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Abstract Objectives Psychiatric symptoms occur frequently in people living with human immunodeficiency virus (PLWH), which may affect quality of life, sexual risk behavior, and adherence to antiretroviral therapy (ART). Data from large cohorts are limited, and symptoms are often analyzed in isolation. Therefore, we applied a network analysis to assess the interrelatedness of mental health indicators in a large cohort of PLWH. Methods We included 1615 PLWH on ART. Participants reported on the severity of depression, anxiety, impulsivity, substance use, quality of life, sexual risk behavior, and ART adherence. An Ising network model was constructed to analyze interrelations between mental health indicators and connections with clinical consequences. Results Our network analysis revealed that symptoms of depression, anxiety, and indicators of impulsivity were interrelated. Substance use was prevalent and strongly connected with sexual risk behavior. Quality of life was most strongly connected with symptoms of depression. Unexpectedly, ART adherence did not display connections with any of the mental health indicators. Conclusion In PLWH, the interrelatedness between symptoms of depression and anxiety and indicators of impulsivity is high. Mainly, depressive symptoms seem to impact quality of life, which warrants attention for depression in PLWH. We did not observe evidence for the common assumption that patients suffering from psychiatric symptoms are less adherent to HIV treatment.

ENGLISH ABSTRACT: Service-Oriented Architecture (SOA) enables organisations to let their business drive their IT strategy, and creates a technology strategy that is aligned with that of the business. SOA is an architectural style that enables the integration of disparate systems, independent of the implementation technology or physical location, through encapsulating and integrating business processes as a collection of coarse grained, loosely-coupled services. This article aims to examine the ability of SOA to satisfy the requirements and concerns posed by financial trading systems, and to present a SOA framework for building an automated trading application.AFRIKAANSE OPSOMMING: Diens-georiënteerde argitektuur (Service-Oriented Architecture (SOA)) stel organisasies in staat om die IT-strategie vanuit die besigheid te bestuur, en skep ‘n tegnologiestrategie wat belyn is met dié van die besigheid. SOA is ‘n argitektoniese styl wat die integrasie van uiteenlopende stelsels moontlik maak, onafhanklik van die implementeringstegnologie of fisiese ligging, deur besigheidsprosesse te enkapsuleer en te integreer as ‘n versameling van losweg-gekoppelde dienste. Die doel van hierdie artikel is om SOA te ondersoek as ‘n moontlikheid om te voldoen aan die vereistes en knelpunte wat finansiële handelstelsels stel, en om ‘n SOA-raamwerk vir die bou van ‘n outomatiese handelsapplikasie te bied.

IntroductionImmunological non-responders (INR) are people living with HIV (PLHIV) who fail to fully restore CD4+ T-cell counts despite complete viral suppression with antiretroviral therapy (ART). INR are at higher risk for non-HIV related morbidity and mortality. Previous research suggest persistent qualitative defects.MethodsThe 2000HIV study (clinical trials NTC03994835) enrolled 1895 PLHIV, divided in a discovery and validation cohort. PLHIV with CD4 T-cell count 500 cells/mm3. Logistic and rank based regression were used to analyze clinical data, extensive innate and adaptive immunophenotyping, and ex vivo monocyte and lymphocyte cytokine production after stimulation with various stimuli.ResultsThe discovery cohort consisted of 62 INR and 1224 IR, the validation cohort of 26 INR and 243 IR. INR were older, had more advanced HIV disease before starting ART and had more frequently a history of non-AIDS related malignancy. INR had lower absolute CD4+ T-cell numbers in all subsets. Activated (HLA-DR+, CD38+) and exhausted (PD1+) subpopulations were proportionally increased in CD4 T-cells. Monocyte and granulocyte immunophenotypes were comparable. INR lymphocytes produced less IL-22, IFN-γ, IL-10 and IL-17 to stimuli. In contrast, monocyte cytokine production did not differ. The proportions of CD4+CD38+HLA-DR+ and CD4+PD1+ subpopulations showed an inversed correlation to lymphocyte cytokine production.ConclusionsINR compared to IR have hyperactivated and exhausted CD4+ T-cells in combination with lymphocyte functional impairment, while innate immune responses were comparable. Our data provide a rationale to consider the use of anti-PD1 therapy in INR.

Abstract Transgenic luciferase-expressing Plasmodium falciparum parasites have been widely used for the evaluation of anti-malarial compounds. Here, to screen for anti-malarial drugs effective against multiple stages of the parasite, we generate a P. falciparum reporter parasite that constitutively expresses NanoLuciferase (NanoLuc) throughout its whole life cycle. The NanoLuc-expressing P. falciparum reporter parasite shows a quantitative NanoLuc signal in the asexual blood, gametocyte, mosquito, and liver stages. We also establish assay systems to evaluate the anti-malarial activity of compounds at the asexual blood, gametocyte, and liver stages, and then determine the 50% inhibitory concentration (IC50) value of several anti-malarial compounds. Through the development of this robust high-throughput screening system, we identify an anti-malarial compound that kills the asexual blood stage parasites. Our study highlights the utility of the NanoLuc reporter line, which may advance anti-malarial drug development through the improved screening of compounds targeting the human malarial parasite at multiple stages.

In people living with HIV (PLHIV), integrase strand transfer inhibitors (INSTIs) are part of the first-line combination antiretroviral therapy (cART), while non-nucleoside reverse transcriptase inhibitor (NNRTI)-based regimens are alternatives. Distinct cART regimens may variably influence the risk for non-AIDS comorbidities. We aimed to compare the metabolome and lipidome of INSTI and NNRTI-based regimens. The 2000HIV study includes asymptomatic PLHIV (n = 1646) on long-term cART, separated into a discovery cohort with 730 INSTI and 617 NNRTI users, and a validation cohort encompassing 209 INSTI and 90 NNRTI users. Baseline plasma samples from INSTI and NNRTI users were compared using mass spectrometry-based untargeted metabolomic (n = 500) analysis. Perturbed metabolic pathways were identified using MetaboAnalyst software. Subsequently, nuclear magnetic resonance spectroscopy was used for targeted lipoprotein and lipid (n = 141) analysis. Metabolome homogeneity was observed between the different types of INSTI and NNRTI. In contrast, higher and lower levels of 59 and 45 metabolites, respectively, were found in the INSTI group compared to NNRTI users, of which 77.9% (81/104) had consistent directionality in the validation cohort. Annotated metabolites belonged mainly to ‘lipid and lipid-like molecules’, ‘organic acids and derivatives’ and ‘organoheterocyclic compounds’. In pathway analysis, perturbed ‘vitamin B1 (thiamin) metabolism’, ‘de novo fatty acid biosynthesis’, ‘bile acid biosynthesis’ and ‘pentose phosphate pathway’ were detected, among others. Lipoprotein and lipid levels in NNRTIs were heterogeneous and could not be compared as a group. INSTIs compared to individual NNRTI types showed that HDL cholesterol was lower in INSTIs compared to nevirapine but higher in INSTIs compared to doravirine. In addition, LDL size was lower in INSTIs and nevirapine compared to doravirine. NNRTIs show more heterogeneous cardiometabolic effects than INSTIs, which hampers the comparison between these two classes of drugs. Targeted lipoproteomic and lipid NMR spectroscopy showed that INSTI use was associated with a more unfavorable lipid profile compared to nevirapine, which was shifted to a more favorable profile for INSTI when substituting nevirapine for doravirine, with evidently higher fold changes. The cardiovascular disease risk profile seems more favorable in INSTIs compared to NNRTIs in untargeted metabolomic analysis using mass-spectrometry.

Background Cardiovascular disease is a major cause of morbidity and mortality in people living with HIV, who are at higher risk than the general population. We assessed, in a large cohort of people living with HIV, which cardiovascular, HIV‐specific, and lipoproteomic markers were associated with carotid intima‐media thickness (cIMT) and carotid plaque presence. We also studied guideline adherence on lipid‐lowering medication in individuals with high and very high risk for cardiovascular disease. Methods and Results In 1814 individuals with a median (interquartile range) age of 53 (44–60) years, we found a carotid plaque in 909 (50.1%) and a median (interquartile range) intima‐media thickness of 0.66 (0.57–0.76) mm. Ultrasonography was used for the assessment of cIMT and plaque presence. Univariable and multivariable regression models were used for associations with cIMT and presence of plaques. Age, Black race, body mass index, type 2 diabetes, and smoking (pack years) were all positively associated with higher cIMT. Levels of high‐density lipoprotein cholesterol, specifically medium and large high‐density lipoprotein subclasses, were negatively associated with higher cIMT. Only age and prior myocardial infarction were positively related to the presence of a carotid plaque. Lipid‐lowering treatment was prescribed in one‐third of people living with HIV, who are at high and very high risk for cardiovascular disease. Conclusions Traditional cardiovascular risk factors were significantly associated with higher cIMT but not with carotid plaques, except for age. HIV‐specific factors were not associated with both ultrasound measurements. Future studies are needed to elucidate which factors contribute to plaque formation. Improvement of guideline adherence on prescription of lipid‐lowering treatment in high‐ and very high‐risk patients for cardiovascular disease is recommended. Registration URL: https://www.clinicaltrials.gov; Unique identifier: NCT03994835.

A high-throughput screening pipeline tests the feasibility of targeting the sporozoite stage of P. falciparum through chemical interventions and identifies gramicidin as a potential sporontocidal agent.

Salivary mucins, encoded by the MUC5B gene, make up a heterogeneous family of molecules, which are secreted by several glands, including the submandibular, sublingual, and palatine glands. Previous studies have shown that heterogeneity in the salivary mucin population is related to its multiglandular origin. In the present study we address the question to what extent the mucin (MUC5B) population from a single human salivary gland is made up of different glycoforms. Using monoclonal antibodies to defined protein and sulfated carbohydrate epitopes specific to MUC5B, we conduct an immunohistochemical study of different salivary gland types, including submandibular, sublingual, and labial glands. In all tissues studied we found a mosaic expression pattern of sulfo-Lewis a antigen, recognized by mAb F2, which in salivary glands is exclusively present on MUC5B. On the other hand, mucous acini were uniformly labeled by mAb EU-MUC5Bb, evoked against a peptide-stretch of the tandem repeat region of MUC5B. Double staining with both antibodies confirmed the presence of MUC5B-positive/sulfo-Lewis a-positive cells, as well as MUC5B-positive/sulfo-Lewis a-negative cells within one glandular unit. These results indicate that one and the same salivary gland synthesizes different MUC5B glycoforms. [ABSTRACT FROM AUTHOR]

Foot-and-mouth disease (FMD) is an acute, highly contagious viral infection of domestic and wild cloven-hoofed animals. The virus is a single-stranded RNA virus that has a high rate of nucleotide mutation and amino acid substitution. In southern Africa the South African Territories (SAT) 1-3 serotypes of FMD virus are maintained by large numbers of African buffaloes (Syncerus caffer), which provide a potential source of infection for domestic livestock and wild animals. During February 2001, an outbreak of SAT-2 was recorded in cattle in the FMD control zone of South Africa, adjacent to the Kruger National Park (KNP). They had not been vaccinated against the disease since they form the buffer between the vaccination and free zones but in the face of the outbreak, they were vaccinated as part of the control measures to contain the disease. The virus was, however, isolated from some of them on several occasions up to May 2001. These isolates were characterized to determine the rate of genetic change in the main antigenic determinant, the 1D/2A gene. Nucleotide substitutions at 12 different sites were identified of which five led to amino acid changes. Three of these occurred in known antigenic sites, viz. the GH-loop and C-terminal part of the protein, and two of these have previously been shown to be subject to positive selection. Likelihood models indicated that the ratio of non-synonymous to synonymous changes among the outbreak sequences recovered from cattle was four times higher than among comparable sequences isolated from wildlife, suggesting that the virus may be under greater selective pressure during rapid transmission events.

Foot-and-mouth disease (FMD) is an economically important disease of cloven-hoofed animals that is primarily controlled by vaccination of susceptible animals and movement restrictions for animals and animal-derived products in South Africa. Vaccination using aluminium hydroxide gel-saponin (AS) adjuvanted vaccines containing the South African Territories (SAT) serotypes has been shown to be effective both in ensuring that disease does not spread from the endemic to the free zone and in controlling outbreaks in the free zone. Various vaccine formulations containing antigens derived from the SAT serotypes were tested in cattle that were challenged 1 year later. Both the AS and ISA 206B vaccines adjuvanted with saponin protected cattle against virulent virus challenge. The oilbased ISA 206B-adjuvanted vaccine with and without stimulators was evaluated in a field trial and both elicited antibody responses that lasted for 1 year. Furthermore, the ISA 206 adjuvanted FMD vaccine protected groups of cattle against homologous virus challenge at very low payloads, while pigs vaccinated with an emergency ISA 206B-based FMD vaccine containing the SAT 1 vaccine strains were protected against the heterologous SAT 1 outbreak strain.

A serological survey of bovine babesiosis and anaplasmosis was conducted in the foot-and-mouth disease buffer zone surrounding the Kruger National Park in South Africa between 2001 and 2003 to determine whether the withdrawal of government-subsidized dipping in certain regions had affected the seroprevalence of these tick-borne diseases. Seroprevalence of Anaplasma marginale and Babesia bovis increased during the study period. This increase was greater in Limpopo Province where farmers had to supply their own acaricide than in Mpumalanga Province where dipping materials were provided by the local Veterinary Services. The number of animals testing positive for B. bigemina decreased in both provinces during the study period, which was attributed to possible vector displacement rather than more effective tick control measures. Responses to a questionnaire on ticks and tick-borne diseases revealed local knowledge on the subject to be highly variable and sometimes incorrect.

The epidemiology of serotype SAT-2 foot-and-mouth disease was investigated in sub-Saharan Africa by phylogenetic analysis using the 1D gene encoding the major antigenic determinant. Fourteen genotypes were identified of which three are novel and belong to East Africa, bringing the total number of genotypes for that region to eight. The genotypes clustered into three lineages that demonstrated surprising links between East, southern and south-western Africa. One lineage was unique to West Africa. These results established numerous incursions across country borders in East Africa and long term conservation of sequences for periods up to 41 years. Ethiopia, Kenya and Uganda have all experienced outbreaks from more than one unrelated strain, demonstrating the potential for new introductions. The amount of variation observed within this serotype nearly equalled that which was found between serotypes; this has severe implications for disease control using vaccination.

Partial 1D gene characterization was used to study phylogenetic relationships between 17 serotype O foot-and-mouth disease (FMD) viruses in Ethiopia as well as with other O-type isolates from Eritrea, Kenya, South and West Africa, the Middle East, Asia and South America. A homologous region of 495 bp corresponding to the C-terminus end of the 1D gene was used for phylogenetic analysis. This study described three lineages, viz. African/Middle East-Asia, Cathay and South American. Within lineage I, three topotypes were defined, viz. East and West Africa and the Middle East-Asia together with the South African isolate. The Ethiopian isolates clustered as part of topotype I, the East African topotype. Two clades (based on < 12% nucleotide difference) A and B were identified within the East African isolates, with clade A being further classified into three significant branches, A1 (80 % bootstrap support), A2 (89 % bootstrap support) and A3 (94 % bootstrap support). Clade B consisted of two Kenyan isolates. Within topotype I, the 17 Ethiopian isolates showed genetic heterogeneity between themselves with sequence differences ranging from 4.6-14 %. Lineage 2 and 3 could be equated to two significant topotypes, viz. Cathay and South America. Comparison of amino acid variability at the immunodominant sites between the vaccine strain (ETH/19/77) and other Ethiopian outbreak isolates revealed variations within these sites. These results encourage further work towards the reassessment of the type O vaccine strain currently being used in Ethiopia to provide protection against field variants of the virus.

During the early 1920's some sporadic (and experimental) commentaries on the Bible in Afrikaans were published. However, after the Bible was translated into Afrikaans in 1933 these drops turned into a stream and soon swelled into a torrent of publications. These publications display a steady growth in Biblical understanding from a literal and even fundamentalistic interpretation to a critical exegesis of Scripture. At least three distinct phases can be demonstrated, namely a focus on the author, then on the text, and finally on the reader. Given the limited number of readers, the publication of Afrikaans commentaries in the future is faced with unique problems and decisions.

Willem Sterrenberg Prinsloo seen from the viewpoint of a colleague: The sudden and tragic death of Professor W S Prinsloo raises many questions that cannot be answered. However, he should be remembered for the huge contribution he has made in the field of Old Testament studies in South Africa. As editor of this journal he was outspoken about the necessity that church and university, theology and science need one another and that theologians are scientific in their research even though it is done from an ecclesiastical viewpoint. As dean he combined strictness with empathy and as a preacher he integrated solid exegesis with spiritual dedication.

The composition and structure of the book Hosea Notwithstanding the composition of a book as revealed by means of historical critical methods, it is necessary to establish the structure of the final text by way of literary criteria and with regard to contents. Such a structure does not necessarily represent the intention of the author(s), but represents a reader�s approach to the text. Most commentaries on the book of Hosea follow the main divisions 1-3; 4-11; 12-14, which has been demonstrated to be the result of formation and the final redactor�s activities. A more viable structure is proposed to facilitate the reading of the text.

In die lig van die Bybel glo ek nie in die Satan nie. ’n Christen glo net in God, en kan dus nie ook in die Satan glo nie, want dit sou vertroue en ’n persoonlike verhouding impliseer. Miskien moet ek eerder saam met Otto Weber sê: "Ons glo teen die duiwel"! Hierdie onderskeid is nodig omdat te veel Christene soveel statuur aan die Satan gee dat dit God verdwerg. In sy Screwtape Letters skryf die gevierde C.S. Lewis:

False expectations exposed: The function of traditions in Jeremiah 7:1-15 In Jeremiah 7:1-15 various traditions are employed in different ways. The expectations based on the Zion Tradition, namely that Jerusalem is inviolable because of Jahweh�s presence, are exposed to be false. This is done with reference to the Sinai Tradition, accentuating that Jahweh's saving acts are not determined by his presence in the temple, but by Juda�s loyalty to the prescriptions of Sinai. Furthermore, the Conquest Tradition is used to describe the extent of the judgment in store for Juda, namely the loss of the land.

A central challenge of antimalarial therapy is the emergence of resistance to the components of artemisinin-based combination therapies (ACTs) and the urgent need for new drugs acting through novel mechanism of action. Over the last decade, compounds identified in phenotypic high throughput screens (HTS) have provided the starting point for six candidate drugs currently in the Medicines for Malaria Venture (MMV) clinical development portfolio. However, the published screening data which provided much of the new chemical matter for malaria drug discovery projects have been extensively mined. Here we present a new screening and selection cascade for generation of hit compounds active against the blood stage of Plasmodium falciparum. In addition, we validate our approach by testing a library of 141,786 compounds not reported earlier as being tested against malaria. The Hit Generation Library 1 (HGL1) was designed to maximise the chemical diversity and novelty of compounds with physicochemical properties associated with potential for further development. A robust HTS cascade containing orthogonal efficacy and cytotoxicity assays, including a newly developed and validated nanoluciferase-based assay was used to profile the compounds. 75 compounds (Screening Active hit rate of 0.05%) were identified meeting our stringent selection criteria of potency in drug sensitive (NF54) and drug resistant (Dd2) parasite strains (IC50 ≤ 2 µM), rapid speed of action and cell viability in HepG2 cells (IC50 ≥ 10 µM). Following further profiling, 33 compounds were identified that meet the MMV Confirmed Active profile and are high quality starting points for new antimalarial drug discovery projects.

The faithful God is my Protector: Psalm 59 The psalm consists of eight strophes, spread over three main sections, that is, verses 2�5a, 5b�11 and 12�18. The last two main sections are parallel, yet in such a way that strong contrasts are emphasized in order to articulate the message, the main thrust being that the Lord is the faithful Protector, the One who is in control of the whole earth and in whom the believer can trust.

Die insinking en oplewing in die geestelike lewe van ou Israel spruit voort uit 'n wye verskeidenheid faktore en kan op verskillende maniere benader word. In hierdie lesing wil ek my bepaal by die belangrikste faktore en hulle vanuit enkele gesigshoeke belig.

Die woord "r�ah� kom byna 400 keer in die Ou Testament voor. In 'n groot aantal gevalle het dit niks met "gees" of "Gees" te doen nie en dikwels is dit moeilik om presies te bepaal wat met "Gees/gees" bedoel word. Hieruit blyk reeds dat die betekenis-aanwending in verskillende kategorie� verdeel kan word.

Die afgelope twintig jaar was daar �n toenemende belangstelling in die aartsvadervertellinge in Genesis. Benewens die talle tydskrifartikels het skrywers soos die volgende ook boeke oor die onderwerp die lig laat sien: A. Parrot (1962), W. M. Clark (1964), R. Kilian (1966), R. E. Clements (1967), N. Lohfink (1967), H. Weidemann (1968), R. Martin-Achard (1969), T. L. Thompson (1974), J. van Seters (1975), J. P. Fokkelman (1975), C. Westermann (1976), R. Rendtorff (1977), J. Bright (1977), W. McKane (1979) en A. R. Millard en D. J. Wiseman (1980).

Reeds 2 500 jaar gelede was "vrede" 'n slagspreuk van welmenende maar verkeerd ingeligte mense. Aangaande hierdie valse hoop en eensydige verkondiging s� die profeet van ouds: "En hulle genees die verbreking van die dogter van my volk op die maklikste manier deur te s�: 'Vrede, vrede' - terwyl daar geen vrede is nie" (Jer 6:14).

BackgroundEven during long-term combination antiretroviral therapy (cART), people living with HIV (PLHIV) have a dysregulated immune system, characterized by persistent immune activation, accelerated immune ageing and increased risk of non-AIDS comorbidities. A multi-omics approach is applied to a large cohort of PLHIV to understand pathways underlying these dysregulations in order to identify new biomarkers and novel genetically validated therapeutic drugs targets.MethodsThe 2000HIV study is a prospective longitudinal cohort study of PLHIV on cART. In addition, untreated HIV spontaneous controllers were recruited. In-depth multi-omics characterization will be performed, including genomics, epigenomics, transcriptomics, proteomics, metabolomics and metagenomics, functional immunological assays and extensive immunophenotyping. Furthermore, the latent viral reservoir will be assessed through cell associated HIV-1 RNA and DNA, and full-length individual proviral sequencing on a subset. Clinical measurements include an ECG, carotid intima-media thickness and plaque measurement, hepatic steatosis and fibrosis measurement as well as psychological symptoms and recreational drug questionnaires. Additionally, considering the developing pandemic, COVID-19 history and vaccination was recorded. Participants return for a two-year follow-up visit. The 2000HIV study consists of a discovery and validation cohort collected at separate sites to immediately validate any finding in an independent cohort.ResultsOverall, 1895 PLHIV from four sites were included for analysis, 1559 in the discovery and 336 in the validation cohort. The study population was representative of a Western European HIV population, including 288 (15.2%) cis-women, 463 (24.4%) non-whites, and 1360 (71.8%) MSM (Men who have Sex with Men). Extreme phenotypes included 114 spontaneous controllers, 81 rapid progressors and 162 immunological non-responders. According to the Framingham score 321 (16.9%) had a cardiovascular risk of >20% in the next 10 years. COVID-19 infection was documented in 234 (12.3%) participants and 474 (25.0%) individuals had received a COVID-19 vaccine.ConclusionThe 2000HIV study established a cohort of 1895 PLHIV that employs multi-omics to discover new biological pathways and biomarkers to unravel non-AIDS comorbidities, extreme phenotypes and the latent viral reservoir that impact the health of PLHIV. The ultimate goal is to contribute to a more personalized approach to the best standard of care and a potential cure for PLHIV.

IntroductionWhy are women (not) romantically attracted to dark personalities or villains, which might be a risk factor for intimate partner violence (IPV) victimization? In the current study, it is opted to investigate how adult attachment, maladaptive personality traits, and acceptance of couple violence in women predict romantic attraction to heroic/villainous characters using structural equation modeling (SEM).MethodFirst, a pilot study was conducted in 122 heterosexual women (aged 16–25) to select male TV characters. This resulted in the selection of six villains and 10 heroes for the main study, in which 194 other heterosexual women (aged 16–25) were asked to rate the pictures of TV characters through an online questionnaire. This was combined with self-report measures of maladaptive personality traits, acceptance of couple violence, and adult attachment. These variables were entered into a SEM model to assess model fit.ResultsOverall, women rated heroes higher on physical appearance (pilot study) and romantic attraction (main study) compared to villains. We found different direct effects of avoidant (negative) and anxious (positive) attachment styles on romantic attraction to heroes. Moreover, maladaptive personality traits fully mediated the positive effect of avoidant attachment style on romantic attraction to villains.DiscussionDespite the limitations of the study design (e.g., low N, low notoriety of the TV characters), this study emphasizes that women are generally more romantically attracted to heroes (vs. villains). Besides, there are different predictors of romantic attraction to heroes and villains, which requires further investigation, especially in the context of IPV.

This work addresses the need for new chemical matter in product development for control of pest insects and vector-borne diseases. We present a barcoding strategy that enables phenotypic screens of blood-feeding insects against small molecules in microtiter plate-based arrays and apply this to discovery of novel systemic insecticides and compounds that block malaria parasite development in the mosquito vector. Encoding of the blood meals was achieved through recombinant DNA-tagged Asaia bacteria that successfully colonised Aedes and Anopheles mosquitoes. An arrayed screen of a collection of pesticides showed that chemical classes of avermectins, phenylpyrazoles, and neonicotinoids were enriched for compounds with systemic adulticide activity against Anopheles. Using a luminescent Plasmodium falciparum reporter strain, barcoded screens identified 48 drug-like transmission-blocking compounds from a 400-compound antimicrobial library. The approach significantly increases the throughput in phenotypic screening campaigns using adult insects and identifies novel candidate small molecules for disease control.

A highly efficacious pre-erythrocytic stage vaccine would be an important tool for the control and elimination of malaria but is currently unavailable. High-level protection in humans can be achieved by experimental immunization with Plasmodium falciparum sporozoites attenuated by radiation or under anti-malarial drug coverage. Immunization with genetically attenuated parasites (GAP) would be an attractive alternative approach. In this study, we present data on safety and protective efficacy using sporozoites with deletions of two genes, that is the newly identified b9 and slarp, which govern independent and critical processes for successful liver-stage development. In the rodent malaria model, PbΔb9ΔslarpGAP was completely attenuated showing no breakthrough infections while efficiently inducing high-level protection. The human PfΔb9ΔslarpGAP generated without drug resistance markers were infective to human hepatocytes in vitro and to humanized mice engrafted with human hepatocytes in vivo but completely aborted development after infection. These findings support the clinical development of a PfΔb9ΔslarpSPZ vaccine.

Genetically-modified mutants are now indispensable Plasmodium gene-function reagents, which are also being pursued as genetically attenuated parasite vaccines. Currently, the generation of transgenic malaria-parasites requires the use of drug-resistance markers. Here we present the development of an FRT/FLP-recombinase system that enables the generation of transgenic parasites free of resistance genes. We demonstrate in the human malaria parasite, P. falciparum, the complete and efficient removal of the introduced resistance gene. We targeted two neighbouring genes, p52 and p36, using a construct that has a selectable marker cassette flanked by FRT-sequences. This permitted the subsequent removal of the selectable marker cassette by transient transfection of a plasmid that expressed a 37°C thermostable and enhanced FLP-recombinase. This method of removing heterologous DNA sequences from the genome opens up new possibilities in Plasmodium research to sequentially target multiple genes and for using genetically-modified parasites as live, attenuated malaria vaccines.