Dual-Luciferase-Based Fast and Sensitive Detection of Malaria Hypnozoites for the Discovery of Antirelapse Compounds.

Efforts to eradicate Plasmodium vivax malaria are hampered by the presence of hypnozoites, persisting stages in the liver that can reactivate after prolonged periods of time enabling further transmission and causing renewed disease. Large-scale drug screening is needed to identify compounds with antihypnozoite activity, but current platforms rely on time-consuming high-content fluorescence imaging as read-out, limiting assay throughput. We here report an ultrafast and sensitive dual-luciferase-based method to differentiate hypnozoites from liver stage schizonts using a transgenic P. cynomolgi parasite line that contains Nanoluc driven by the constitutive hsp70 promoter, as well as firefly luciferase driven by the schizont-specific lisp2 promoter. The transgenic parasite line showed similar fitness and drug sensitivity profiles of selected compounds to wild type. We demonstrate robust bioluminescence-based detection of hypnozoites in 96-well and 384-well plate formats, setting the stage for implementation in large scale drug screens.

A dual fluorescent Plasmodium cynomolgi reporter line reveals in vitro malaria hypnozoite reactivation.

Plasmodium vivax malaria is characterized by repeated episodes of blood stage infection (relapses) resulting from activation of dormant stages in the liver, so-called hypnozoites. Transition of hypnozoites into developing schizonts has never been observed. A barrier for studying this has been the lack of a system in which to monitor growth of liver stages. Here, exploiting the unique strengths of the simian hypnozoite model P. cynomolgi, we have developed green-fluorescent (GFP) hypnozoites that turn on red-fluorescent (mCherry) upon activation. The transgenic parasites show full liver stage development, including merozoite release and red blood cell infection. We demonstrate that individual hypnozoites actually can activate and resume development after prolonged culture, providing the last missing evidence of the hypnozoite theory of relapse. The few events identified indicate that hypnozoite activation in vitro is infrequent. This system will further our understanding of the mechanisms of hypnozoite activation and may facilitate drug discovery approaches.

Transcriptomic analysis reveals reduced transcriptional activity in the malaria parasite Plasmodium cynomolgi during progression into dormancy.

Relapses of Plasmodium dormant liver hypnozoites compromise malaria eradication efforts. New radical cure drugs are urgently needed, yet the vast gap in knowledge of hypnozoite biology impedes drug discovery. We previously unraveled the transcriptome of 6 to 7 day-old P. cynomolgi liver stages, highlighting pathways associated with hypnozoite dormancy (Voorberg-van der Wel et al., 2017). We now extend these findings by transcriptome profiling of 9 to 10 day-old liver stage parasites, thus revealing for the first time the maturation of the dormant stage over time. Although progression of dormancy leads to a 10-fold decrease in transcription and expression of only 840 genes, including genes associated with housekeeping functions, we show that pathways involved in quiescence, energy metabolism and maintenance of genome integrity remain the prevalent pathways active in mature hypnozoites.

PI4 Kinase Is a Prophylactic but Not Radical Curative Target in Plasmodium vivax-Type Malaria Parasites.

Two Plasmodium PI4 kinase (PI4K) inhibitors, KDU691 and LMV599, were selected for in vivo testing as causal prophylactic and radical-cure agents for Plasmodium cynomolgi sporozoite-infected rhesus macaques, based on their in vitro activity against liver stages. Animals were infected with P. cynomolgi sporozoites, and compounds were dosed orally. Both the KDU691 and LMV599 compounds were fully protective when administered prophylactically, and the more potent compound LMV599 achieved protection as a single oral dose of 25 mg/kg of body weight. In contrast, when tested for radical cure, five daily doses of 20 mg/kg of KDU691 or 25 mg/kg of LMV599 did not prevent relapse, as all animals experienced a secondary infection due to the reactivation of hypnozoites in the liver. Pharmacokinetic data show that LMV599 achieved plasma exposure that was sufficient to achieve efficacy based on our in vitro data. These findings indicate that Plasmodium PI4K is a potential drug target for malaria prophylaxis but not radical cure. Longer in vitro culture systems will be required to assess these compounds' activity on established hypnozoites and predict radical cure in vivo.

Psychometric properties of self-sufficiency assessment tools in adolescents in vocational education.

BACKGROUND: Self-sufficiency is the realisation of an acceptable level of functioning either by the person him/herself or through the adequate organisation of help from informal or formal care providers. Assessment of self-sufficiency for determining an individual's functional strengths and areas for improvement is increasingly being applied among adolescents in vocational education, a group considered vulnerable with high school dropout rates and often characterised by an accumulation of problems. This study examined the psychometric properties of two instruments, i.e. a self-report questionnaire assessing self-sufficiency and the Self-Sufficiency Matrix for professionals (SSM-D) conducted among adolescents in vocational education. METHODS: The self-report questionnaire used to assess self-sufficiency was completed by 581 adolescents. Professionals completed the SSM-D for 224 of the 581 adolescents. Furthermore, constructs related to the domains of self-sufficiency were assessed with self-report questionnaires and information about school absenteeism was monitored via the school registration system. RESULTS: For both self-report and professional-report ratings, the internal consistency was satisfactory (Cronbach'α > 0.70) and various minor to strong correlations were found between the domains of self-sufficiency and related constructs. For most of the domains, there was little or no agreement between professionals and adolescents. CONCLUSIONS: Both the self-report questionnaire assessing self-sufficiency and the SSM-D applied in this study seem to possess adequate psychometric properties. The results indicated that adolescents and professionals provide different views of adolescents' self-sufficiency, which merits further study. In the meantime, we recommend assessment of adolescents' self-sufficiency by using both the self-report questionnaire and the SSM-D to get a comprehensive measure of adolescents' self-sufficiency. TRIAL REGISTRATION: Netherlands Trial Register: NTR3545 ; 30 July 2012.

Depressive symptoms and clustering of risk behaviours among adolescents and young adults attending vocational education: a cross-sectional study.

BACKGROUND: Depressive symptoms and risk behaviours often do not occur in isolation among adolescents and young adults. In order to improve intervention programmes, more research is needed to elucidate the clustering of risk behaviours, the association with depressive symptoms, and demographic variables. Therefore, this study examined the clustering of risk behaviours, the association with depressive symptoms, and demographic variables among adolescents and young adults in vocational education. Furthermore, the prevalence of depressive symptoms and risk behaviours was examined. METHODS: This study included 584 students (mean age 18.3 years) attending vocational education in the Netherlands. Depressive symptoms and risk behaviours (binge drinking, cannabis use, smoking, delinquency and incurring debts) were assessed with self-report questionnaires. Truancy was monitored via the school registration system. Principal Components Analysis (PCA) was conducted to assess the factor structure of the risk behaviours (i.e. clustering). Linear regression analyses with a bootstrapping method were performed to assess the associations. RESULTS: Binge drinking was reported by 50.5% and cannabis use by 14.2% of the students (both in the past 4 weeks), whereas 37.7% reported currently being a smoker. More than 10% reported having been questioned at a police station in the past year. Furthermore, 82.2% had been truanting in the first two months of education, 21.0% reported having debts and 29.2% reported clinically-relevant depressive symptoms. The PCA indicated two clusters. The 'substance use' cluster consisted of the risk behaviours: binge drinking, cannabis use and smoking. The 'problem behaviours' cluster consisted of the risk behaviours: delinquency, truancy and incurring debts. Both clusters were associated with depressive symptoms. Various demographic variables were associated with both clusters. CONCLUSIONS: Risk behaviours formed two clusters, both of which were associated with depressive symptoms. These findings underscore the importance of screening adolescents and young adults at lower educational levels for multiple risk behaviours and depressive symptoms and of focusing on multiple risk behaviours in interventions simultaneously.

Your health, an intervention at senior vocational schools to promote adolescents' health and health behaviors.

UNLABELLED: This study evaluates the appreciation, application and effects of an intervention (Your Health), in which adolescents received a consultation with the school nurse. A cluster randomized controlled trial with an intervention and control group (care-as-usual) was conducted among first-grade senior vocational students. Adolescents (n = 418) completed a questionnaire at baseline and 6-month follow-up assessing health and health behaviors. School absenteeism was monitored via the school registration system. After the consultation, adolescents and nurses evaluated the consultation by questionnaire. Adolescents appreciated being invited for the consultation and gave the consultation a positive mean rating of 8.78 on a 10-point scale. Adolescents rated the other nine items on the appreciation of the consultation also high. In 36.8% of the adolescents, nurses suspected problems. Most often these adolescents were given tailored advice (59.3%) or they were referred to another professional (40.7%). No statistically significant effects of the intervention were found on the health and health behaviors of adolescents. This study supports the use of Your Health as a promising intervention to reach senior vocational students. Future research is needed to evaluate long-term effects and the effects and appreciation of the subsequent help that is offered. TRIAL REGISTRATION: www.trialregister.nl, NTR3545.

KAI407, a potent non-8-aminoquinoline compound that kills Plasmodium cynomolgi early dormant liver stage parasites in vitro.

Preventing relapses of Plasmodium vivax malaria through a radical cure depends on use of the 8-aminoquinoline primaquine, which is associated with safety and compliance issues. For future malaria eradication strategies, new, safer radical curative compounds that efficiently kill dormant liver stages (hypnozoites) will be essential. A new compound with potential radical cure activity was identified using a low-throughput assay of in vitro-cultured hypnozoite forms of Plasmodium cynomolgi (an excellent and accessible model for Plasmodium vivax). In this assay, primary rhesus hepatocytes are infected with P. cynomolgi sporozoites, and exoerythrocytic development is monitored in the presence of compounds. Liver stage cultures are fixed after 6 days and stained with anti-Hsp70 antibodies, and the relative proportions of small (hypnozoite) and large (schizont) forms relative to the untreated controls are determined. This assay was used to screen a series of 18 known antimalarials and 14 new non-8-aminoquinolines (preselected for blood and/or liver stage activity) in three-point 10-fold dilutions (0.1, 1, and 10 µM final concentrations). A novel compound, designated KAI407 showed an activity profile similar to that of primaquine (PQ), efficiently killing the earliest stages of the parasites that become either primary hepatic schizonts or hypnozoites (50% inhibitory concentration [IC50] for hypnozoites, KAI407, 0.69 µM, and PQ, 0.84 µM; for developing liver stages, KAI407, 0.64 µM, and PQ, 0.37 µM). When given as causal prophylaxis, a single oral dose of 100 mg/kg of body weight prevented blood stage parasitemia in mice. From these results, we conclude that KAI407 may represent a new compound class for P. vivax malaria prophylaxis and potentially a radical cure.

Transgenic fluorescent Plasmodium cynomolgi liver stages enable live imaging and purification of Malaria hypnozoite-forms.

A major challenge for strategies to combat the human malaria parasite Plasmodium vivax is the presence of hypnozoites in the liver. These dormant forms can cause renewed clinical disease after reactivation through unknown mechanisms. The closely related non-human primate malaria P. cynomolgi is a frequently used model for studying hypnozoite-induced relapses. Here we report the generation of the first transgenic P. cynomolgi parasites that stably express fluorescent markers in liver stages by transfection with novel DNA-constructs containing a P. cynomolgi centromere. Analysis of fluorescent liver stages in culture identified, in addition to developing liver-schizonts, uninucleate persisting parasites that were atovaquone resistant but primaquine sensitive, features associated with hypnozoites. We demonstrate that these hypnozoite-forms could be isolated by fluorescence-activated cell sorting. The fluorescently-tagged parasites in combination with FACS-purification open new avenues for a wide range of studies for analysing hypnozoite biology and reactivation.