Parasite Viability as a Superior Measure of Antimalarial Drug Activity in Humans.

BACKGROUND: Artemisinin derivatives are the leading class of antimalarial drugs due to their rapid onset of action and rapid clearance of circulating parasites. The parasite clearance half-life measures the rate of loss of parasites from blood after treatment, and this is currently used to assess antimalarial activity of novel agents and to monitor resistance. However, a number of recent studies have challenged the use of parasite clearance to measure drug activity, arguing that many circulating parasites may be nonviable. METHODS: Plasmodium falciparum-infected subjects (n = 10) in a malaria volunteer infection study were administered a single dose of artesunate (2 mg/kg). Circulating parasite concentration was assessed by means of quantitative polymerase chain reaction (qPCR). Parasite viability after artesunate administration was estimated by mathematical modeling of the ex vivo growth of parasites collected from subjects. RESULTS: We showed that in artemisinin-sensitive infection, viable parasites declined to <0.1% of baseline within 8 hours after artesunate administration, while the total number of circulating parasites measured with quantitative polymerase chain reaction remained unchanged. In artemisinin-resistant infections over the same interval, viable parasites declined to 51.4% (standard error of the mean, 4.6%) of baseline. CONCLUSIONS: These results demonstrate that in vivo drug activity of artesunate is faster than is indicated by the parasite clearance half-life.

Safety and feasibility of apheresis to harvest and concentrate parasites from subjects with induced blood stage Plasmodium vivax infection.

BACKGROUND: In the absence of a method to culture Plasmodium vivax, the only way to source parasites is ex vivo. This hampers many aspects of P. vivax research. This study aimed to assess the safety of apheresis, a method for selective removal of specific components of blood as a means of extracting and concentrating P. vivax parasites. METHODS: An iterative approach was employed across four non-immune healthy human subjects in single subject cohorts. All four subjects were inoculated with ~ 564 blood stage P. vivax (HMP013-Pv) and subjected to apheresis 10 to 11 days later. Blood samples collected during apheresis (haematocrit layers 0.5% to 11%) were tested for the presence and concentration of P. vivax by microscopy, flow cytometry, 18S rDNA qPCR for total parasites, and pvs25 qRT-PCR for female gametocyte transcripts. Safety was determined by monitoring adverse events. Malaria transmission to mosquitoes was assessed by membrane feeding assays. RESULTS: There were no serious adverse events and no significant safety concerns. Apheresis concentrated asexual parasites by up to 4.9-fold (range: 0.9-4.9-fold) and gametocytes by up to 1.45-fold (range: 0.38-1.45-fold) compared to pre-apheresis densities. No single haematocrit layer contained > 40% of all the recovered P. vivax asexual parasites. Ex vivo concentration of parasites by Percoll gradient centrifugation of whole blood achieved greater concentration of gametocytes than apheresis. Mosquito transmission was enhanced by up to fivefold in a single apheresis sample compared to pre-apheresis. CONCLUSION: The modest level of parasite concentration suggests that the use of apheresis may not be an ideal method for harvesting P. vivax. Trial Registration Australia New Zealand Clinical Trials Registry (ANZCTR) Trial ID: ACTRN12617001502325 registered on 19th October 2017. https://www.anzctr.org.au/Trial/Registration/TrialReview.aspx?id=373812.

Safety and parasite clearance of artemisinin-resistant Plasmodium falciparum infection: A pilot and a randomised volunteer infection study in Australia.

BACKGROUND: Artemisinin resistance is threatening malaria control. We aimed to develop and test a human model of artemisinin-resistant (ART-R) Plasmodium falciparum to evaluate the efficacy of drugs against ART-R malaria. METHODS AND FINDINGS: We conducted 2 sequential phase 1, single-centre, open-label clinical trials at Q-Pharm, Brisbane, Australia, using the induced blood-stage malaria (IBSM) model, whereby healthy participants are intravenously inoculated with blood-stage parasites. In a pilot study, participants were inoculated (Day 0) with approximately 2,800 viable P. falciparum ART-R parasites. In a comparative study, participants were randomised to receive approximately 2,800 viable P. falciparum ART-R (Day 0) or artemisinin-sensitive (ART-S) parasites (Day 1). In both studies, participants were administered a single approximately 2 mg/kg oral dose of artesunate (AS; Day 9). Primary outcomes were safety, ART-R parasite infectivity, and parasite clearance. In the pilot study, 2 participants were enrolled between April 27, 2017, and September 12, 2017, and included in final analyses (males n = 2 [100%], mean age = 26 years [range, 23-28 years]). In the comparative study, 25 participants were enrolled between October 26, 2017, and October 18, 2018, of whom 22 were inoculated and included in final analyses (ART-R infected participants: males n = 7 [53.8%], median age = 22 years [range, 18-40 years]; ART-S infected participants: males n = 5 [55.6%], median age = 28 years [range, 22-35 years]). In both studies, all participants inoculated with ART-R parasites became parasitaemic. A total of 36 adverse events were reported in the pilot study and 277 in the comparative study. Common adverse events in both studies included headache, pyrexia, myalgia, nausea, and chills; none were serious. Seven participants experienced transient severe falls in white cell counts and/or elevations in liver transaminase levels which were considered related to malaria. Additionally, 2 participants developed ventricular extrasystoles that were attributed to unmasking of a predisposition to benign fever-induced tachyarrhythmia. In the comparative study, parasite clearance half-life after AS was significantly longer for ART-R infected participants (n = 13, 6.5 hours; 95% confidence interval [CI] 6.3-6.7 hours) compared with ART-S infected participants (n = 9, 3.2 hours; 95% CI 3.0-3.3 hours; p < 0.001). The main limitation of this study was that the ART-R and ART-S parasite strains did not share the same genetic background. CONCLUSIONS: We developed the first (to our knowledge) human model of ART-R malaria. The delayed clearance profile of ART-R parasites after AS aligns with field study observations. Although based on a relatively small sample size, results indicate that this model can be safely used to assess new drugs against ART-R P. falciparum. TRIAL REGISTRATION: The studies were registered with the Australian New Zealand Clinical Trials Registry: ACTRN12617000244303 (https://www.anzctr.org.au/Trial/Registration/TrialReview.aspx?id=372357) and ACTRN12617001394336 (https://www.anzctr.org.au/Trial/Registration/TrialReview.aspx?id=373637).

Molecular analysis of asymptomatic bacteriuria Escherichia coli strain VR50 reveals adaptation to the urinary tract by gene acquisition.

Urinary tract infections (UTIs) are among the most common infectious diseases of humans, with Escherichia coli responsible for >80% of all cases. One extreme of UTI is asymptomatic bacteriuria (ABU), which occurs as an asymptomatic carrier state that resembles commensalism. To understand the evolution and molecular mechanisms that underpin ABU, the genome of the ABU E. coli strain VR50 was sequenced. Analysis of the complete genome indicated that it most resembles E. coli K-12, with the addition of a 94-kb genomic island (GI-VR50-pheV), eight prophages, and multiple plasmids. GI-VR50-pheV has a mosaic structure and contains genes encoding a number of UTI-associated virulence factors, namely, Afa (afimbrial adhesin), two autotransporter proteins (Ag43 and Sat), and aerobactin. We demonstrated that the presence of this island in VR50 confers its ability to colonize the murine bladder, as a VR50 mutant with GI-VR50-pheV deleted was attenuated in a mouse model of UTI in vivo. We established that Afa is the island-encoded factor responsible for this phenotype using two independent deletion (Afa operon and AfaE adhesin) mutants. E. coli VR50afa and VR50afaE displayed significantly decreased ability to adhere to human bladder epithelial cells. In the mouse model of UTI, VR50afa and VR50afaE displayed reduced bladder colonization compared to wild-type VR50, similar to the colonization level of the GI-VR50-pheV mutant. Our study suggests that E. coli VR50 is a commensal-like strain that has acquired fitness factors that facilitate colonization of the human bladder.

An antioxidant role for catecholate siderophores in Salmonella.

Iron acquisition is an important aspect of the host-pathogen interaction. In the case of Salmonella it is established that catecholate siderophores are important for full virulence. In view of their very high affinity for ferric iron, functional studies of siderophores have been almost exclusively focused on their role in acquisition of iron from the host. In the present study, we investigated whether the siderophores (enterobactin and salmochelin) produced by Salmonella enterica sv. Typhimurium could act as antioxidants and protect from the oxidative stress encountered after macrophage invasion. Our results show that the ability to produce siderophores enhanced the survival of Salmonella in the macrophage mainly at the early stages of infection, coincident with the oxidative burst. Using siderophore biosynthetic and siderophore receptor mutants we demonstrated that salmochelin and enterobactin protect S. Typhimurium against ROS (reactive oxygen species) in vitro and that siderophores must be intracellular to confer full protection. We also investigated whether other chemically distinct siderophores (yersiniabactin and aerobactin) or the monomeric catechol 2,3-dihydroxybenzoate could provide protection against oxidative stress and found that only catecholate siderophores have this property. Collectively, the results of the present study identify additional functions for siderophores during host-pathogen interactions.

Escherichia coli 83972 expressing a P fimbriae oligosaccharide receptor mimic impairs adhesion of uropathogenic E. coli.

Urinary tract infections (UTIs) caused by uropathogenic Escherichia coli (UPEC) are a significant health concern, exacerbated by the rapid emergence of multidrug resistant strains refractory to antibiotic treatment. P fimbriae are strongly associated with upper urinary tract colonization due to specific binding to α-D-galactopyranosyl-(1-4)-ß-D-galactopyranoside receptors in the kidneys. Thus, inhibiting P-fimbrial adhesion may reduce the incidence of UPEC-mediated UTI. E. coli 83972 is an asymptomatic bacteriuria isolate successfully used as a prophylactic agent to prevent UTI in human studies. We constructed a recombinant E. coli 83972 strain displaying a surface-located oligosaccharide P fimbriae receptor mimic that bound to P-fimbriated E. coli producing any of the 3 PapG adhesin variants. The recombinant strain, E. coli 83972::lgtCE, impaired P fimbriae-mediated adhesion to human erythrocytes and kidney epithelial cells. Additionally, E. coli 83972::lgtCE impaired urine colonization by UPEC in a mouse UTI model, demonstrating its potential as a prophylactic agent to prevent UTI.

Contribution of siderophore systems to growth and urinary tract colonization of asymptomatic bacteriuria Escherichia coli.

The molecular mechanisms that define asymptomatic bacteriuria (ABU) Escherichia coli colonization of the human urinary tract remain to be properly elucidated. Here, we utilize ABU E. coli strain 83972 as a model to dissect the contribution of siderophores to iron acquisition, growth, fitness, and colonization of the urinary tract. We show that E. coli 83972 produces enterobactin, salmochelin, aerobactin, and yersiniabactin and examine the role of these systems using mutants defective in siderophore biosynthesis and uptake. Enterobactin and aerobactin contributed most to total siderophore activity and growth in defined iron-deficient medium. No siderophores were detected in an 83972 quadruple mutant deficient in all four siderophore biosynthesis pathways; this mutant did not grow in defined iron-deficient medium but grew in iron-limited pooled human urine due to iron uptake via the FecA ferric citrate receptor. In a mixed 1:1 growth assay with strain 83972, there was no fitness disadvantage of the 83972 quadruple biosynthetic mutant, demonstrating its capacity to act as a "cheater" and utilize siderophores produced by the wild-type strain for iron uptake. An 83972 enterobactin/salmochelin double receptor mutant was outcompeted by 83972 in human urine and the mouse urinary tract, indicating a role for catecholate receptors in urinary tract colonization.

Escherichia coli isolates causing asymptomatic bacteriuria in catheterized and noncatheterized individuals possess similar virulence properties.

Urinary tract infections (UTIs) are among the most common infectious diseases of humans, with Escherichia coli being responsible for >80% of all cases. Asymptomatic bacteriuria (ABU) occurs when bacteria colonize the urinary tract without causing clinical symptoms and can affect both catheterized patients (catheter-associated ABU [CA-ABU]) and noncatheterized patients. Here, we compared the virulence properties of a collection of ABU and CA-ABU nosocomial E. coli isolates in terms of antibiotic resistance, phylogenetic grouping, specific UTI-associated virulence genes, hemagglutination characteristics, and biofilm formation. CA-ABU isolates were similar to ABU isolates with regard to the majority of these characteristics; exceptions were that CA-ABU isolates had a higher prevalence of the polysaccharide capsule marker genes kpsMT II and kpsMT K1, while more ABU strains were capable of mannose-resistant hemagglutination. To examine biofilm growth in detail, we performed a global gene expression analysis with two CA-ABU strains that formed a strong biofilm and that possessed a limited adhesin repertoire. The gene expression profile of the CA-ABU strains during biofilm growth showed considerable overlap with that previously described for the prototype ABU E. coli strain, 83972. This is the first global gene expression analysis of E. coli CA-ABU strains. Overall, our data suggest that nosocomial ABU and CA-ABU E. coli isolates possess similar virulence profiles.

Virulence properties of asymptomatic bacteriuria Escherichia coli.

In asymptomatic bacteriuria (ABU), bacteria colonize the urinary tract without provoking symptoms. Here, we compared the virulence properties of a collection of ABU Escherichia coli strains to cystitis and pyelonephritis strains. Specific urinary tract infection (UTI)-associated virulence genes, hemagglutination characteristics, siderophore production, hemolysis, biofilm formation, and the ability of strains to adhere to and induce cytokine responses in epithelial cells were analyzed. ABU strains were phylogenetically related to strains that cause symptomatic UTI. However, the virulence properties of the ABU strains were variable and dependent on a combination of genotypic and phenotypic factors. Most ABU strains adhered poorly to epithelial cells; however, we also identified a subgroup of strongly adherent strains that were unable to stimulate an epithelial cell IL-6 cytokine response. Poor immune activation may represent one mechanism whereby ABU E. coli evade immune detection after the establishment of bacteriuria.