suggested as evidence for cross-reactivity of responses against the erythrocyte surface. The role of these relatively cross-reactive antibodies in protection from clinical malaria is currently unknown. We studied the incidence of clinical malaria amongst children living on the coast of Kenya through one high transmission season. By categorising individuals according to their pre-season parasite status and antibody response to the surface of erythrocytes infected with four parasite isolates we were able to identify a group of children, those who failed to make a concomitant antibody response in the presence of an asymptomatic parasitaemia, at increased susceptibility to clinical malaria in the subsequent 6 months. The fact that this susceptible group was identified regardless of the parasite isolate tested infers a cross-reactive or conserved target is present on the surface of infected erythrocytes. Identification of this target will significantly aid understanding of naturally acquired immunity to clinical malaria amongst children in endemic areas.

Positive replication and linkage disequilibrium mapping of the chromosome 21q22.1 malaria susceptibility locus.

Four cytokine receptor genes are located on Chr21q22.11, encoding the alpha and beta subunits of the interferon-alpha receptor (IFNAR1 and IFNAR2), the beta subunit of the interleukin 10 receptor (IL10RB) and the second subunit of the interferon-gamma receptor (IFNGR2). We previously reported that two variants in IFNAR1 were associated with susceptibility to malaria in Gambians. We now present an extensive fine-scale mapping of the associated region utilizing 45 additional genetic markers obtained from public databases and by sequencing a 44 kb region in and around the IFNAR1 gene in 24 Gambian children (12 cases/12 controls). Within the IFNAR1 gene, a newly studied C --> G single-nucleotide polymorphism (IFNAR1 272354c-g) at position -576 relative to the transcription start was found to be more strongly associated with susceptibility to severe malaria. Association was observed in three populations: in Gambian (P=0.002), Kenyan (P=0.022) and Vietnamese (P=0.005) case-control studies. When all three studies were combined, using the Mantel-Haenszel test, the presence of IFNAR1 -576G was associated with a substantially elevated risk of severe malaria (N=2444, OR=1.38, 95% CI: 1.17-1.64; P=1.7 x 10(-4)). This study builds on previous work to further highlight the importance of the type-I interferon pathway in malaria susceptibility and illustrates the utility of typing SNPs within regions of high linkage disequilibrium in multiple populations to confirm initial positive associations.

Sickle cell disease in Africa: burden and research priorities.

Sickle cell disease (SCD) has recently been recognised as a problem of major public-health significance by the World Health Organization. Despite the fact that >70% of sufferers live in Africa, expenditure on the related care and research in the continent is negligible, and most advances in the understanding and management of this condition have been based on research conducted in the North. In order to target limited resources, African countries need to focus research and interventions on areas that will lead to the maximum impact. This review details the epidemiological and clinical background of SCD, with an emphasis on Africa, before identifying the research priorities that will provide the necessary evidence base for improving the management of African patients. Malaria, bacterial and viral infections and cerebrovascular accidents are areas in which further research may lead to a significant improvement in SCD-related morbidity and mortality. As patients with high concentrations of foetal haemoglobin (HbF) appear to be protected from all but mild SCD, the various factors and pharmacological agents that might increase HbF levels need to be assessed in Africa, as options for interventions that would improve quality of life and reduce mortality.

Cytokine mRNA expression and iron status in children living in a malaria endemic area.

Iron deficiency has been reported to affect both malaria pathogenesis and cell-mediated immune responses; however, it is unclear whether the protection afforded by iron deficiency is mediated through direct effects on the parasite, through immune effector functions or through both. We have determined cytokine mRNA expression levels in 59 children living in a malaria endemic area on the coast of Kenya who we selected on the basis of their biochemical iron status. Real-time quantitative reverse transcriptase polymerase chain reaction analysis of cytokine mRNA levels of peripheral blood mononuclear cells (PBMC) obtained from these children showed an association between interleukin-4 (IL-4) mRNA levels and all the biochemical indices of iron that we measured. Furthermore, IL-10 mRNA was higher in parasite blood smear-positive children than in blood smear-negative children irrespective of their iron status. This study suggests that IL-4 expression by PBMC may be affected by iron status.

HLA class-I and class-II allele frequencies and two-locus haplotypes in Melanesians of Vanuatu and New Caledonia.

HLA class-I and class-II allele frequencies and two-locus haplotypes were examined in 367 unrelated Melanesians living on the islands of Vanuatu and New Caledonia. Diversity at all HLA class-I and class-II loci was relatively limited. In class-I loci, three HLA-A allelic groups (HLA-A*24, HLA-A*34 and HLA-A*11), seven HLA-B alleles or allelic groups (HLA-B*1506, HLA-B*5602, HLA-B*13, HLA-B*5601, HLA-B*4001, HLA-B*4002 and HLA-B*2704) and four HLA-C alleles or allelic groups (HLA-Cw*04, HLA-Cw*01, HLA-Cw*0702 and HLA-Cw*15) constituted more than 90% of the alleles observed. In the class-II loci, four HLA-DRB1 alleles (HLA-DRB1*15, HLA-DRB1*11, HLA-DRB1*04 and HLA-DRB1*16), three HLA-DRB3-5 alleles (HLA-DRB3*02, HLA-DRB4*01 and HLA-DRB5*01/02) and five HLA-DQB1 alleles (HLA-DQB1*0301, HLA-DQB1*04, HLA-DQB1*05, HLA-DQB1*0601 and HLA-DQB1*0602) constituted over 93, 97 and 98% of the alleles observed, respectively. Homozygosity showed significant departures from expected levels for neutrality based on allele frequency (i.e. excess diversity) at the HLA-B, HLA-Cw, HLA-DQB1 and HLA-DRB3/5 loci on some islands. The locus with the strongest departure from neutrality was HLA-DQB1, homozygosity being significantly lower than expected on all islands except New Caledonia. No consistent pattern was demonstrated for any HLA locus in relation to malaria endemicity.

Reevaluation of flow cytometry for investigating antibody binding to the surface of Plasmodium falciparum trophozoite-infected red blood cells.

BACKGROUND: The acquisition of antibodies directed toward variant surface antigens (VSAs) expressed on the surface of the trophozoite-infected red blood cell is an important determinant of natural immunity to Plasmodium falciparum malaria. In recent years, flow cytometry has been used increasingly to investigate these responses, but few systematic assessments of this method are available in the published literature. METHODS: We developed a highly standardized experimental protocol and used parasites of the A4 laboratory clone, a monoclonal antibody to the VSA expressed by this clone (monoclonal antibody BC6), and a single pool of hyperimmune plasma to explore the parameters responsible for variations in VSA antibody responses measured by flow cytometry. RESULTS: Despite strenuous efforts to standardize our flow cytometric assay, we found marked variability in our assay readout, even between repeat experiments using identical antibody and parasite combinations. We found no remediable cause for much of this variability. However, we identified three major factors that we considered important contributors: antibody concentration, nonspecific antibody binding to uninfected red blood cells, and parasite agglutination. CONCLUSIONS: A number of potential pitfalls should be considered when designing and interpreting studies using this technique. In particular, we suggest that comparisons between assays conducted on different occasions can be made only through reference to carefully selected standards. We anticipate that a better appreciation of the factors that lead to assay variation will assist the design of improved experimental protocols.

Genetic restriction of Plasmodium falciparum in an area of stable transmission: an example of island evolution?

To date, a high degree of polymorphism has been demonstrated at both the MSP1 and MSP2 loci in parasites from areas of stable malaria transmission. As a consequence, in such areas it is rare to find parasites of the same 2-locus genotype in more than 1 subject. We have studied MSP1 and MSP2 diversity in parasites collected from subjects with both symptomatic (n = 86) and asymptomatic (34) malaria living on the island of Santo, Vanuatu, an area of stable malaria transmission. Polymorphism at the MSP1 and MSP2 loci was considerably less than previously reported: only 5 MSP1 and 5 MSP2 alleles were detected and these showed no size variation within alleles. Santo is unique amongst the areas studied so far in that it is a small island at the limit of the malaria belt in the South Pacific. Thus, the evolution of the parasite population may have been affected by the small size and isolation of this island population. Moreover, limited parasite diversity may explain the unusually mild nature of Plasmodium falciparum disease on Santo. Islands have fascinated biologists for centuries and fuelled the advancement of evolutionary theory, since they are natural laboratories for the study of evolution. The simplicity of the Vanuatu P. falciparum population may facilitate the use and interpretation of sequence level analyses to address the mechanisms by which genetic diversity is generated and maintained in natural populations.

Reduced soluble transferrin receptor concentrations in acute malaria in Vanuatu.

Soluble transferrin receptor (sTfR) concentration is a sensitive index of iron deficiency when used in conjunction with ferritin measurements in adults. One advantage of this assay is that unlike ferritin it does not appear to be affected by a range of infectious and inflammatory conditions or by pregnancy, rendering it a promising adjunct to the diagnosis of iron deficiency in tropical populations. We have measured plasma sTfR concentrations in a group of malaria patients (n = 21) and asymptomatic (18) and aparasitemic (76) controls in Vanuatu. Plasma sTfR concentration was significantly reduced in individuals with acute malaria (P = 0.003). While this observation provides evidence that erythropoeitic suppression may be an important etiologic component in malarial anemia, it also suggests that malaria may be a confounding factor when interpreting sTfR concentrations in such populations. The role of sTfR in the diagnosis of iron deficiency in tropical populations remains to be established.

Alpha thalassaemia is associated with increased soluble transferrin receptor levels.

Although alpha+ thalassaemia is the commonest haemoglobinopathy in the world, it is not known if it is associated with significant ineffective erythropoiesis, a fact of importance in interpreting its complex interaction with malaria. To study this problem, we have measured the concentrations of soluble transferrin receptor (sTIR) and ferritin in 181 children from Vanuatu with heterozygous (68) and homozygous (46) alpha+ thalassaemia, and normal controls (67). sTfR concentrations were significantly higher in both homozygotes (mean 3.1 mg/l, range 2.8-3.4) and heterozygotes (2.86 mg/l, 2.6-3.2) compared to the normal controls (2.48 mg/l, 2.3-2.7), suggesting that although globin chain imbalance is minimal, there is ineffective erythropoiesis in both these conditions. Age was also shown to significantly affect sTfR, with peak levels occurring in the 5-9 years age group. Ferritin concentrations showed a similar trend, being higher in the thalassaemic groups, although this did not reach statistical significance. No individuals had low ferritin concentrations, although two had significantly elevated sTfR levels. These observations suggest that the alpha+ thalassaemia phenotype includes an expansion of the erythron, and may suggest possible mechanisms for the increased susceptibility in babies with alpha thalassaemia to both P. falciparum and P. vivax malaria.