Automated wash and reuse of disposable pipette tips in a SARS-CoV-2 RT-qPCR diagnostic pipeline.

The Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) pandemic led to global shortages in laboratory consumables, in particular for automated PCR. The Technical University of Denmark supported Danish hospitals from 2020 to 2022, conducting SARS-CoV-2 RT-qPCR on around 10,000 patient samples daily. We encountered shortages of disposable pipette tips used with automated liquid handlers that transferred oropharyngeal swab samples to 96-well microplates before RNA extraction. To enable tip reuse, we developed an automated protocol for washing tips with a 0.5 % sodium hypochlorite solution. This effectively eliminated carry-over of genomic material and the wash solution remained effective when stored in an open reservoir at ambient temperatures for 24 h. A three-day validation setup demonstrated the robustness of the tip wash protocol. Reducing the number of tips used for transferring samples to 96-well microplates from 96 to 8 enabled us to mitigate pipette tip shortages, lower costs, and minimize plastic waste generation.

A generic protocol to immobilize lipopolysaccharides on microbeads for multiplex analysis.

Bead-based multiplex serodiagnostics enables simultaneous analysis of antibodies against several antigens. Binding of the antigens onto the surface of the bead, preserving the antigenicity of the antigen is a pivotal step to ensure high sensitivity and selectivity of the assay. Here, a generic method for immobilization of lipopolysaccharide (LPS) antigens from different Gram-negative bacteria to microbeads using non-covalent conjugation has been developed and tested. The method involves coupling of N,N-diethylethylenediamine (DEDA) and derivatives to microbeads. This enhances non-covalent interactions so that LPS is easily immobilized. LPS antigens from the Gram-negative bacteria Actinobacillus pleuropneumoniae (APP) and Salmonella enterica serogroup B (Sal. B) were immobilized on the DEDA-coupled microbeads. In parallel, the same LPS antigens were coupled to beads using two previously reported methods. The performance of microbeads coupled with antigen using the different methods was compared by measuring antibodies in positive and negative serum samples from pigs. DEDA-beads coupled with LPS detected pathogen specific serum antibodies with equal or higher sensitivity and specificity compared to the other coupling methods used in this study. Furthermore, derivatives of DEDA, where the tertiary amine was alkylated with a methyl (m-DEDA) and ethyl group (e-DEDA) to give a positively charged tetraalkylammonium group, were compared with DEDA for the binding of LPS antigens. Here, it was concluded that the DEDA-modified bead was most efficient in the binding of LPS antigens from two Actinobacillus pleuropneumoniae serovars and Salmonella enterica serogroup B.

Plasmodium falciparum expressing domain cassette 5 type PfEMP1 (DC5-PfEMP1) bind PECAM1.

Members of the Plasmodium falciparum Erythrocyte Membrane protein 1 (PfEMP1) family expressed on the surface of malaria-infected erythrocytes mediate binding of the parasite to different receptors on the vascular lining. This process drives pathologies, and severe childhood malaria has been associated with the expression of particular subsets of PfEMP1 molecules. PfEMP1 are grouped into subtypes based on upstream sequences and the presence of semi-conserved PfEMP1 domain compositions named domain cassettes (DCs). Earlier studies have indicated that DC5-containing PfEMP1 (DC5-PfEMP1) are more likely to be expressed in children with severe malaria disease than in children with uncomplicated malaria, but these PfEMP1 subtypes only dominate in a relatively small proportion of the children with severe disease. In this study, we have characterised the genomic sequence characteristic for DC5, and show that two genetically different parasite lines expressing DC5-PfEMP1 bind PECAM1, and that anti-DC5-specific antibodies inhibit binding of DC5-PfEMP1-expressing parasites to transformed human bone marrow endothelial cells (TrHBMEC). We also show that antibodies against each of the four domains characteristic for DC5 react with native PfEMP1 expressed on the surface of infected erythrocytes, and that some of these antibodies are cross-reactive between the two DC5-containing PfEMP1 molecules tested. Finally, we confirm that anti-DC5 antibodies are acquired early in life by individuals living in malaria endemic areas, that individuals having high levels of these antibodies are less likely to develop febrile malaria episodes and that the antibody levels correlate positively with hemoglobin levels.

Severe malaria is associated with parasite binding to endothelial protein C receptor.

Sequestration of Plasmodium falciparum-infected erythrocytes in host blood vessels is a key triggering event in the pathogenesis of severe childhood malaria, which is responsible for about one million deaths every year. Sequestration is mediated by specific interactions between members of the P. falciparum erythrocyte membrane protein 1 (PfEMP1) family and receptors on the endothelial lining. Severe childhood malaria is associated with expression of specific PfEMP1 subtypes containing domain cassettes (DCs) 8 and 13 (ref. 3), but the endothelial receptor for parasites expressing these proteins was unknown. Here we identify endothelial protein C receptor (EPCR), which mediates the cytoprotective effects of activated protein C, as the endothelial receptor for DC8 and DC13 PfEMP1. We show that EPCR binding is mediated through the amino-terminal cysteine-rich interdomain region (CIDRα1) of DC8 and group A PfEMP1 subfamilies, and that CIDRα1 interferes with protein C binding to EPCR. This PfEMP1 adhesive property links P. falciparum cytoadhesion to a host receptor involved in anticoagulation and endothelial cytoprotective pathways, and has implications for understanding malaria pathology and the development of new malaria interventions.

Plasmodium falciparum erythrocyte membrane protein 1 domain cassettes 8 and 13 are associated with severe malaria in children.

The clinical outcome of Plasmodium falciparum infections ranges from asymptomatic parasitemia to severe malaria syndromes associated with high mortality. The virulence of P. falciparum infections is associated with the type of P. falciparum erythrocyte membrane protein 1 (PfEMP1) expressed on the surface of infected erythrocytes to anchor these to the vascular lining. Although var2csa, the var gene encoding the PfEMP1 associated with placental malaria, was discovered in 2003, the identification of the var/PfEMP1 variants associated with severe malaria in children has remained elusive. To identify var/PfEMP1 variants associated with severe disease outcome, we compared var transcript levels in parasites from 88 children with severe malaria and 40 children admitted to the hospital with uncomplicated malaria. Transcript analysis was performed by RT-quantitative PCR using a set of 42 primer pairs amplifying var subtype-specific loci covering most var/PfEMP1 subtypes. In addition, we characterized the near-full-length sequence of the most prominently expressed var genes in three patients diagnosed with severe anemia and/or cerebral malaria. The combined analysis showed that severe malaria syndromes, including severe anemia and cerebral malaria, are associated with high transcript levels of PfEMP1 domain cassette 8-encoding var genes. Transcript levels of group A var genes, including genes encoding domain cassette 13, were also significantly higher in patients with severe syndromes compared with those with uncomplicated malaria. This study specifies the var/PfEMP1 types expressed in severe malaria in children, and thereby provides unique targets for future efforts to prevent and treat severe malaria infections.

Evidence for in vitro and in vivo expression of the conserved VAR3 (type 3) plasmodium falciparum erythrocyte membrane protein 1.

BACKGROUND: Members of the Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) adhesion antigen family are major contributors to the pathogenesis of P. falciparum malaria infections. The PfEMP1-encoding var genes are among the most diverse sequences in nature, but three genes, var1, var2csa and var3 are found conserved in most parasite genomes. The most severe forms of malaria disease are caused by parasites expressing a subset of antigenically conserved PfEMP1 variants. Thus the ubiquitous and conserved VAR3 PfEMP1 is of particular interest to the research field. Evidence of VAR3 expression on the infected erythrocyte surface has never been presented, and var3 genes have been proposed to be transcribed and expressed differently from the rest of the var gene family members. METHODS: In this study, parasites expressing VAR3 PfEMP1 were generated using anti-VAR3 antibodies and the var transcript and PfEMP1 expression profiles of the generated parasites were investigated. The IgG reactivity by plasma from children living in malaria-endemic Tanzania was tested to parasites and recombinant VAR3 protein. Parasites from hospitalized children were isolated and the transcript level of var3 was investigated. RESULTS: Var3 is transcribed and its protein product expressed on the surface of infected erythrocytes. The VAR3-expressing parasites were better recognized by children´s IgG than a parasite line expressing a Group B var gene. Two in 130 children showed increased recognition of parasites expressing VAR3 and to the recombinant VAR3 protein after a malaria episode and the isolated parasites showed high levels of var3 transcripts. CONCLUSIONS: Collectively, the presented data suggest that var3 is transcribed and its protein product expressed on the surface of infected erythrocytes in the same manner as seen for other var genes both in vitro and in vivo. Only very few children exhibit seroconversion to VAR3 following a malaria episode requiring hospitalization, supporting the previous conclusion drawn from var3 transcript analysis of parasites collected from children hospitalized with malaria, that VAR3 is not associated with severe anaemia or cerebral malaria syndromes in children.

Surface co-expression of two different PfEMP1 antigens on single plasmodium falciparum-infected erythrocytes facilitates binding to ICAM1 and PECAM1.

The Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) antigens play a major role in cytoadhesion of infected erythrocytes (IE), antigenic variation, and immunity to malaria. The current consensus on control of variant surface antigen expression is that only one PfEMP1 encoded by one var gene is expressed per cell at a time. We measured var mRNA transcript levels by real-time Q-PCR, analysed var gene transcripts by single-cell FISH and directly compared these with PfEMP1 antigen surface expression and cytoadhesion in three different antibody-selected P. falciparum 3D7 sub-lines using live confocal microscopy, flow cytometry and in vitro adhesion assays. We found that one selected parasite sub-line simultaneously expressed two different var genes as surface antigens, on single IE. Importantly, and of physiological relevance to adhesion and malaria pathogenesis, this parasite sub-line was found to bind both CD31/PECAM1 and CD54/ICAM1 and to adhere twice as efficiently to human endothelial cells, compared to infected cells having only one PfEMP1 variant on the surface. These new results on PfEMP1 antigen expression indicate that a re-evaluation of the molecular mechanisms involved in P. falciparum adhesion and of the accepted paradigm of absolutely mutually exclusive var gene transcription is required.