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Determining the causal role of malaria in elevating blood pressure and pulse wave velocity in Kenyan adolescents and adults.

INTRODUCTION: High blood pressure is recognized as a leading risk factor for stroke and death in sub-Saharan Africa (sSA). While many studies have examined the role of established risk factors such as obesity and salt consumption, less is known about other factors, such as infection, that could be of particular importance in sSA. Ambulatory blood pressure measurement has emerged as the optimal method in recent years in Western settings, but there has been limited use to date in sSA. This work presents the results of a study investigating whether malaria, which is widespread in sSA could contribute to the development of high blood pressure using ambulatory measurements. METHODS: Preliminary work involved determining the prevalence of hypertension in Kilifi, Kenya and examining the population-level effects of using ambulatory blood pressure monitoring (ABPM) for diagnosing hypertension. A literature review outlining the basis of the malaria-high blood pressure hypothesis and the Mendelian randomization method for testing the hypothesis was conducted. Sickle cell trait and alpha (+) thalassemia were chosen as instrumental variables to represent malaria exposure because they protect against malaria. Two studies were performed in Nairobi, Kenya among the same cohort to confirm that sickle-cell trait and alpha-thalassemia do not influence blood pressure in the absence of malaria and were therefore valid instrumental variables to test the malaria-high blood pressure hypothesis in Kilifi where there is malaria transmission. A Mendelian randomization study was then conducted in Kilifi, Kenya where 24-hour blood pressure and arterial stiffness indices were compared in individuals with and without sickle cell trait and alpha thalassemia. RESULTS: The prevalence of hypertension in Kilifi, a rural area, was found to be as high as in urban areas of Kenya despite the low frequency of classical risk factors such as obesity and excessive salt consumption. Use of ambulatory blood pressure monitoring for diagnosing hypertension was found to improve the accuracy of detection of high blood pressure. Neither Sickle-cell trait (SCT) nor alpha+ thalassemia influenced blood pressure or arterial stiffness indices among adolescents that had been lifelong residents of Nairobi, where there is no malaria transmission. Among individuals that had been lifelong residents of Kilifi, Kenya where there has been on-going malaria transmission, blood pressure was found to be lower among individuals with SCT, which protects against malaria episodes compared to those without SCT. The difference in BP by SCT status was larger in women than in men. There were no significant differences in arterial stiffness based on SCT status. CONCLUSION: This work suggests that malaria contributes to the burden of hypertension in sSA, and the control of malaria may lead to a reduction in blood pressure in this group. Future work should focus on confirming the findings using alternative study designs such as examining blood pressure in cohorts born before and after complete malaria elimination in parts of the world where this has been achieved. Subsequent work would involve delineating the pathophysiological mechanisms involved in malaria induced BP elevation with a view to generating new drugs to control hypertension.
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Correction to: An exploration of mortality risk factors in non-severe pneumonia in children using clinical data from Kenya

CORRECTION: The original article contains an omission in the Acknowledgements sub-section of the Declarations.
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An exploration of mortality risk factors in non-severe pneumonia in children using clinical data from Kenya

BACKGROUND: Childhood pneumonia is the leading infectious cause of mortality in children younger than 5 years old. Recent updates to World Health Organization pneumonia guidelines recommend outpatient care for a population of children previously classified as high risk. This revision has been challenged by policymakers in Africa, where mortality related to pneumonia is higher than in other regions and often complicated by comorbidities. This study aimed to identify factors that best discriminate inpatient mortality risk in non-severe pneumonia and explore whether these factors offer any added benefit over the current criteria used to identify children with pneumonia requiring inpatient care. METHODS: We undertook a retrospective cohort study of children aged 2-59 months admitted with a clinical diagnosis of pneumonia at 14 public hospitals in Kenya between February 2014 and February 2016. Using machine learning techniques, we analysed whether clinical characteristics and common comorbidities increased the risk of inpatient mortality for non-severe pneumonia. The topmost risk factors were subjected to decision curve analysis to explore if using them as admission criteria had any net benefit above the current criteria. RESULTS: Out of 16,162 children admitted with pneumonia during the study period, 10,687 were eligible for subsequent analysis. Inpatient mortality within this non-severe group was 252/10,687 (2.36%). Models demonstrated moderately good performance; the partial least squares discriminant analysis model had higher sensitivity for predicting mortality in comparison to logistic regression. Elevated respiratory rate (>/=70 bpm), age 2-11 months and weight-for-age Z-score (WAZ) < -3SD were highly discriminative of mortality. These factors ranked consistently across the different models. For a risk threshold probability of 7-14%, there is a net benefit to admitting the patient sub-populations with these features as additional criteria alongside those currently used to classify severe pneumonia. Of the population studied, 70.54% met at least one of these criteria. Sensitivity analyses indicated that the overall results were not significantly affected by variations in pneumonia severity classification criteria. CONCLUSIONS: Children with non-severe pneumonia aged 2-11 months or with respiratory rate >/= 70 bpm or very low WAZ experience risks of inpatient mortality comparable to severe pneumonia. Inpatient care is warranted in these high-risk groups of children.
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Does audit and feedback improve the adoption of recommended practices? Evidence from a longitudinal observational study of an emerging clinical network in Kenya

Background: Audit and feedback (A&F) is widely used in healthcare but there are few examples of how to deploy it at scale in low-income countries. Establishing the Clinical Information Network (CIN) in Kenya provided an opportunity to examine the effect of A&F delivered as part of a wider set of activities to promote paediatric guideline adherence. Methods: We analysed data collected from medical records on discharge for children aged 2-59 months from 14 Kenyan hospitals in the CIN. Hospitals joined CIN in phases and for each we analysed their initial 25 months of participation that occurred between December 2013 and March 2016. A total of 34 indicators of adherence to recommendations were selected for evaluation each classified by form of feedback (passive, active and none) and type of task (simple or difficult documentation and those requiring cognitive work). Performance change was explored graphically and using generalised linear mixed models with attention given to the effects of time and use of a standardised paediatric admission record (PAR) form. Results: Data from 60 214 admissions were eligible for analysis. Adherence to recommendations across hospitals significantly improved for 24/34 indicators. Improvements were not obviously related to nature of feedback, may be related to task type and were related to PAR use in the case of documentation indicators. There was, however, marked variability in adoption and adherence to recommended practices across sites and indicators. Hospital-specific factors, low baseline performance and specific contextual changes appeared to influence the magnitude of change in specific cases. Conclusion: Our observational data suggest some change in multiple indicators of adherence to recommendations (aspects of quality of care) can be achieved in low-resource hospitals using A&F and simple job aides in the context of a wider network approach.
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Proteomic analysis of extracellular vesicles from a Plasmodium falciparum Kenyan clinical isolate defines a core parasite secretome

BACKGROUND: Many pathogens secrete effector molecules to subvert host immune responses, to acquire nutrients, and/or to prepare host cells for invasion. One of the ways that effector molecules are secreted is through extracellular vesicles (EVs) such as exosomes. Recently, the malaria parasite P. falciparum has been shown to produce EVs that can mediate transfer of genetic material between parasites and induce sexual commitment. Characterizing the content of these vesicles may improve our understanding of P. falciparum pathogenesis and virulence. METHODS: Previous studies of P. falciparum EVs have been limited to long-term adapted laboratory isolates. In this study, we isolated EVs from a Kenyan P. falciparum clinical isolate adapted to in vitro culture for a short period and characterized their protein content by mass spectrometry (data are available via ProteomeXchange, with identifier PXD006925). RESULTS: We show that P. falciparum extracellular vesicles ( PfEVs) are enriched in proteins found within the exomembrane compartments of infected erythrocytes such as Maurer's clefts (MCs), as well as the secretory endomembrane compartments in the apical end of the merozoites, suggesting that these proteins play a role in parasite-host interactions. Comparison of this novel clinically relevant dataset with previously published datasets helps to define a core secretome present in Plasmodium EVs. CONCLUSIONS: P. falciparum extracellular vesicles contain virulence-associated parasite proteins. Therefore, analysis of PfEVs contents from a range of clinical isolates, and their functional validation may improve our understanding of the virulence mechanisms of the parasite, and potentially identify targets for interventions or diagnostics.
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Proteomic analysis of extracellular vesicles from a Plasmodium falciparum Kenyan clinical isolate defines a core parasite secretome

BACKGROUND: Many pathogens secrete effector molecules to subvert host immune responses, to acquire nutrients, and/or to prepare host cells for invasion. One of the ways that effector molecules are secreted is through extracellular vesicles (EVs) such as exosomes. Recently, the malaria parasite P. falciparum has been shown to produce EVs that can mediate transfer of genetic material between parasites and induce sexual commitment. Characterizing the content of these vesicles may improve our understanding of P. falciparum pathogenesis and virulence. METHODS: Previous studies of P. falciparum EVs have been limited to long-term adapted laboratory isolates. In this study, we isolated EVs from a Kenyan P. falciparum clinical isolate adapted to in vitro culture for a short period and characterized their protein content by mass spectrometry (data are available via ProteomeXchange, with identifier PXD006925). RESULTS: We show that P. falciparum extracellular vesicles ( PfEVs) are enriched in proteins found within the exomembrane compartments of infected erythrocytes such as Maurer's clefts (MCs), as well as the secretory endomembrane compartments in the apical end of the merozoites, suggesting that these proteins play a role in parasite-host interactions. Comparison of this novel clinically relevant dataset with previously published datasets helps to define a core secretome present in Plasmodium EVs. CONCLUSIONS: P. falciparum extracellular vesicles contain virulence-associated parasite proteins. Therefore, analysis of PfEVs contents from a range of clinical isolates, and their functional validation may improve our understanding of the virulence mechanisms of the parasite, and potentially identify targets for interventions or diagnostics.
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Public antibodies to malaria antigens generated by two LAIR1 insertion modalities

In two previously described donors, the extracellular domain of LAIR1, a collagen-binding inhibitory receptor encoded on chromosome 19 (ref. 1), was inserted between the V and DJ segments of an antibody. This insertion generated, through somatic mutations, broadly reactive antibodies against RIFINs, a type of variant antigen expressed on the surface of Plasmodium falciparum-infected erythrocytes. To investigate how frequently such antibodies are produced in response to malaria infection, we screened plasma from two large cohorts of individuals living in malaria-endemic regions. Here we report that 5-10% of malaria-exposed individuals, but none of the European blood donors tested, have high levels of LAIR1-containing antibodies that dominate the response to infected erythrocytes without conferring enhanced protection against febrile malaria. By analysing the antibody-producing B cell clones at the protein, cDNA and gDNA levels, we characterized additional LAIR1 insertions between the V and DJ segments and discovered a second insertion modality whereby the LAIR1 exon encoding the extracellular domain and flanking intronic sequences are inserted into the switch region. By exon shuffling, this mechanism leads to the production of bispecific antibodies in which the LAIR1 domain is precisely positioned at the elbow between the VH and CH1 domains. Additionally, in one donor the genomic DNA encoding the VH and CH1 domains was deleted, leading to the production of a camel-like LAIR1-containing antibody. Sequencing of the switch regions of memory B cells from European blood donors revealed frequent templated inserts originating from transcribed genes that, in rare cases, comprised exons with orientations and frames compatible with expression. These results reveal different modalities of LAIR1 insertion that lead to public and dominant antibodies against infected erythrocytes and suggest that insertion of templated DNA represents an additional mechanism of antibody diversification that can be selected in the immune response against pathogens and exploited for B cell engineering.
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Public antibodies to malaria antigens generated by two LAIR1 insertion modalities

In two previously described donors, the extracellular domain of LAIR1, a collagen-binding inhibitory receptor encoded on chromosome 19 (ref. 1), was inserted between the V and DJ segments of an antibody. This insertion generated, through somatic mutations, broadly reactive antibodies against RIFINs, a type of variant antigen expressed on the surface of Plasmodium falciparum-infected erythrocytes. To investigate how frequently such antibodies are produced in response to malaria infection, we screened plasma from two large cohorts of individuals living in malaria-endemic regions. Here we report that 5-10% of malaria-exposed individuals, but none of the European blood donors tested, have high levels of LAIR1-containing antibodies that dominate the response to infected erythrocytes without conferring enhanced protection against febrile malaria. By analysing the antibody-producing B cell clones at the protein, cDNA and gDNA levels, we characterized additional LAIR1 insertions between the V and DJ segments and discovered a second insertion modality whereby the LAIR1 exon encoding the extracellular domain and flanking intronic sequences are inserted into the switch region. By exon shuffling, this mechanism leads to the production of bispecific antibodies in which the LAIR1 domain is precisely positioned at the elbow between the VH and CH1 domains. Additionally, in one donor the genomic DNA encoding the VH and CH1 domains was deleted, leading to the production of a camel-like LAIR1-containing antibody. Sequencing of the switch regions of memory B cells from European blood donors revealed frequent templated inserts originating from transcribed genes that, in rare cases, comprised exons with orientations and frames compatible with expression. These results reveal different modalities of LAIR1 insertion that lead to public and dominant antibodies against infected erythrocytes and suggest that insertion of templated DNA represents an additional mechanism of antibody diversification that can be selected in the immune response against pathogens and exploited for B cell engineering.
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Plasmodium falciparum malaria parasite var gene expression is modified by host antibodies: longitudinal evidence from controlled infections of Kenyan adults with varying natural exposure

BACKGROUND: The PfEMP1 family of Plasmodium falciparum antigens play a key role in pathogenesis of severe malaria through their insertion into the surface of parasite infected erythrocytes, and adhesion to host cells. Previous studies have suggested that parasites expressing PfEMP1 subclasses group A and DC8, associated with severe malaria, may have a growth advantage in immunologically naive individuals. However, this idea has not been tested in longitudinal studies. METHODS: Here we assessed expression of the var genes encoding PfEMP1, in parasites sampled from volunteers with varying prior exposure to malaria, following experimental infection by sporozoites (PfSPZ). Using qPCR, we tested for associations between the expression of various var subgroups in surviving parasite populations from each volunteer and 1) the levels of participants' antibodies to infected erythrocytes before challenge infection and 2) the apparent in vivo parasite multiplication rate. RESULTS: We show that 1) expression of var genes encoding for group A and DC8-like PfEMP1 were associated with low levels of antibodies to infected erythrocytes (alphaIE) before challenge, and 2) expression of a DC8-like CIDRalpha1.1 domain was associated with higher apparent parasite multiplication rate in a manner that was independent of levels of prior antibodies to infected erythrocytes. CONCLUSIONS: This study provides insight into the role of antibodies to infected erythrocytes surface antigens in the development of naturally acquired immunity and may help explain why specific PfEMP1 variants may be associated with severe malaria. TRIAL REGISTRATION: Pan African Clinical Trial Registry: PACTR201211000433272 . Date of registration: 10th October 2012.
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Plasmodium falciparum malaria parasite var gene expression is modified by host antibodies: longitudinal evidence from controlled infections of Kenyan adults with varying natural exposure

BACKGROUND: The PfEMP1 family of Plasmodium falciparum antigens play a key role in pathogenesis of severe malaria through their insertion into the surface of parasite infected erythrocytes, and adhesion to host cells. Previous studies have suggested that parasites expressing PfEMP1 subclasses group A and DC8, associated with severe malaria, may have a growth advantage in immunologically naive individuals. However, this idea has not been tested in longitudinal studies. METHODS: Here we assessed expression of the var genes encoding PfEMP1, in parasites sampled from volunteers with varying prior exposure to malaria, following experimental infection by sporozoites (PfSPZ). Using qPCR, we tested for associations between the expression of various var subgroups in surviving parasite populations from each volunteer and 1) the levels of participants' antibodies to infected erythrocytes before challenge infection and 2) the apparent in vivo parasite multiplication rate. RESULTS: We show that 1) expression of var genes encoding for group A and DC8-like PfEMP1 were associated with low levels of antibodies to infected erythrocytes (alphaIE) before challenge, and 2) expression of a DC8-like CIDRalpha1.1 domain was associated with higher apparent parasite multiplication rate in a manner that was independent of levels of prior antibodies to infected erythrocytes. CONCLUSIONS: This study provides insight into the role of antibodies to infected erythrocytes surface antigens in the development of naturally acquired immunity and may help explain why specific PfEMP1 variants may be associated with severe malaria. TRIAL REGISTRATION: Pan African Clinical Trial Registry: PACTR201211000433272 . Date of registration: 10th October 2012.
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