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Levels of human proteins in plasma as indicators for acute severe pediatric malaria
KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science.ORCID iD: 0000-0003-0344-8049
KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Protein Science.
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(English)Manuscript (preprint) (Other academic)
Abstract [en]

Background

Existing low resource diagnostics for malaria infection suffer from sensitivity and specificity issues while lacking sufficient prognostic value. Identifying human host proteins could improve the possibilities to predict the risk of development of acute severe malaria. This will possible enable improved treatment and thereby lead to a decrease in mortality of malaria infected children. Furthermore, discovering host proteins with altered levels during active infection could generate leads to better understand host-parasite interaction.

Results

Here, we have analyzed a total of 541 pediatric plasma samples that were collected from community controls and individuals with mild or severe malaria in Rwanda. Protein profiles of these plasma samples were generated with an antibody-based suspension bead array containing 255 antibodies targeting 115 human proteins. We present 22 proteins with a strong discriminatory capacity (adjusted p-values below 10-19) for separating malaria cases from community controls. This panel of proteins contains among others acute phase proteins and proteins connected to cell adhesion and migration. Among these, three proteins showed lower plasma levels in the group of malaria-infected individuals compared to the control group. One of these proteins is the anti-adhesive secreted protein acidic and cysteine rich (SPARC) with possible connections to parasite cytoadhesion. A multi-protein panel of six proteins, including SPARC, could differentiate between controls and malaria cases with an AUC of 0.98. Furthermore, a panel of 37 proteins, including proteins associated to erythrocyte membranes, was identified as candidates for separation of mild and severe malaria patients (adjusted pvalues below 0.05).

Conclusion

The herein identified set of human proteins has a significant discriminatory capacity between community controls and malaria cases. We also present proteins offering the possibility to enable stratification and risk prediction for the development of severe malaria. This constitutes an important set that could enable enhanced understanding and thereby also possibilities for better treatment of acute severe pediatric malaria.

 

Keywords [en]
affinity proteomics; human plasma profiling; malaria; Plasmodium falciparum, suspension bead arrays; cytoadhesion; Secreted protein acidic and cysteine rich, SPARC, Osteonectin
National Category
Medical Biotechnology
Identifiers
URN: urn:nbn:se:kth:diva-227161OAI: oai:DiVA.org:kth-227161DiVA, id: diva2:1203275
Funder
EU, European Research Council, 615458.Available from: 2018-05-03 Created: 2018-05-03 Last updated: 2018-05-03Bibliographically approved
In thesis
1. Development of array systems for molecular diagnostic assays
Open this publication in new window or tab >>Development of array systems for molecular diagnostic assays
2018 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

For molecular diagnostics, assays detecting biomarkers can be used to provide information to medical questions. Array formats such as microarrays and suspension bead arrays allow for multiplex assays, analyzing hundreds or thousands of analytes simultaneously in one single sample. There is a growing demand of multiplex assays, using panels of biomarkers, for the use in Point-of-Care (POC) diagnostic tests. These diagnostic tests require little or no equipment and relies on easy read-out systems. Lateral flow assays (LFAs) are well-known, paper-based POC assays, with advantageous features, which include performing the assay by capillary forces, stable reagents storage and naked-eye read-out. Vertical Flow Microarrays (VFMs) have previously been presented as an alternative to LFAs, circumventing downstream effects by vertically applying sample and reagents. In Paper I and II of this thesis, VFM arrays have been applied in the development of assays for molecular diagnosis, while Paper III explores Suspension Bead Array (SBA) format for biomarker discovery.

 

In Paper I, we have developed a DNA-VFM assay towards POC testing of Neisseria meningitidis, one of the major meningitis causing bacteria. Here, the target gene of N. meningitidis was amplified using Recombinase Polymerase Amplification (RPA). The amplified DNA was digested into ssDNA and hybridized to multiple VFM probes, for multiplex detection of different segments of the target gene. Optimization of the assay resulted in a Limit Of Detection (LOD) of 4.4 nM for amplification of synthetic DNA.

 

In Paper II, a VFM for reverse phase detection of IgE was developed. The assay was optimized using IgE spiked into IgE-negative serum, resulting in a LOD of 1.9 μg/mL. Optimized conditions were then used to screen a cohort of serum samples, including patients with rare primary immunodeficiency Hyper IgE syndrome and healthy controls. A comparative, traditional reverse phase assay was also performed on the same serum samples, showing comparable results to the reverse phase VFM.

 

In Paper III, a pediatric cohort from Plasmodium falciparum endemic Rwanda was analyzed using SBAs for proteins involved during various stages of malaria infection. Large, significant differences between cases and controls were found for 22 of the analyzed proteins. The majority of the candidate proteins presented validated previous work, nevertheless, several proteins were identified with no previously known link to malaria pathogenesis. Proteins discriminating between mild and severe malaria infection were also identified, showing minor separation between the two sample groups.

 

In Paper IV, a focus group discussion of using POC tests with Ugandan health care personnel was conducted. Health care personnel at different levels were interviewed about their perception of using POC tests in the health care system, including strengths and weaknesses. This study was designed to bridge some of the knowledge gap between the POC test developers and end-users.

Place, publisher, year, edition, pages
Stockholm: KTH Royal Institute of Technology, 2018. p. 71
Series
TRITA-CBH-FOU ; 2018:5
National Category
Medical Biotechnology
Research subject
Biotechnology
Identifiers
urn:nbn:se:kth:diva-227159 (URN)978-91-7729-717-8 (ISBN)
Public defence
2018-05-25, Gardaulan, Folkhälsomyndigheten, Nobels väg 18, Solna, 10:00 (English)
Opponent
Funder
EU, European Research Council, 615458
Note

QC 20180503

Available from: 2018-05-03 Created: 2018-05-03 Last updated: 2018-05-03Bibliographically approved

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