Seroprevalence of SARS-CoV-2 antibodies among children and adolescents recruited in a malariometric survey in north-eastern Tanzania July 2021.

BACKGROUND: African countries stand out globally as the region seemingly least affected by the COVID-19 pandemic, caused by the virus SARS-CoV-2. Besides a younger population and potential pre-existing immunity to a SARS-CoV-2-like virus, it has been hypothesized that co-infection or recent history of Plasmodium falciparum malaria may be protective of COVID-19 severity and mortality. The number of COVID-19 cases and deaths, however, may be vastly undercounted. Very little is known about the extent to which the Tanzanian population has been exposed to SARS-CoV-2. Here, we investigated the seroprevalence of IgG to SARS-CoV-2 spike protein in two Tanzanian rural communities 1½ years into the pandemic and the association of coinciding malaria infection and exposure. METHODS: During a malariometric survey in July 2021 in two villages in north-eastern Tanzania, blood samples were taken from 501 participants (0-19 years old). Malaria was detected by mRDT and microscopy. Levels of IgG against the spike protein of SARS-CoV-2 were measured by ELISA as well as IgG against five different antigens of P. falciparum; CIDRα1.1, CIDRα1.4 and CIDRα1.5 of PfEMP1 and GLURP and MSP3. RESULTS: The seroprevalence of SARS-CoV-2 IgG was 39.7% (106/267) in Kwamasimba and 32.5% (76/234) in Mkokola. In both villages the odds of being seropositive increased significantly with age (AOR = 1.12, 95% CI 1.07-1.17, p < 0.001). P. falciparum malaria prevalence by blood smear microscopy was 7.9% in Kwamasimba and 2.1% in Mkokola. 81.3% and 70.5% in Kwamasimba and Mkokola, respectively, showed recognition of minimum one malaria antigen. Residing in Kwamasimba was associated with a broader recognition (AOR = 1.91, 95% CI 1.34-2.71, p < 0.001). The recognition of malaria antigens increased significantly with age in both villages (AOR = 1.12; 95% CI 1.08-1.16, p < 0.001). Being SARS-CoV-2 seropositive did not associate with the breadth of malaria antigen recognition when adjusting for age (AOR = 0.99; 95% CI 0.83-1.18; p = 0.91). CONCLUSION: More than a third of the children and adolescents in two rural communities in Tanzania had antibodies to SARS-CoV-2. In particular, the adolescents were seropositive but being seropositive did not associate with the status of coinciding malaria infections or previous exposure. In Tanzania, natural immunity may have developed fast, potentially protecting a substantial part of the population from later variants.

Human IgG responses to Aedes mosquito salivary peptide Nterm-34kDa and its comparison to Anopheles salivary antigen (gSG6-P1) IgG responses measured among individuals living in Lower Moshi, Tanzania.

BACKGROUND: The level of human exposure to arbovirus vectors, the Aedes mosquitoes, is mainly assessed by entomological methods which are labour intensive, difficult to sustain at a large scale and are affected if transmission and exposure levels are low. Alternatively, serological biomarkers which detect levels of human exposure to mosquito bites may complement the existing epidemiologic tools as they seem cost-effective, simple, rapid, and sensitive. This study explored human IgG responses to an Aedes mosquito salivary gland peptide Nterm-34kDa in Lower Moshi, a highland area with evidence of circulating arboviruses and compared the Aedes IgG responses to Anopheles mosquitoes' salivary antigen (GSG6-P1) IgG responses. METHODS: Three cross-sectional surveys were conducted in 2019: during the first dry season in March, at the end of the rainy season in June and during the second dry season in September in five villages located in Lower Moshi. Blood samples were collected from enrolled participants above six months of age (age span: 7 months to 94 years) and analysed for the presence of anti-Nterm-34kDa IgG antibodies. Possible associations between Nterm-34kDa seroprevalence and participants' characteristics were determined. Levels of IgG responses and seroprevalence were correlated and compared to the already measured IgG responses and seroprevalence of Anopheles mosquitoes' salivary antigen, GSG6-P1. RESULTS: During the first dry season, Nterm-34kDa seroprevalence was 34.1% and significantly increased at the end of the rainy season to 45.3% (Chi square (χ2) = 6.42 p = 0.011). During the second dry season, the seroprevalence significantly declined to 26.5% (χ2 = 15.12 p<0.001). During the rainy season, seroprevalence was significantly higher among residents of Oria village (adjusted odds ratio (AOR) = 2.86; 95% CI = 1.0-7.8; p = 0.041) compared to Newland. Moreover, during the rainy season, the risk of exposure was significantly lower among individuals aged between 16 and 30 years (AOR = 0.25; 95% CI = 0.1 = 0.9; p = 0.036) compared to individuals aged between 0 and 5 years. There was weak to moderate negative correlation between N-term 34kDa IgG and gSG6-P1 antigens. N-term 34kDa seroprevalence were higher compared to gSG6-P1 seroprevalence. CONCLUSION: The findings of this study support that IgG antibody responses towards the Aedes mosquito salivary peptide Nterm-34kDa are detectable among individuals living in lower Moshi and vary with season and geographical area. More individuals are exposed to Aedes mosquito bites than Anopheles mosquito and those exposed to Aedes bites are not necessarily exposed to Anopheles mosquitoes.

Prevalence of asymptomatic malaria, submicroscopic parasitaemia and anaemia in Korogwe District, north-eastern Tanzania.

BACKGROUND: Asymptomatic malaria infections largely remain undetected and act as a reservoir for continuous transmission. The study assessed the prevalence of submicroscopic asymptomatic malaria infections and anaemia in two rural low (300 m above sea level) and highland (700 m asl) settings of Korogwe District north-eastern Tanzania. METHODS: A cross-sectional malariometric survey involving individuals aged 0-19 years was conducted in June 2018 in the two rural villages. Venous blood was collected from eligible study participants for estimation of haemoglobin level, detection of malaria by rapid diagnostic test (RDT), quantification of malaria parasitaemia by microscopy, as well as dried blood spot (DBS) for determining submicroscopic infections by PCR targeting the small subunit of the ribosomal ribonucleic acid (ssrRNA) of human Plasmodium. RESULTS: Out of 565 individuals tested, 211 (37.3%) were malaria positive based on RDT, whereas only 81 (14.3%) were positive by microscopy. There was no significant difference in the prevalence between the highland and the lowland village, p = 0.19 and p = 0.78 microscopy and RDT, respectively. Three out of 206 (1.5%) RDT/microscopy negative samples were P. falciparum positive by PCR. Of the 211 RDT and 81 microscopy positive, 130 (61.6%) and 33 (40.7%), respectively, were defined as being asymptomatic. Of the 565 individuals, 135 (23.9%) were anaemic (haemoglobin < 11 g/dL) out of which 5.2% were severely anaemic. The risk of being anaemic was significantly higher among individuals with asymptomatic malaria as compared to those without malaria as confirmed by RDT (AOR = 2.06 (95% CI 1.32-3.20) while based on microscopic results there was no significant differences observed (AOR = 2.09, 95% CI 0.98-4.47). Age and altitude had no effect on the risk of anaemia even after adjusting for asymptomatic malaria. CONCLUSIONS: Asymptomatic malaria is associated with an increased risk of having anaemia in the study communities. The findings highlight the need for targeted interventions focusing on asymptomatic infections which is an important risks factor for anaemia in the community and act as a source of continued transmission of malaria in the study area.

Use of anti-gSG6-P1 IgG as a serological biomarker to assess temporal exposure to Anopheles' mosquito bites in Lower Moshi.

BACKGROUND: Malaria prevalence in the highlands of Northern Tanzania is currently below 1% making this an elimination prone setting. As climate changes may facilitate increasing distribution of Anopheles mosquitoes in such settings, there is a need to monitor changes in risks of exposure to ensure that established control tools meet the required needs. This study explored the use of human antibodies against gambiae salivary gland protein 6 peptide 1 (gSG6-P1) as a biomarker of Anopheles exposure and assessed temporal exposure to mosquito bites in populations living in Lower Moshi, Northern Tanzania. METHODS: Three cross-sectional surveys were conducted in 2019: during the dry season in March, at the end of the rainy season in June and during the dry season in September. Blood samples were collected from enrolled participants and analysed for the presence of anti-gSG6-P1 IgG. Mosquitoes were sampled from 10% of the participants' households, quantified and identified to species level. Possible associations between gSG6-P1 seroprevalence and participants' characteristics were determined. RESULTS: The total number of Anopheles mosquitoes collected was highest during the rainy season (n = 1364) when compared to the two dry seasons (n = 360 and n = 1075, respectively). The gSG6-P1 seroprevalence increased from 18.8% during the dry season to 25.0% during the rainy season (χ2 = 2.66; p = 0.103) followed by a significant decline to 11.0% during the next dry season (χ2 = 12.56; p = 0.001). The largest number of mosquitoes were collected in one village (Oria), but the seroprevalence was significantly lower among the residents as compared to the rest of the villages (p = 0.039), explained by Oria having the highest number of participants owning and using bed nets. Both individual and household gSG6-P1 IgG levels had no correlation with numbers of Anopheles mosquitoes collected. CONCLUSION: Anti-gSG6-P1 IgG is a potential tool in detecting and distinguishing temporal and spatial variations in exposure to Anopheles mosquito bites in settings of extremely low malaria transmission where entomological tools may be obsolete. However studies with larger sample size and extensive mosquito sampling are warranted to further explore the association between this serological marker and abundance of Anopheles mosquito.

Complement C1s cleaves PfEMP1 at interdomain conserved sites inhibiting Plasmodium falciparum cytoadherence.

Cytoadhesion of Plasmodium falciparum-infected erythrocytes (IEs) to the endothelial lining of blood vessels protects parasites from splenic destruction, but also leads to detrimental inflammation and vessel occlusion. Surface display of the P. falciparum erythrocyte membrane protein 1 (PfEMP1) adhesion ligands exposes them to host antibodies and serum proteins. PfEMP1 are important targets of acquired immunity to malaria, and through evolution, the protein family has expanded and diversified to bind a select set of host receptors through antigenically diversified receptor-binding domains. Here, we show that complement component 1s (C1s) in serum cleaves PfEMP1 at semiconserved arginine motifs located at interdomain regions between the receptor-binding domains, rendering the IE incapable of binding the two main PfEMP1 receptors, CD36 and endothelial protein C receptor (EPCR). Bioinformatic analyses of PfEMP1 protein sequences from 15 P. falciparum genomes found the C1s motif was present in most PfEMP1 variants. Prediction of C1s cleavage and loss of binding to endothelial receptors was further corroborated by testing of several different parasite lines. These observations suggest that the parasites have maintained susceptibility for cleavage by the serine protease, C1s, and provides evidence for a complex relationship between the complement system and the P. falciparum cytoadhesion virulence determinant.

Ten years of monitoring malaria trend and factors associated with malaria test positivity rates in Lower Moshi.

BACKGROUND: High altitude settings in Eastern Africa have been reported to experience increased malaria burden due to vector habitat expansion. This study explored possible associations between malaria test positivity rates and its predictors including malaria control measures and meteorological factors at a high-altitude, low malaria transmission setting, south of Mount Kilimanjaro. METHODS: Malaria cases reported at the Tanganyika Plantation Company (TPC) hospital's malaria registers, meteorological data recorded at TPC sugar factory and data on bed nets distributed in Lower Moshi from 2009 to 2018 were studied. Correlation between bed nets distributed and malaria test positivity rates were explored by using Pearson correlation analysis and the associations between malaria test positivity rates and demographic and meteorological variables were determined by logistic regression and negative binomial regression analyses, respectively. RESULTS: Malaria cases reported at TPC hospital ranged between 0.48 and 2.26% per year and increased slightly at the introduction of malaria rapid diagnostic tests. The risk of testing positive for malaria were significantly highest among individuals aged between 6 and 15 years (OR = 1.65; 1.65 CI = 1.28-2.13; p = 0.001) and 16-30 years (OR = 1.49; CI = 1.17-1.89; p = 0.001) and when adjusted for age, the risk were significantly higher among male individuals when compared to female individuals (OR = 1.54; 1.00-1.31; p = 0.044). Malaria test positivity rates were positively associated with average monthly minimum temperatures and negatively associated with average monthly maximum temperatures (incidence rate ratio (IRR) = 1.37, 95% confidence interval (CI) = 1.05-1.78, p = 0.019 and IRR = 0.72, 95% CI = 0.58-0.91, p = 0.005, respectively). When analysed with one month lag for predictor variables, malaria test positivity rates were still significantly associated with average monthly minimum and maximum temperatures (IRR = 1.67, 95% CI = 1.28-2.19, p = 0.001 and IRR = 0.68, 95% CI = 0.54-0.85, p = 0.001, respectively). Average monthly rainfall and relative humidity with or without a one month lag was not associated with malaria test positivity rates in the adjusted models. Explopring possible associations between distribution of long-lasting insecticidal nets, (LLINs) and malaria test positivity rates showed no apparent correlation between numbers of LLINs distributed in a particular year and malaria test positivity rates. CONCLUSION: In Lower Moshi, the risk of being tested positive for malaria was highest for older children and male individuals. Higher minimum and lower maximum temperatures were the strongest climatic predictors for malaria test positivity rates. In areas with extensive irrigation activity as in Lower Moshi, vector abundance and thus malaria transmission may be less dependent on rainfall patterns and humidity. Mass distribution of LLINs did not have an effect in this area with already very low malaria transmission.

In vitro selection for adhesion of Plasmodium falciparum-infected erythrocytes to ABO antigens does not affect PfEMP1 and RIFIN expression.

Plasmodium falciparum causes the most severe form of malaria in humans. The adhesion of the infected erythrocytes (IEs) to endothelial receptors (sequestration) and to uninfected erythrocytes (rosetting) are considered major elements in the pathogenesis of the disease. Both sequestration and rosetting appear to involve particular members of several IE variant surface antigens (VSAs) as ligands, interacting with multiple vascular host receptors, including the ABO blood group antigens. In this study, we subjected genetically distinct P. falciparum parasites to in vitro selection for increased IE adhesion to ABO antigens in the absence of potentially confounding receptors. The selection resulted in IEs that adhered stronger to pure ABO antigens, to erythrocytes, and to various human cell lines than their unselected counterparts. However, selection did not result in marked qualitative changes in transcript levels of the genes encoding the best-described VSA families, PfEMP1 and RIFIN. Rather, overall transcription of both gene families tended to decline following selection. Furthermore, selection-induced increases in the adhesion to ABO occurred in the absence of marked changes in immune IgG recognition of IE surface antigens, generally assumed to target mainly VSAs. Our study sheds new light on our understanding of the processes and molecules involved in IE sequestration and rosetting.

Meta-analysis of Plasmodium falciparum var Signatures Contributing to Severe Malaria in African Children and Indian Adults.

The clinical presentation of severe Plasmodium falciparum malaria differs between children and adults, but the mechanistic basis for this remains unclear. Contributing factors to disease severity include total parasite biomass and the diverse cytoadhesive properties mediated by the polymorphic var gene parasite ligand family displayed on infected erythrocytes. To explore these factors, we performed a multicohort analysis of the contribution of var expression and parasite biomass to severe malaria in two previously published pediatric cohorts in Tanzania and Malawi and an adult cohort in India. Machine learning analysis revealed independent and complementary roles for var adhesion types and parasite biomass in adult and pediatric severe malaria and showed that similar var profiles, including upregulation of group A and DC8 var, predict severe malaria in adults and children. Among adults, patients with multiorgan complications presented infections with significantly higher parasite biomass without significant differences in var adhesion types. Conversely, pediatric patients with specific complications showed distinct var signatures. Cerebral malaria patients showed broadly increased expression of var genes, in particular group A and DC8 var, while children with severe malaria anemia were classified based on high transcription of DC8 var only. This study represents the first large multisite meta-analysis of var expression, and it demonstrates the presence of common var profiles in severe malaria patients of different ages across distant geographical sites, as well as syndrome-specific disease signatures. The complex associations between parasite biomass, var adhesion type, and clinical presentation revealed here represent the most comprehensive picture so far of the relationship between cytoadhesion, parasite load, and clinical syndrome.IMPORTANCEP. falciparum malaria can cause multiple disease complications that differ by patient age. Previous studies have attempted to address the roles of parasite adhesion and biomass in disease severity; however, these studies have been limited to single geographical sites, and there is limited understanding of how parasite adhesion and biomass interact to influence disease manifestations. In this meta-analysis, we compared parasite disease determinants in African children and Indian adults. This study demonstrates that parasite biomass and specific subsets of var genes are independently associated with detrimental outcomes in both childhood and adult malaria. We also explored how parasite var adhesion types and biomass play different roles in the development of specific severe malaria pathologies, including childhood cerebral malaria and multiorgan complications in adults. This work represents the largest study to date of the role of both var adhesion types and biomass in severe malaria.

Cerebral malaria is associated with differential cytoadherence to brain endothelial cells.

Sequestration of Plasmodium falciparum-infected erythrocytes (IE) within the brain microvasculature is a hallmark of cerebral malaria (CM). Using a microchannel flow adhesion assay with TNF-activated primary human microvascular endothelial cells, we demonstrate that IE isolated from Malawian paediatric CM cases showed increased binding to brain microvascular endothelial cells compared to IE from uncomplicated malaria (UM) cases. Further, UM isolates showed significantly greater adhesion to dermal than to brain microvascular endothelial cells. The major mediator of parasite adhesion is P. falciparum erythrocyte membrane protein 1, encoded by var genes. Higher levels of var gene transcripts predicted to bind host endothelial protein C receptor (EPCR) and ICAM-1 were detected in CM isolates. These data provide further evidence for differential tissue binding in severe and uncomplicated malaria syndromes, and give additional support to the hypothesis that CM pathology is based on increased cytoadherence of IE in the brain microvasculature.

Corrigendum: Immune evasion of Plasmodium falciparum by RIFIN via inhibitory receptors.

This corrects the article DOI: 10.1038/nature24994.

Immune evasion of Plasmodium falciparum by RIFIN via inhibitory receptors.

Malaria is among the most serious infectious diseases affecting humans, accounting for approximately half a million deaths each year. Plasmodium falciparum causes most life-threatening cases of malaria. Acquired immunity to malaria is inefficient, even after repeated exposure to P. falciparum, but the immune regulatory mechanisms used by P. falciparum remain largely unknown. Here we show that P. falciparum uses immune inhibitory receptors to achieve immune evasion. RIFIN proteins are products of a polymorphic multigene family comprising approximately 150-200 genes per parasite genome that are expressed on the surface of infected erythrocytes. We found that a subset of RIFINs binds to either leucocyte immunoglobulin-like receptor B1 (LILRB1) or leucocyte-associated immunoglobulin-like receptor 1 (LAIR1). LILRB1-binding RIFINs inhibit activation of LILRB1-expressing B cells and natural killer (NK) cells. Furthermore, P. falciparum-infected erythrocytes isolated from patients with severe malaria were more likely to interact with LILRB1 than erythrocytes from patients with non-severe malaria, although an extended study with larger sample sizes is required to confirm this finding. Our results suggest that P. falciparum has acquired multiple RIFINs to evade the host immune system by targeting immune inhibitory receptors.

Parasites Causing Cerebral Falciparum Malaria Bind Multiple Endothelial Receptors and Express EPCR and ICAM-1-Binding PfEMP1.

Background: Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) mediates the binding and accumulation of infected erythrocytes (IE) to blood vessels and tissues. Specific interactions have been described between PfEMP1 and human endothelial proteins CD36, intercellular adhesion molecule-1 (ICAM-1), and endothelial protein C receptor (EPCR); however, cytoadhesion patterns typical for pediatric malaria syndromes and the associated PfEMP1 members are still undefined. Methods: In a cohort of 94 hospitalized children with malaria, we characterized the binding properties of IE collected on admission, and var gene transcription using quantitative polymerase chain reaction. Results: IE from patients with cerebral malaria were more likely to bind EPCR and ICAM-1 than IE from children with uncomplicated malaria (P = .007). The level of transcripts encoding CIDRα1.4 and CIDRα1.5 domain subclasses was higher in patients with severe disease (P < .05). IE populations exhibiting binding to all 3 receptors had higher levels of transcripts encoding PfEMP1 with CIDRα1.4 and Duffy binding-like (DBL)-ß3 domains than parasites, which only bound CD36. Conclusions: These results underpin the significance of EPCR binding in pediatric malaria patients that require hospital admission, and support the notion that complementary receptor interactions of EPCR binding PfEMP1with ICAM-1 amplifies development of severe malaria symptoms.

Structure-Guided Identification of a Family of Dual Receptor-Binding PfEMP1 that Is Associated with Cerebral Malaria.

Cerebral malaria is a deadly outcome of infection by Plasmodium falciparum, occurring when parasite-infected erythrocytes accumulate in the brain. These erythrocytes display parasite proteins of the PfEMP1 family that bind various endothelial receptors. Despite the importance of cerebral malaria, a binding phenotype linked to its symptoms has not been identified. Here, we used structural biology to determine how a group of PfEMP1 proteins interacts with intercellular adhesion molecule 1 (ICAM-1), allowing us to predict binders from a specific sequence motif alone. Analysis of multiple Plasmodium falciparum genomes showed that ICAM-1-binding PfEMP1s also interact with endothelial protein C receptor (EPCR), allowing infected erythrocytes to synergistically bind both receptors. Expression of these PfEMP1s, predicted to bind both ICAM-1 and EPCR, is associated with increased risk of developing cerebral malaria. This study therefore reveals an important PfEMP1-binding phenotype that could be targeted as part of a strategy to prevent cerebral malaria.

Cellulose filtration of blood from malaria patients for improving ex vivo growth of Plasmodium falciparum parasites.

BACKGROUND: Establishing in vitro Plasmodium falciparum culture lines from patient parasite isolates can offer deeper understanding of geographic variations of drug sensitivity and mechanisms of malaria pathogenesis and immunity. Cellulose column filtration of blood is an inexpensive, rapid and effective method for the removal of host factors, such as leucocytes and platelets, significantly improving the purification of parasite DNA in a blood sample. METHODS: In this study, the effect of cellulose column filtration of venous blood on the initial in vitro growth of P. falciparum parasite isolates from Tanzanian children admitted to hospital was tested. The parasites were allowed to expand in culture without subcultivation until 5 days after admission or the appearance of dead parasites and parasitaemia was determined daily. To investigate whether the filtration had an effect on clonality, P. falciparum merozoite surface protein 2 genotyping was performed using nested PCR on extracted genomic DNA, and the var gene transcript levels were investigated, using quantitative PCR on extracted RNA, at admission and 4 days of culture. RESULTS: The cellulose-filtered parasites grew to higher parasitaemia faster than non-filtered parasites seemingly due to a higher development ratio of ring stage parasites progressing into the late stages. Cellulose filtration had no apparent effect on clonality or var gene expression; however, evident differences were observed after only 4 days of culture in both the number of clones and transcript levels of var genes compared to the time of admission. CONCLUSIONS: Cellulose column filtration of parasitized blood is a cheap, applicable method for improving cultivation of P. falciparum field isolates for ex vivo based assays; however, when assessing phenotype and genotype of cultured parasites, in general, assumed to represent the in vivo infection, caution is advised.

The Severity of Plasmodium falciparum Infection Is Associated with Transcript Levels of var Genes Encoding Endothelial Protein C Receptor-Binding P. falciparum Erythrocyte Membrane Protein 1.

By attaching infected erythrocytes to the vascular lining, Plasmodium falciparum parasites leave blood circulation and avoid splenic clearance. This sequestration is central to pathogenesis. Severe malaria is associated with parasites expressing an antigenically distinct P. falciparum erythrocyte membrane protein 1 (PfEMP1) subset mediating binding to endothelial receptors. Previous studies indicate that PfEMP1 adhesins with so-called CIDRα1 domains capable of binding endothelial protein C receptor (EPCR) constitute the PfEMP1 subset associated with severe pediatric malaria. To analyze the relative importance of different subtypes of CIDRα1 domains, we compared Pfemp1 transcript levels in children with severe malaria (including 9 fatal and 114 surviving cases), children hospitalized with uncomplicated malaria (n = 42), children with mild malaria not requiring hospitalization (n = 10), and children with parasitemia and no ongoing fever (n = 12). High levels of transcripts encoding EPCR-binding PfEMP1 were found in patients with symptomatic infections, and the abundance of these transcripts increased with disease severity. The compositions of CIDRα1 subtype transcripts varied markedly between patients, and none of the subtypes were dominant. Transcript-level analyses targeting other domain types indicated that subtypes of DBLß or DBLζ domains might mediate binding phenomena that, in conjunction with EPCR binding, could contribute to pathogenesis. These observations strengthen the rationale for targeting the PfEMP1-EPCR interaction by vaccines and adjunctive therapies. Interventions should target EPCR binding of all CIDRα1 subtypes.

Molecular Evidence of Malaria and Zoonotic Diseases Among Rapid Diagnostic Test-Negative Febrile Patients in Low-Transmission Season, Mali.

From November to December 2012 in Sélingué-Mali, blood samples from 88 febrile patients who tested negative by malaria Paracheck® rapid diagnostic tests (RDTs) were used to assess the presence of sub-RDT Plasmodium falciparum as well as Borrelia, Coxiella burnetii, and Babesia applying molecular tools. Plasmodium sp. was present among 57 (60.2%) of the 88 malaria RDT-negative patients, whereas the prevalence of Borrelia, C. burnetii, and Babesia were 3.4% (N = 3), 1.1% (N = 1), and 0.0%, respectively. The additional diagnostic use of polymerase chain reaction (PCR) identified a high proportion of Plasmodium sp.-positive samples and although this may be a concern for malaria control, the respective PCR-identified malaria infections were less likely responsible for the observed fevers given the low parasite density. Also, the low infection levels of Borrelia and C. burnetii and lack of Babesia among the febrile patients call for further studies to assess the causes of fever among malaria RDT-negative patients in Sélingué.

Cytoadhesion to gC1qR through Plasmodium falciparum Erythrocyte Membrane Protein 1 in Severe Malaria.

Cytoadhesion of Plasmodium falciparum infected erythrocytes to gC1qR has been associated with severe malaria, but the parasite ligand involved is currently unknown. To assess if binding to gC1qR is mediated through the P. falciparum erythrocyte membrane protein 1 (PfEMP1) family, we analyzed by static binding assays and qPCR the cytoadhesion and var gene transcriptional profile of 86 P. falciparum isolates from Mozambican children with severe and uncomplicated malaria, as well as of a P. falciparum 3D7 line selected for binding to gC1qR (Pf3D7gC1qR). Transcript levels of DC8 correlated positively with cytoadhesion to gC1qR (rho = 0.287, P = 0.007), were higher in isolates from children with severe anemia than with uncomplicated malaria, as well as in isolates from Europeans presenting a first episode of malaria (n = 21) than Mozambican adults (n = 25), and were associated with an increased IgG recognition of infected erythrocytes by flow cytometry. Pf3D7gC1qR overexpressed the DC8 type PFD0020c (5.3-fold transcript levels relative to Seryl-tRNA-synthetase gene) compared to the unselected line (0.001-fold). DBLß12 from PFD0020c bound to gC1qR in ELISA-based binding assays and polyclonal antibodies against this domain were able to inhibit binding to gC1qR of Pf3D7gC1qR and four Mozambican P. falciparum isolates by 50%. Our results show that DC8-type PfEMP1s mediate binding to gC1qR through conserved surface epitopes in DBLß12 domain which can be inhibited by strain-transcending functional antibodies. This study supports a key role for gC1qR in malaria-associated endovascular pathogenesis and suggests the feasibility of designing interventions against severe malaria targeting this specific interaction.

Plasma Ang2 and ADAM17 levels are elevated during clinical malaria; Ang2 level correlates with severity and expression of EPCR-binding PfEMP1.

The pathogenesis of Plasmodium falciparum malaria involves a complex interplay between parasite adhesion and inflammatory response that includes release of cytokines and activation of the endothelium with accompanying release of Angiopoitin 2 (Ang2) to the plasma. A-disintegrin and metalloproteinase 17 (ADAM17) is a protein responsible for releasing cytokines, including Tumor Necrosis Factor α (TNFα), and shedding of adhesion proteins. In this study, we show that plasma levels of ADAM17 are increased in Tanzanian children hospitalized with a malaria infection compared with asymptomatic children but similar to children hospitalized with other infectious diseases. The plasma levels of ADAM17 decreased during recovery after an acute malaria episode. Plasma levels of Ang2 were associated with markers of malaria severity and levels of var transcripts encoding P. falciparum Erythrocyte Membrane Protein 1 (PfEMP1) containing Cysteine Rich Inter Domain Region α1 (CIDRα1) domains predicted to bind Endothelial Protein C receptor (EPCR). ADAM17 levels were not associated with expression of var genes encoding different PfEMP1 types when controlling for age. These data are the first to report ADAM17 plasma levels in malaria-exposed individuals, and support the notion that parasite sequestration mediated by EPCR-binding PfEMP1 is associated with endothelial activation and pathology in severe paediatric malaria.

Plasmodium falciparum var genes expressed in children with severe malaria encode CIDRα1 domains.

Most severe Plasmodium falciparum infections are experienced by young children. Severe symptoms are precipitated by vascular sequestration of parasites expressing a particular subset of the polymorphic P. falciparum erythrocyte membrane protein 1 (PfEMP1) adhesion molecules. Parasites binding human endothelial protein C receptor (EPCR) through the CIDRα1 domain of certain PfEMP1 were recently associated with severe malaria in children. However, it has remained unclear to which extend the EPCR-binding CIDRα1 domains epitomize PfEMP1 expressed in severe malaria. Here, we characterized the near full-length transcripts dominating the var transcriptome in children with severe malaria and found that the only common feature of the encoded PfEMP1 was CIDRα1 domains. Such genes were highly and dominantly expressed in both children with severe malarial anaemia and cerebral malaria. These observations support the hypothesis that the CIDRα1-EPCR interaction is key to the pathogenesis of severe malaria and strengthen the rationale for pursuing a vaccine or adjunctive treatment aiming at inhibiting or reducing the damaging effects of this interaction.

Adhesion of Plasmodium falciparum infected erythrocytes in ex vivo perfused placental tissue: a novel model of placental malaria.

BACKGROUND: Placental malaria occurs when Plasmodium falciparum infected erythrocytes sequester in the placenta. Placental parasite isolates bind to chondroitin sulphate A (CSA) by expression of VAR2CSA on the surface of infected erythrocytes, but may sequester by other VAR2CSA mediated mechanisms, such as binding to immunoglobulins. Furthermore, other parasite antigens have been associated with placental malaria. These findings have important implications for placental malaria vaccine design. The objective of this study was to adapt and describe a biologically relevant model of parasite adhesion in intact placental tissue. RESULTS: The ex vivo placental perfusion model was modified to study adhesion of infected erythrocytes binding to CSA, endothelial protein C receptor (EPCR) or a transgenic parasite where P. falciparum erythrocyte membrane protein 1 expression had been shut down. Infected erythrocytes expressing VAR2CSA accumulated in perfused placental tissue whereas the EPCR binding and the transgenic parasite did not. Soluble CSA and antibodies specific against VAR2CSA inhibited binding of infected erythrocytes. CONCLUSION: The ex vivo model provides a novel way of studying receptor-ligand interactions and antibody mediated inhibition of binding in placental malaria.