BackgroundSequestration of parasitized red blood cells from the peripheral circulation during an infection with Plasmodium falciparum is caused by an interaction between the parasite protein PfEMP1 and receptors on the surface of host endothelial cells, known as cytoadherence. Several lines of evidence point to a link between the pathology of severe malaria and cytoadherence, therefore blocking adhesion receptors involved in this process could be a good target to inhibit pRBC sequestration and prevent disease. In a malaria endemic setting this is likely to be used as an adjunct therapy by reversing existing cytoadherence. Two well-characterized parasite lines plus three recently derived patient isolates were tested for their cytoadherence to purified receptors (CD36 and ICAM-1) as well as endothelial cells. Monoclonal antibodies against human CD36 and ICAM-1 were used to inhibit and reverse infected erythrocyte binding in static and flow-based adhesion assays.ResultsAnti-ICAM-1 and CD36 monoclonal antibodies were able to inhibit and reverse P. falciparum binding of lab and recently adapted patient isolates in vitro. However, reversal of binding was incomplete and varied in its efficiency between parasite isolates.ConclusionsThe results show that, as a proof of concept, disturbing existing ligand–receptor interactions is possible and could have potential therapeutic value for severe malaria. The variation seen in the degree of reversing existing binding with different parasite isolates and the incomplete nature of reversal, despite the use of high affinity inhibitors, suggest that anti-adhesion approaches as adjunct therapies for severe malaria may not be effective, and the focus may need to be on inhibitory approaches such as vaccines.

BackgroundThe ability of mature forms of Plasmodium falciparum infected erythrocytes to bind to a range of host receptors including those displayed on endothelial cells has been associated with the pathology of this infection. Investigations into this adhesive phenomenon have used protein and cell-based adhesion assays to quantify the ability of infected red blood cells to bind. These adhesion assays tend to have relatively high inherent variability and so require multiple experiments in order to provide good quantitation. This means that investigators doing these experiments must count many fields of adherent parasites, a task that is time-consuming and laborious. To address this issue and to facilitate cytoadherence research, developed automated protocols were developed for counting parasite adhesion.MethodsParasite adhesion assays were mainly carried out under static conditions using purified receptors, which is the simplest form of these assays and is translatable to the field. Two different software platforms were used, one commercial (Image Pro-Plus (Media Cybernetics)) and one available in the public domain (ImageSXM) based on the freely available NIH Image software. The adhesion assays were performed and parasite binding quantified using standard manual techniques. Images were also captured using video microscopy and analysed using the two automated systems. The results generated by each system were compared using the Bland and Altman method for assessing the agreement between two methods.ResultsBoth automated counting programs showed concordance compared to the 'gold standard' manual counting within the normal range of adhesion seen with these assays, although the ImageSXM technique had some systematic bias. There was some fall-off in accuracy at very high parasite densities, but this can be resolved through good design of the experiments. Cell based assays were also used as inputs to one of the automated systems (ImageSXM) and produced variable, but encouraging, results.ConclusionsThe automated counting programs are an accurate and practical way of quantifying static parasite binding assays to purified proteins. They are less accurate when applied to cell based systems, but can still provide a reasonable level of accuracy to give a semi-quantitative readout.

A feature of mature Plasmodium falciparum parasitized red blood cells is their ability to bind surface molecules of the microvascular endothelium via the parasite-derived surface protein Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1). This ligand is associated with the cytoadherence pathology observed in severe malaria. As pRBC treated with effective anti-malarial drugs are still able to cytoadhere, there is therefore a need to find an adjunct treatment that can inhibit and reverse the adhesion process. One semi-synthetic, sulfated polysaccharide has been identified that is capable of inhibiting and reversing sequestration of pRBC on endothelial cells in vitro under physiological flow conditions. Furthermore, it exhibits low toxicity in the intrinsic (APTT assay) and extrinsic (PT assay) clotting pathways, as well as exhibiting minimal effects on cell (HUVEC) viability (MTT proliferation assay). These findings suggest that carbohydrate-based anti-adhesive candidates may provide potential leads for therapeutics for severe malaria.

Dibutyl phthalate (DBP) produced by Streptomyces sp. H11809 exerted inhibitory activity against human GSK-3β (Hs GSK-3β) and Plasmodiumfalciparum 3D7 (Pf 3D7) malaria parasites. The current study aimed to determine DBP’s plausible mode of action against Hs GSK-3β and Pf 3D7. Molecular docking analysis indicated that DBP has a higher binding affinity to the substrate-binding site (pocket 2; −6.9 kcal/mol) than the ATP-binding site (pocket 1; −6.1 kcal/mol) of Hs GSK-3β. It was suggested that the esters of DBP play a pivotal role in the inhibition of Hs GSK-3β through the formation of hydrogen bonds with Arg96/Glu97 amino acid residues in pocket 2. Subsequently, an in vitro Hs GSK-3β enzymatic assay revealed that DBP inhibits the activity of Hs GSK-3β via mixed inhibition inhibitory mechanisms, with a moderate IC50 of 2.0 µM. Furthermore, the decrease in Km value with an increasing DBP concentration suggested that DBP favors binding on free Hs GSK-3β over its substrate-bound state. However, the antimalarial mode of action of DBP remains unknown since the generation of a Pf 3D7 DBP-resistant clone was not successful. Thus, the molecular target of DBP might be indispensable for Pf survival. We also identified nocardamine as another active compound from Streptomyces sp. H11809 chloroform extract. It showed potent antimalarial activity with an IC50 of 1.5 μM, which is ~10-fold more potent than DBP, but with no effect on Hs GSK-3β. The addition of ≥12.5 µM ferric ions into the Pf culture reduced nocardamine antimalarial activity by 90% under in vitro settings. Hence, the iron-chelating ability of nocardamine was shown to starve the parasites from their iron source, eventually inhibiting their growth.

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Melioidosis is a severe disease caused by Burkholderia pseudomallei (B. pseudomallei), a Gram-negative environmental bacterium. It is endemic in Southeast Asia and Northern Australia, but it is underreported in many other countries. The principal routes of entry for B. pseudomallei are skin penetration, inhalation, and ingestion. It mainly affects immunocompromised populations, especially patients with type 2 diabetes mellitus. The laboratory diagnosis of melioidosis is challenging due to its non-specific clinical manifestations, which mimic other severe infections. The culture method is considered an imperfect gold standard for the diagnosis of melioidosis due to its low sensitivity. Antibody detection has low sensitivity and specificity due to the high seropositivity among healthy people in endemic regions. Antigen detection using various proteins has been tested for the rapid determination of B. pseudomallei; however, it presents certain limitations in terms of its sensitivity and specificity. Therefore, this review aims to frame the present knowledge of a potential target known as the Burkholderia invasion protein D (BipD), including future directions for its detection using an aptamer-based sensor (aptasensor).

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Typhoid fever, also known as typhoid, is a life-threatening bacterial infection that remains a global health concern. The infection is associated with a significant morbidity and mortality rate, resulting in an urgent need for specific and rapid detection tests to aid prevention and management of the disease. The present review aims to assess the specificity and sensitivity of the available literature on the immunodiagnostics of typhoid fever. A literature search was conducted using three databases (PubMed, ProQuest and Scopus) and manual searches through the references of identified full texts to retrieve relevant literature published between 1 January 2011 and 31 December 2020. Of the 577 studies identified in our search, 12 were included in further analysis. Lipopolysaccharides (LPS) and hemolysin E (HlyE) were the most frequently studied antigens. The specimens examined in these studies included serum and saliva. Using blood culture as the gold standard, anti-LPS IgA gave the highest sensitivity of 96% (95% CI: 93–99) and specificity of 96% (95% CI: 93–99) for distinguishing between typhoid cases and healthy controls, whereas the combination of anti-LPS and anti-flagellin total IgGAM gave the highest sensitivity of 93% (95% CI: 86–99) and specificity of 95% (95% CI: 89–100) for distinguishing typhoid cases and other febrile infections. A comparably high sensitivity of 92% (95% CI: 86–98) and specificity of 89% (95% CI: 78–100) were shown in testing based on detection of the combination of anti-LPS (IgA and IgM) and anti-HlyE IgG as well as a slightly lower sensitivity of 91% (95% CI: 74–100) in the case of anti-50kDa IgA. Anti-50kDa IgM had the lowest sensitivity of 36% (95% CI: 6–65) against both healthy and febrile controls. The development of a rapid diagnostic test targeting antibodies against lipopolysaccharides combined with flagellin appeared to be a suitable approach for the rapid detection test of typhoid fever. Saliva is added benefit for rapid typhoid diagnosis since it is less invasive. As a result, further studies could be done to develop additional approaches for adopting such samples.