A G358S mutation in the Plasmodium falciparum Na⁺ pump PfATP4 confers clinically-relevant resistance to cipargamin

Diverse compounds target the Plasmodium falciparum Na(+) pump PfATP4, with cipargamin and (+)-SJ733 the most clinically-advanced. In a recent clinical trial for cipargamin, recrudescent parasites emerged, with most having a G358S mutation in PfATP4. Here, we show that PfATP4(G358S) parasites can withstand micromolar concentrations of cipargamin and (+)-SJ733, while remaining susceptible to antimalarials that do not target PfATP4. The G358S mutation in PfATP4, and the equivalent mutation in Toxoplasma gondii ATP4, decrease the sensitivity of ATP4 to inhibition by cipargamin and (+)-SJ733, thereby protecting parasites from disruption of Na(+) regulation. The G358S mutation reduces the affinity of PfATP4 for Na(+) and is associated with an increase in the parasite’s resting cytosolic [Na(+)]. However, no defect in parasite growth or transmissibility is observed. Our findings suggest that PfATP4 inhibitors in clinical development should be tested against PfATP4(G358S) parasites, and that their combination with unrelated antimalarials may mitigate against resistance development

Influence of fecal collection conditions and 16S rRNA gene sequencing at two centers on human gut microbiota analysis

To optimise fecal sampling for reproducible analysis of the gut microbiome, we compared different methods of sample collection and sequencing of 16S rRNA genes at two centers. Samples collected from six individuals on three consecutive days were placed in commercial collection tubes (OMNIgeneGut OMR-200) or in sterile screw-top tubes in a home fridge or home freezer for 6-24 h, before transfer and storage at-80 °C. Replicate samples were shipped to centers in Australia and the USA for DNA extraction and sequencing by their respective PCR protocols, and analysed with the same bioinformatic pipeline. Variation in gut microbiome was dominated by differences between individuals. Minor differences in the abundance of taxa were found between collection-processing methods and day of collection, and between the two centers. We conclude that collection with storage and transport at 4 °C within 24 h is adequate for 16S rRNA analysis of the gut microbiome. Other factors including differences in PCR and sequencing methods account for relatively minor variation compared to differences between individuals

Safety, infectivity and immunogenicity of a genetically attenuated blood-stage malaria vaccine

Background There is a clear need for novel approaches to malaria vaccine development. We aimed to develop a genetically attenuated blood-stage vaccine and test its safety, infectivity, and immunogenicity in healthy volunteers. Our approach was to target the gene encoding the knob-associated histidine-rich protein (KAHRP), which is responsible for the assembly of knob structures at the infected erythrocyte surface. Knobs are required for correct display of the polymorphic adhesion ligand P. falciparum erythrocyte membrane protein 1 (PfEMP1), a key virulence determinant encoded by a repertoire of var genes. Methods The gene encoding KAHRP was deleted from P. falciparum 3D7 and a master cell bank was produced in accordance with Good Manufacturing Practice. Eight malaria naïve males were intravenously inoculated (day 0) with 1800 (2 subjects), 1.8 × 105 (2 subjects), or 3 × 106 viable parasites (4 subjects). Parasitemia was measured using qPCR; immunogenicity was determined using standard assays. Parasites were rescued into culture for in vitro analyses (genome sequencing, cytoadhesion assays, scanning electron microscopy, var gene expression). Results None of the subjects who were administered with 1800 or 1.8 × 105 parasites developed parasitemia; 3/4 subjects administered 3× 106 parasites developed significant parasitemia, first detected on days 13, 18, and 22. One of these three subjects developed symptoms of malaria simultaneously with influenza B (day 17; 14,022 parasites/mL); one subject developed mild symptoms on day 28 (19,956 parasites/mL); and one subject remained asymptomatic up to day 35 (5046 parasites/mL). Parasitemia rapidly cleared with artemether/lumefantrine. Parasitemia induced a parasite-specific antibody and cell-mediated immune response. Parasites cultured ex vivo exhibited genotypic and phenotypic properties similar to inoculated parasites, although the var gene expression profile changed during growth in vivo. Conclusions This study represents the first clinical investigation of a genetically attenuated blood-stage human malaria vaccine. A P. falciparum 3D7 kahrp– strain was tested in vivo and found to be immunogenic but can lead to patent parasitemia at high doses

Exploration and characterization of the antimalarial activity of cyclopropyl carboxamides that target the mitochondrial protein, cytochrome b.

Drug resistance against antimalarials is rendering them increasingly ineffective and so there is a need for the development of new antimalarials. To discover new antimalarial chemotypes a phenotypic screen of the Janssen Jumpstarter library against the P. falciparum asexual stage was undertaken, uncovering the cyclopropyl carboxamide structural hit class. Structure-activity analysis revealed that each structural moiety was largely resistant to change, although small changes led to the frontrunner compound, WJM280, which has potent asexual stage activity (EC50 40 nM) and no human cell cytotoxicity. Forward genetics uncovered that cyclopropyl carboxamide resistant parasites have mutations and an amplification in the cytochrome b gene. Cytochrome b was then verified as the target with profiling against cytochrome b drug-resistant parasites and a mitochondrial oxygen consumption assay. Accordingly, the cyclopropyl carboxamide class was shown to have slow-acting asexual stage activity and activity against male gametes and exoerythrocytic forms. Enhancing metabolic stability to attain efficacy in malaria mouse models remains a challenge in the future development of this antimalarial chemotype

Adverse effects of isoniazid preventative therapy for latent tuberculosis infection: a prospective cohort study

Introduction: Isoniazid preventative therapy (IPT) is a widely used intervention for treatment of latent tuberculosis infection (LTBI), particularly in patients at high risk for reactivation. While treatment-limiting adverse effects have been well studied, few prospective studies have considered the range of adverse effects that patients may experience with IPT.\ud \ud Methods: All patients commencing treatment for LTBI were prospectively enrolled in an ongoing database of LTBI treatment outcomes particularly related to adverse effects, treatment adherence, and treatment completion.\ud \ud Results: Data on the first 100 patients who were prescribed IPT are presented. Fifty-six patients reported at least one adverse effect at some stage during treatment, with six experiencing at least one World Health Organization (WHO) Grade 3–4 adverse effect. Increased age was significantly associated with risk of adverse effects (odds ratio [OR] =1.05 per year; confidence interval [CI] of 1.02–1.08=95%). Eighty-five patients had documented completion of therapy locally, with ten patients ceasing IPT due to adverse effects.\ud \ud Discussion: This report highlights a variety of somatic adverse effects that occurred in a real-world cohort of patients receiving IPT. While adverse effects were frequently identified in this study, the considerable majority were low grade and transient. Despite frequent adverse effects of LTBI in our treatment cohort, the study demonstrated high levels of treatment adherence and completion

Have it at 8 a.m.! This comic was drawn using Comix I/O (http://cmx.io/).

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Journal club attendance over time versus reading alone.

<p>Journal club attendance over time versus reading alone.</p

How to find contentious articles.

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Why our journal club beats a “normal” journal club.

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