STRIDE: a command-line HMM-based identifier and sub-classifier of Plasmodium falciparum RIFIN and STEVOR variant surface antigen families

Background RIFINs and STEVORs are variant surface antigens expressed by P. falciparum that play roles in severe malaria pathogenesis and immune evasion. These two highly diverse multigene families feature multiple paralogs, making their classification challenging using traditional bioinformatic methods. Results STRIDE (STevor and RIfin iDEntifier) is an HMM-based, command-line program that automates the identification and classification of RIFIN and STEVOR protein sequences in the malaria parasite Plasmodium falciparum. STRIDE is more sensitive in detecting RIFINs and STEVORs than available PFAM and TIGRFAM tools and reports RIFIN subtypes and the number of sequences with a FHEYDER amino acid motif, which has been associated with severe malaria pathogenesis. Conclusions STRIDE will be beneficial to malaria research groups analyzing genome sequences and transcripts of clinical field isolates, providing insight into parasite biology and virulence.

Cross-sectional study to predict subnational levels of health workers’ knowledge about severe malaria treatment in Kenya

ObjectivesThis study applied a Bayesian hierarchical ecological spatial model beyond predictor analysis to test for the best fitting spatial effects model to predict subnational levels of health workers’ knowledge of severe malaria treatment policy, artesunate dosing, and preparation.SettingCounty referral government and major faith-based hospitals across 47 counties in Kenya in 2019.Design and participantsA secondary analysis of cross-sectional survey data from 345 health workers across 89 hospitals with inpatient departments who were randomly selected and interviewed.Outcome measuresThree ordinal outcome variables for severe malaria treatment policy, artesunate dose and preparation were considered, while 12 individual and contextual predictors were included in the spatial models.ResultsA third of the health workers had high knowledge levels on artesunate treatment policy; almost three-quarters had high knowledge levels on artesunate dosing and preparation. The likelihood of having high knowledge on severe malaria treatment policy was lower among nurses relative to clinicians (adjusted OR (aOR)=0.48, 95% CI 0.25 to 0.87), health workers older than 30 years were 61% less likely to have high knowledge about dosing compared with younger health workers (aOR=0.39, 95% CI 0.22 to 0.67), while health workers exposed to artesunate posters had 2.4-fold higher odds of higher knowledge about dosing compared with non-exposed health workers (aOR=2.38, 95% CI 1.22 to 4.74). The best model fitted with spatially structured random effects and spatial variations of the knowledge level across the 47 counties exhibited neighbourhood influence.ConclusionsKnowledge of severe malaria treatment policies is not adequately and optimally available among health workers across Kenya. The factors associated with the health workers’ level of knowledge were cadre, age and exposure to artesunate posters. The spatial maps provided subnational estimates of knowledge levels for focused interventions.

Starting at the community: Treatment seeking pathways of children with suspected severe malaria in Uganda

IntroductionCommunity health workers (CHW) usually refer children with suspected severe malaria to the nearest public health facility or a designated public referral health facility (RHF). Caregivers do not always follow this recommendation. This study aimed at identifying post-referral treatment-seeking pathways that lead to appropriate antimalarial treatment for children less than five years with suspected severe malaria.MethodsAn observational study in Uganda enrolled children below five years presenting to CHWs with signs of severe malaria. Children were followed up 28 days after enrolment to assess their condition and treatment-seeking history, including referral advice and provision of antimalarial treatment from visited providers.ResultsOf 2211 children included in the analysis, 96% visited a second provider after attending a CHW. The majority of CHWs recommended caregivers to take their child to a designated RHF (65%); however, only 59% followed this recommendation. Many children were brought to a private clinic (33%), even though CHWs rarely recommended this type of provider (3%). Children who were brought to a private clinic were more likely to receive an injection than children brought to a RHF (78% vs 51%, p<0.001) and more likely to receive the second or third-line injectable antimalarial (artemether: 22% vs. 2%, p<0.001, quinine: 12% vs. 3%, p<0.001). Children who only went to non-RHF providers were less likely to receive an artemisinin-based combination therapy (ACT) than children who attended a RHF (odds ratio [OR] = 0.64, 95% CI 0.51–0.79, p<0.001). Children who did not go to any provider after seeing a CHW were the least likely to receive an ACT (OR = 0.21, 95% CI 0.14–0.34, p<0.001).ConclusionsHealth policies should recognise local treatment-seeking practices and ensure adequate quality of care at the various public and private sector providers where caregivers of children with suspected severe malaria actually seek care.

Ameliorative Effects of Dietary Ellagic Acid Against Severe Malaria Pathogenesis by Reducing Cytokine Storms and Oxidative Stress

Ellagic acid (EA), a fruit- and vegetable-derived flavonoid, has been reported for multiple pharmacological activities, which encouraged us to examine its useful effect in severe malaria pathogenesis, especially malaria-induced cytokine storms and oxidative stress linked to damage in major organs. Malaria was induced by injecting Plasmodium berghei–infected RBCs intraperitoneally into the mice. EA was given orally (5, 10, and 20 mg/kg) following Peter’s 4-day suppression test. EA exhibited the suppression of parasitemia, production of inflammatory cytokine storms and oxidative stress marker level quantified from vital organs significantly and an increase in hemoglobin, blood glucose, and mean survival time compared to the vehicle-treated infected group. EA administration also restored the blood–brain barrier integrity evidenced through Evans blue staining. Furthermore, we demonstrated the protecting effect of EA in LPS-induced inflammatory cytokine storms and oxidative stress in glial cells. The present study conclude that ellagic acid is able to alleviate severe malaria pathogenesis by reducing cytokine storms and oxidative stress–induced by malarial parasites. It also attributed promising antimalarial activity and afforded to improve the blood glucose and hemoglobin levels in treated mice. These research findings suggested the suitability of ellagic acid as a useful bioflavonoid for further study for the management of severe malaria pathogenesis.

The prognostic and diagnostic value of intraleukocytic malaria pigment: an individual patient data pooled meta-analysis of 32,000 patients with severe falciparum malaria in Africa and Asia

BackgroundSevere falciparum malaria is a major cause of death in tropical countries, particularly in African children. Accurate diagnosis and prognostic assessment are critical to clinical management.MethodsThe prognostic value of the malaria parasite count, and the proportions of polymorphonuclear leukocytes (PMNs) and monocytes (PMMs) containing malaria pigment in peripheral blood films were assessed in three randomized controlled trials conducted in severe malaria patients; two in Southeast Asia (AQ Vietnam; n=483 and SEAQUAMAT; n=1,330) and one in Africa (AQUAMAT; n=4,211). Following a systematic review of the literature, we incorporated these data into an individual patient data meta-analysis including published data from the Severe Malaria in African children (SMAC) network (n=25,845) and a study from Mali (n=166).FindingsThe proportion of pigment containing PMNs on peripheral blood films was strongly positively correlated with prognosis (odds-ratio for in-hospital mortality for a tenfold increase: 2.53 [95% CI: 2.13-3.00], p = 10−26). The meta-analytic odds-ratio estimate for in-hospital death in patients with >5% pigment containing PMNs compared with lower values was 2.67 (95% CI: 2.08-3.42; p = 10−14). Particularly in African children, the proportion of pigment containing PMNs added substantially to the prognostic assessment from simple bedside examination, and also to the conventional parasite count. In all analyses, the proportion of pigment containing monocytes had a lower prognostic value.InterpretationMicroscopy assessment of the proportion of pigment containing PMNs in a blood film is simple and rapid, and should be performed in all patients hospitalised with suspected severe malaria. Patients with >5% pigment containing PMNs have more than double the risk of death.OtherFunded by Wellcome. The systematic review was registered prospectively on PROS-PERO, number CRD42021284527Research in contextEvidence before this studySevere falciparum malaria remains a major cause of preventable childhood mortality in sub-Saharan Africa. In 2019 there were an estimated 274,000 deaths in children under 5 years. Rapidly identifying patients at the greatest risk of death and providing effective treatment is essential to saving lives. Based on data from our prospective studies of strictly defined severe falciparum malaria in Vietnamese adults, the proportions of peripheral blood neutrophils and monocytes containing malaria pigment (haemozoin) was proposed as a prognostic factor for mortality. We carried out a systematic review on PubMed of all articles published between database inception and October 11, 2021, using search terms “intraleukocytic pigment” and “severe malaria”. In addition to papers published by our research group, we found two other studies that reported the prognostic value of intraleukocytic pigment counts in severe malaria cohorts of at least 100 patients: the SMAC network study, the largest published cohort study conducted in over 25,000 African children with suspected severe malaria, and a cohort of 172 children from Mali. The SMAC study reported that intraleukocytic malaria pigment counts were not a useful predictor of outcome in African children diagnosed with severe malaria. This differed from the results from the Malian study and our original study in Vietnamese adults.Added value of this studyWe provide new data on the prognostic value of intraleukocytic malaria pigment counts in over 6,000 adults and children with a strict diagnosis of severe falciparum malaria studied prospectively in Asia and Africa. These patients were enrolled in three of the largest randomised controlled trials in severe malaria. These randomised trials have provided the main evidence base for current global therapeutic recommendations. Our data show that there is substantial prognostic value in counting intraleukocytic malaria pigment. This was significantly greater for neutrophil rather than monocyte associated pigment. Pooling all the individual patient data showed that the prognostic value was consistent across studies and countries, despite the substantial differences in study populations and study designs. Having more than 5% pigment containing neutrophils was associated with over double the risk of death from severe falciparum malaria.Implications of all the available evidenceIntraleukocytic malaria pigment counts have sub-stantial prognostic value in severe falciparum malaria. The proportion of neutrophils containing malaria pigment should be counted in thin blood films in all patients with suspected severe malaria. Patients with over 5% of pigment containing neutrophils have a high risk of death.

Identifying targets of protective antibodies against severe malaria in Papua, Indonesia using locally expressed domains of Plasmodium falciparum Erythrocyte Membrane Protein 1.

Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1), a diverse family of multi-domain proteins expressed on the surface of malaria-infected erythrocytes, is an important target of protective immunity against malaria. Our group recently studied transcription of the var genes encoding PfEMP1 in individuals from Papua, Indonesia with severe or uncomplicated malaria. We cloned and expressed domains from 32 PfEMP1s including 22 that were upregulated in severe malaria and 10 that were upregulated in uncomplicated malaria, using a wheat germ cell-free expression system. We used Luminex technology to measure IgG antibodies to these 32 domains and control proteins in 63 individuals (11 children). At presentation to hospital, levels of antibodies to PfEMP1 domains were either higher in uncomplicated malaria or were not significantly different between groups. Using principal components analysis, antibodies to three of 32 domains were highly discriminatory between groups. These included two domains upregulated in severe malaria, a DBLβ13 domain and a CIDRα1.6 domain (which has been previously implicated in severe malaria pathogenesis), and a DBLδ domain that was upregulated in uncomplicated malaria. Antibody to control non-PfEMP1 antigens did not differ with disease severity. Antibodies to PfEMP1 domains differ with malaria severity. Lack of antibodies to locally expressed PfEMP1 types, including both domains previously associated with severe malaria and newly identified targets, may in part explain malaria severity in Papuan adults. Importance Severe Plasmodium falciparum malaria kills many African children, and lack of antibody immunity predisposes to severe disease. A critical antibody target is the P. falciparum erythrocyte membrane 1 (PfEMP1) family of multidomain proteins, which are expressed on the infected erythrocyte surface and mediate parasite sequestration in deep organs. We previously identified var genes encoding PfEMP1 that were differentially expressed between severe and uncomplicated malaria in Papua, Indonesia. Here, we have expressed domains from 32 of these PfEMP1s and measured IgG antibody responses to them in Papuan adults and children. Using Principal Component Analysis, IgG antibodies to three domains distinguished between severe and uncomplicated malaria and were higher in uncomplicated malaria. Domains included CIDRα1.6, implicated in severe malaria; a DBLβ13 domain; and a DBLδ domain of unknown function. Immunity to locally relevant PfEMP1 domains may protect from severe malaria. Targets of immunity show important overlap between Asian adults and African children.

The Impact of Covid-19 on Malaria Services in Three High Endemic Districts in Rwanda – A Mixed-Method Study

Background: Rwanda has achieved impressive reductions in malaria morbidity and mortality over the past two decades. However, the disruption of essential services due to the current Covid-19 pandemic can lead to a reversal of these gains in malaria control unless targeted, evidence-based interventions are implemented to mitigate the impact of the pandemic. The extent to which malaria services have been disrupted has not been fully characterized. In this study, we thus assessed the impact of Covid-19 on malaria services in Rwanda. Methods: We conducted a mixed-methods study in three purposively selected districts in Rwanda. The quantitative data included malaria aggregated data reported at the health facility level and the community level. The data included the number of malaria tests, uncomplicated malaria cases, severe malaria cases, and malaria deaths. We collected qualitative data using focus group discussions with community members and community health workers, as well as in-depth interviews with health care providers and staff working in the malaria program. We conducted interrupted time series analysis to compare changes in malaria presentations between the pre-Covid-19 period (January 2019 to February 2020) and Covid-19 periods (from March 2020 to November 2020). We used the constant comparative method in qualitative thematic analysis. Results: Compared to pre-Covid-19 period, there was a monthly reduction in patients tested in health facilities of 5.09 per 1000 population and a monthly increase in patients tested in the community of 3.13 per 1000 population during the Covid-19 period. There was no change in presentation rate for uncomplicated malaria or severe malaria. Additionally, although healthcare providers continued to provide malaria services, they were fearful that this would expose them and their families to Covid-19. Covid-19 mitigation measures limited the availability of transportation options for the community to seek care in health facilities and delayed the implementation of some key malaria interventions. The focus on Covid-19-related communication also reduced the amount of health information for other diseases provided to community members. Conclusion The Covid-19 pandemic resulted in patients increasingly seeking care in the community and poses challenges to maintaining delivery of malaria services in Rwanda. Interventions to mitigate these challenges should focus on strengthening programming for community and home-based care models and integrating malaria messages into Covid-19-related communication. Additionally, implementation of the interrupted interventions should be timed and overlap with the malaria transmission season to mitigate Covid-19 consequences on malaria.

Treatment-seeking for children with suspected severe malaria attending community health workers and primary health centres in Adamawa State, Nigeria

BackgroundThe Community Access to Rectal Artesunate for Malaria project investigated the feasibility of introducing pre-referral rectal artesunate into existing community-based health services. In that study, the case fatality rate of children visiting primary health centres (PHCs) was 19% compared to 6% in children first visiting community health workers, locally called Community Oriented Resource Persons (CORPs). As case management practices did not fully explain this finding, this publication investigates other reasons underlying the observed difference in case fatality.MethodsThe observational study enrolled 589 children under the age of five years with fever and danger signs indicative of severe malaria attending CORPs and PHCs in Adamawa State, Nigeria, between June 2018 and July 2020. After 28 days, follow-up visits were conducted with caregivers to understand background characteristics, severity of symptoms, home treatment administration, and treatment seeking practices during the child’s illness. These factors were compared between children visiting CORPs versus those visiting PHCs as their first health provider.ResultsChildren visiting PHCs were more likely to display danger signs indicative of central nervous system involvement (90% vs. 74%, p < 0.01) and have four or more danger signs (50% vs. 39%, p = 0.02). The delay between illness onset and visiting the community-based provider did not differ between children attending a CORP and children attending a PHC. PHC attendances more often lived in urban areas (16% vs 4%, p=0.01) and travelled farther to their first health provider, which was usually a community-based provider. Although practicing home treatment was common, especially among children attending PHCs (42% vs 33%, p=0.04), almost none of the children were given an antimalarial. PHCs were visited for their professionalism and experience while CORPs were visited for their low cost and because caregivers personally knew and trusted the provider.ConclusionsOur comparison of children with suspected severe malaria seeking care from two kinds of community-based health care providers in Nigeria suggest that illness severity may be the primary driver behind the observed difference in case fatality rate.

Identification of ATP2B4 regulatory element containing functional genetic variants associated with severe malaria

Genome-wide association studies (GWAS) for severe malaria have identified 30 genetic variants that are mostly located in non-coding regions, with only a few associations replicated in independent populations. In this study, we aimed at identifying potential causal genetic variants located in these loci and demonstrate their functional activity. We systematically investigated the regulatory effect of the SNPs in linkage disequilibrium with the tagSNPs associated with severe malaria in several populations. Annotating and prioritizing genetic variants led to the identification of a regulatory region containing 5 ATP2B4 SNPs in linkage disequilibrium with the tagSNP rs10900585. We confirmed the association of rs10900585 and also found significant associations of severe malaria with our candidate SNPs (rs11240734, rs1541252, rs1541253, rs1541254, and rs1541255) in a Senegalese population. Then, we showed that this region had both promoter and enhancer activities and that both individual SNPs and the combination of SNPs had regulatory effects using luciferase reporter assays. Moreover, CRISPR/Cas9-mediated deletion of this region decreased ATP2B4 transcript and protein levels and increased Ca2+ intracellular concentration in the K562 cell line. Taken together, our data show that severe malaria-associated genetic variants alter the activity of a promoter with enhancer function. We showed that this regulatory element controls the expression of ATP2B4 that encodes a plasma membrane calcium-transporting ATPase 4 (PMCA4), which is the major calcium pump on red blood cells. Altering the activity of this regulatory element affects the risk of severe malaria probably through calcium concentration effect on parasitaemia.