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Plasmodium vivax lactate dehydrogenase (PvLDH) is an essential enzyme in the glycolytic pathway of P. vivax. It is widely used as a diagnostic biomarker and a measure of total-body parasite biomass in vivax malaria. However, the dynamics of PvLDH remains poorly understood. Here, we developed mathematical models that capture parasite and matrix PvLDH dynamics in ex vivo culture and the human host. We estimated key biological parameters characterising in vivo PvLDH dynamics based on longitudinal data of parasitemia and PvLDH concentration collected from P. vivax-infected humans, with the estimates informed by the ex vivo data as prior knowledge in a Bayesian hierarchical framework. We found that the in vivo accumulation rate of intraerythrocytic PvLDH peaks at 10-20 h post-invasion (late ring stage) with a median estimate of intraerythrocytic PvLDH mass at the end of the life cycle to be 9.4 × 10-3ng. We also found that the median estimate of in vivo PvLDH half-life was approximately 21.9 h. Our findings provide a foundation with which to advance our quantitative understanding of P. vivax biology and will facilitate the improvement of PvLDH-based diagnostic tools.

Neisseria gonorrhoeae infection is an important public health issue, with an annual global incidence of 87 million. N. gonorrhoeae infection causes significant morbidity and can have serious long-term impacts on reproductive and neonatal health and may rarely cause life-threatening disease. Global rates of N. gonorrhoeae infection have increased over the past 20 years. Importantly, rates of antimicrobial resistance to key antimicrobials also continue to increase, with the United States Centers for Disease Control and Prevention identifying drug-resistant N. gonorrhoeae as an urgent threat to public health. This review summarizes the current evidence for N. gonorrhoeae vaccines, including historical clinical trials, key N. gonorrhoeae vaccine preclinical studies, and studies of the impact of Neisseria meningitidis vaccines on N. gonorrhoeae infection. A comprehensive survey of potential vaccine antigens, including those identified through traditional vaccine immunogenicity approaches, as well as those identified using more contemporary reverse vaccinology approaches, are also described. Finally, the potential epidemiological impacts of a N. gonorrhoeae vaccine and research priorities for further vaccine development are described.

Commercial point-of-care tests remain insufficient for accurately detecting and differentiating low-level malaria infections in regions co-endemic with multiple non-falciparum species, including zoonotic Plasmodium knowlesi (Pk). A 5-plex chemiluminescent assay simultaneously measures pan-Plasmodium lactate dehydrogenase (pLDH), P. falciparum (Pf)-LDH, P. vivax (Pv)-LDH, Pf-histidine-rich protein-2 (HRP2), and C-reactive protein. We assessed its diagnostic performance on whole blood (WB) samples from 102 healthy controls and 306 PCR-confirmed clinical cases of Pf, Pv, Pk, P. malariae (Pm) and P. ovale (Po) mono-infections from Southeast-Asia. We confirm its excellent HRP2-based detection of Pf. Cross-reactivity of Pf-LDH with all non-falciparum species tested was observed (specificity 57.3%). Pv-LDH performance was suboptimal for Pv (93.9% sensitivity and 73.9% specificity). Poor specificity was driven by strong Pk cross-reactivity, with Pv-LDH detecting 93.9% of Pk infections. The pan-LDH-to-Pf-LDH ratio was capable of discerning Pv from Pk, and robustly differentiated Pf from Pm or Po infection, useful in regions with hrp2/3 deletions. We tested the platform's performance in plasma for the first time, with WB outperforming plasma for all analytes except Pv-LDH for Pk. The platform is a promising tool for WB malaria diagnosis, although further development is warranted to improve its utility in regions co-endemic for multiple non-falciparum species.

Cerebral malaria is the most serious manifestation of severe falciparum malaria. Sequestration of infected red blood cells and microvascular dysfunction are key contributing processes. Whether these processes occur in early stage disease prior to clinical manifestations is unknown. To help localize and understand these processes during the early stages of infection, we performed 18-F fluorodeoxyglucose positron emission tomography/magnetic resonance imaging in volunteers with Plasmodium falciparum induced blood stage malaria (IBSM) infection, and compared results to individuals with P. vivax infection, in whom coma is rare. Seven healthy, malaria-naïve participants underwent imaging at baseline, and at early symptom onset a median 9 days following inoculation (n = 4 P. falciparum, n = 3 P. vivax). Participants with P. falciparum infection demonstrated marked lability in radiotracer uptake across all regions of the brain, exceeding expected normal variation (within subject coefficient of variation (wCV): 14.4%) compared to the relatively stable uptake in participants with P. vivax infection (wCV: 3.5%). No consistent imaging changes suggestive of microvascular dysfunction were observed in either group. Neuroimaging in early IBSM studies is safe and technically feasible, with preliminary results suggesting that differences in brain tropism between P. falciparum and P. vivax may occur very early in infection.

Despite repeated malaria infection, individuals living in areas where malaria is endemic remain vulnerable to reinfection. The Janus kinase (JAK1/2) inhibitor ruxolitinib could potentially disrupt the parasite-induced dysfunctional immune response when administered with antimalarial therapy. This randomized, single-blind, placebo-controlled, single-center phase 1 trial investigated the safety, tolerability, and pharmacokinetic and pharmacodynamic profile of ruxolitinib and the approved antimalarial artemether-lumefantrine in combination. Ruxolitinib pharmacodynamics were assessed by inhibition of phosphorylation of signal transducer and activator of transcription 3 (pSTAT3). Eight healthy male and female participants ages 18 to 55 years were randomized to either ruxolitinib (20 mg) (n = 6) or placebo (n = 2) administered 2 h after artemether-lumefantrine (80/480 mg) twice daily for 3 days. Mild adverse events occurred in six participants (four ruxolitinib; two placebo). The combination of artemether-lumefantrine and ruxolitinib was well tolerated, with adverse events and pharmacokinetics consistent with the known profiles of both drugs. The incidence of adverse events and artemether, dihydroartemisinin (the major active metabolite of artemether), and lumefantrine exposure were not affected by ruxolitinib coadministration. Ruxolitinib coadministration resulted in a 3-fold-greater pSTAT3 inhibition compared to placebo (geometric mean ratio = 3.01 [90% confidence interval = 2.14 to 4.24]), with a direct and predictable relationship between ruxolitinib plasma concentrations and %pSTAT3 inhibition. This study supports the investigation of the combination of artemether-lumefantrine and ruxolitinib in healthy volunteers infected with Plasmodium falciparum malaria. (This study has been registered at ClinicalTrials.gov under registration no. NCT04456634.).

The emergence and spread of parasite resistance to currently available antimalarials has highlighted the importance of developing novel antimalarials. This scoping review provides an overview of antimalarial drug candidates undergoing phase I and II studies between 1 January 2016 and 28 April 2021. PubMed, Web of Science, Embase, clinical trial registries, and reference lists were searched for relevant studies. Information regarding antimalarial compound details, clinical trial characteristics, study population, and drug pharmacokinetics and pharmacodynamics (PK-PD) were extracted. A total of 50 studies were included, of which 24 had published their results and 26 were unpublished. New antimalarial compounds were evaluated as monotherapy (28 studies, 14 drug candidates) and combination therapy (9 studies, 10 candidates). Fourteen active compounds were identified in the current antimalarial drug development pipeline together with 11 compounds that are inactive, 6 due to insufficient efficacy. PK-PD data were available from 24 studies published as open-access articles. Four unpublished studies have made their results publicly available on clinical trial registries. The terminal elimination half-life of new antimalarial compounds ranged from 14.7 to 483 h. The log10 parasite reduction ratio over 48 h and parasite clearance half-life for Plasmodium falciparum following a single-dose monotherapy were 1.55 to 4.1 and 3.4 to 9.4 h, respectively. The antimalarial drug development landscape has seen a number of novel compounds, with promising PK-PD properties, evaluated in phase I and II studies over the past 5 years. Timely public disclosure of PK-PD data is crucial for informative decision-making and drug development strategy.

BACKGROUND: Interventions that effectively target Plasmodium vivax are critical for the future control and elimination of malaria. We conducted a P. vivax volunteer infection study to characterize the antimalarial activity of artefenomel, a new drug candidate. METHODS: Eight healthy, malaria-naive participants were intravenously inoculated with blood-stage P. vivax and subsequently received a single oral 200-mg dose of artefenomel. Blood samples were collected to monitor the development and clearance of parasitemia, and plasma artefenomel concentration. Mosquito feeding assays were conducted before artefenomel dosing to investigate parasite transmissibility. RESULTS: Initial parasite clearance occurred in all participants after artefenomel administration (log10 parasite reduction ratio over 48 hours, 1.67; parasite clearance half-life, 8.67 hours). Recrudescence occurred in 7 participants 11-14 days after dosing. A minimum inhibitory concentration of 0.62 ng/mL and minimum parasiticidal concentration that achieves 90% of maximum effect of 0.83 ng/mL were estimated, and a single 300-mg dose was predicted to clear 109 parasites per milliliter with 95% certainty. Gametocytemia developed in all participants and was cleared 4-8 days after dosing. At peak gametocytemia, 75% of participants were infectious to mosquitoes. CONCLUSIONS: The in vivo antimalarial activity of artefenomel supports its further clinical development as a treatment for P. vivax malaria. CLINICAL TRIALS REGISTRATION: NCT02573857.

BACKGROUND: Artemisinin-based combination therapy (ACT) has been a mainstay for malaria prevention and treatment. However, emergence of drug resistance has incentivised development of new drugs. Defining the kinetics with which circulating parasitized red blood cells (pRBC) are lost after drug treatment, referred to as the "parasite clearance curve", has been critical for assessing drug efficacy; yet underlying mechanisms remain partly unresolved. The clearance curve may be shaped both by the rate at which drugs kill parasites, and the rate at which drug-affected parasites are removed from circulation. METHODS: In this context, two anti-malarials, SJ733, and an ACT partner drug, pyronaridine were compared against sodium artesunate in mice infected with Plasmodium berghei (strain ANKA). To measure each compound's capacity for pRBC removal in vivo, flow cytometric monitoring of a single cohort of fluorescently-labelled pRBC was employed, and combined with ex vivo parasite culture to assess parasite maturation and replication. RESULTS: These three compounds were found to be similarly efficacious in controlling established infection by reducing overall parasitaemia. While sodium artesunate acted relatively consistently across the life-stages, single-dose SJ733 elicited a biphasic effect, triggering rapid, partly phagocyte-dependent removal of trophozoites and schizonts, followed by arrest of residual ring-stages. In contrast, pyronaridine abrogated maturation of younger parasites, with less pronounced effects on mature parasites, while modestly increasing pRBC removal. CONCLUSIONS: Anti-malarials SJ733 and pyronaridine, though similarly efficacious in reducing overall parasitaemia in mice, differed markedly in their capacity to arrest replication and remove pRBC from circulation. Thus, similar parasite clearance curves can result for anti-malarials with distinct capacities to inhibit, kill and clear parasites.

BACKGROUND: SJ733, a newly developed inhibitor of P. falciparum ATP4, has a favorable safety profile and rapid antiparasitic effect but insufficient duration to deliver a single-dose cure of malaria. We investigated the safety, tolerability, and pharmacokinetics of a multidose SJ733 regimen and a single-dose pharmacoboost approach using cobicistat to inhibit CYP3A4, thereby increasing exposure. METHODS: Two multidose unboosted cohorts (n = 9) (SJ733, 300 mg and 600 mg daily for 3 days) followed by three single-dose boosted cohorts combining SJ733 (n = 18) (75-, 300-, or 600-mg single dose) with cobicistat (150-mg single dose) as a pharmacokinetic booster were evaluated in healthy volunteers (ClinicalTrials.gov: NCT02661373). FINDINGS: All participants tolerated SJ733 well, with no serious adverse events (AEs), dose-limiting toxicity, or clinically significant electrocardiogram or laboratory test findings. All reported AEs were Grade 1, clinically insignificant, and considered unlikely or unrelated to SJ733. Compared to unboosted cohorts, the SJ733/cobicistat-boosted cohorts showed a median increase in area under the curve and maximum concentration of 3·9 × and 2·6 ×, respectively, and a median decrease in the ratio of the major CYP3A-produced metabolite SJ506 to parent drug of 4·6 × . Incorporating these data in a model of parasite dynamics indicated that a 3-day regimen of SJ733/cobicistat (600 mg/150 mg daily) relative to a single 600-mg dose ± cobicistat would increase parasite clearance from 106 to 1012 parasites/µL. INTERPRETATION: The multidose and pharmacoboosted approaches to delivering SJ733 were well-tolerated and significantly increased drug exposure and prediction of cure. This study supports the further development of SJ733 and demonstrates an innovative pharmacoboost approach for an antimalarial. FUNDING: Global Health Innovative Technology Fund, Medicines for Malaria Venture, National Institutes of Health, and American Lebanese Syrian Associated Chari

BACKGROUND: Rapid diagnostic tests (RDTs) that rely on the detection of Plasmodium falciparum histidine-rich protein 2 (PfHRP2) have become key tools for diagnosing P. falciparum infection. The utility of RDTs can be limited by PfHRP2 persistence, however it can be a potential benefit in low transmission settings where detection of persistent PfHRP2 using newer ultra-sensitive PfHRP2 based RDTs can serve as a surveillance tool to identify recent exposure. Better understanding of the dynamics of PfHRP2 over the course of a malaria infection can inform optimal use of RDTs. METHODS: A previously published mathematical model was refined to mimic the production and decay of PfHRP2 during a malaria infection. Data from 15 individuals from volunteer infection studies were used to update the original model and estimate key model parameters. The refined model was applied to a cohort of patients from Namibia who received treatment for clinical malaria infection for whom longitudinal PfHRP2 concentrations were measured. RESULTS: The refinement of the PfHRP2 dynamic model indicated that in malaria naïve hosts, P. falciparum parasites of the 3D7 strain produce 33.6 × 10-15 g (95% CI 25.0-42.1 × 10-15 g) of PfHRP2 in vivo per parasite replication cycle, with an elimination half-life of 1.67 days (95% CI 1.11-3.40 days). The refined model included these updated parameters and incorporated individualized body fluid volume calculations, which improved predictive accuracy when compared to the original model. The performance of the model in predicting clearance of PfHRP2 post treatment in clinical samples from six adults with P. falciparum infection in Namibia improved when using a longer elimination half-life of 4.5 days, with 14% to 67% of observations for each individual within the predicted range. CONCLUSIONS: The updated mathematical model can predict the growth and clearance of PfHRP2 during the production and decay of a mono-infection with P. falciparum, increasing the understa

The rate at which parasitemia declines in a host after treatment with an antimalarial drug is a major metric for assessment of antimalarial drug activity in preclinical models and in early clinical trials. However, this metric does not distinguish between viable and nonviable parasites. Thus, enumeration of parasites may result in underestimation of drug activity for some compounds, potentially confounding its use as a metric for assessing antimalarial activity in vivo. Here, we report a study of the effect of artesunate on Plasmodium falciparum viability in humans and in mice. We first measured the drug effect in mice by estimating the decrease in parasite viability after treatment using two independent approaches to estimate viability. We demonstrate that, as previously reported in humans, parasite viability declines much faster after artesunate treatment than does the decline in parasitemia (termed parasite clearance). We also observed that artesunate kills parasites faster at higher concentrations, which is not discernible from the traditional parasite clearance curve and that each subsequent dose of artesunate maintains its killing effect. Furthermore, based on measures of parasite viability, we could accurately predict the in vivo recrudescence of infection. Finally, using pharmacometrics modeling, we show that the apparent differences in the antimalarial activity of artesunate in mice and humans are partly explained by differences in host removal of dead parasites in the two hosts. However, these differences, along with different pharmacokinetic profiles, do not fully account for the differences in activity. (This study has been registered with the Australian New Zealand Clinical Trials Registry under identifier ACTRN12617001394336.).

BACKGROUND: Scabies was added to the WHO NTD portfolio in 2017 and targets for the control of scabies were included in the 2021-2030 WHO NTD roadmap. A major component of scabies control efforts a strategy based on mass drug administration (MDA) with ivermectin. Currently diagnosis of scabies relies on clinical examination with a limited role for diagnostic testing. Under the recommendation of the WHO Diagnostic Technical Advisory Group (DTAG) for Neglected Tropical Diseases, a working group was assembled and tasked with agreeing on priority use cases for and developing target product profiles (TPPs) for new diagnostics tools for scabies. METHODOLOGY AND PRINCIPAL FINDINGS: The working group convened three times and established two use cases: establishing if the 10% threshold for mass drug administration had been reached and if the 2% threshold for stopping mass drug administration has been achieved. One subgroup assessed the current diagnostic landscape for scabies and a second subgroup determined the test requirements for both use cases. Draft TPPs were sent out for input from stakeholders and experts. Both TPPs considered the following parameters: product use, design, performance, configuration, cost, access and equity. The group considered the use of the tests as a single step process or as part of a two step process following initial clinical examination. When used a single step test (the ideal scenario) for starting MDA a new diagnostic required a sensitivity of ≥92% and a specificity of ≥98%. When used a single step test (the ideal scenario) for stopping MDA a new diagnostic required a sensitivity of ≥80% and a specificity of ≥99%. CONCLUSIONS: The TPPs developed will provide test developers with guidance to ensure that novel diagnostic tests meet identified public health needs.

Analytical methods for the quantification of the new 8-aminoquinoline antimalarial tafenoquine (TQ) in human blood, plasma and urine, and the 5,6-orthoquinone tafenoquine metabolite (5,6-OQTQ) in human plasma and urine have been validated. The procedure involved acetonitrile extraction of samples followed by ultra-high-performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS). Chromatography was performed using a Waters Atlantis T3 column with a gradient of 0.1% formic acid and acetonitrile at a flow rate of 0.5 mL per minute for blood and plasma. Urine analysis was the same but with methanol containing 0.1% formic acid replacing acetonitrile mobile phase. The calibration range for TQ and 5,6-OQTQ in plasma was 1 to 1200 ng/mL, and in urine was 10 to 1000 ng/mL. Blood calibration range for TQ was 1 to 1200 ng/mL. Blood could not be validated for 5,6-OQTQ due to significant signal suppression. The inter-assay precision (coefficient of variation %) was 9.9% for TQ at 1 ng/mL in blood (n = 14) and 8.2% for TQ and 7.1% for 5,6-OQTQ at 1 ng/mL in plasma (n = 14). For urine, the inter-assay precision was 8.2% for TQ and 6.4% for 5,6-OQTQ at 10 ng/mL (n = 14). TQ and 5,6-OQTQ are stable in blood, plasma and urine for at least three months at both -80 °C and -20 °C. Once validated, the analytical methods were applied to samples collected from healthy volunteers who were experimentally infected with Plasmodium falciparum to evaluate the blood stage antimalarial activity of TQ and to determine the therapeutic dose estimates for TQ, the full details of which will be published elsewhere. In this study, the measurement of TQ and 5,6-OQTQ concentrations in samples from one of the four cohorts of participants is reported. Interestingly, TQ urine concentrations were proportional to parasite recrudescence times post dosing To our knowledge, this is the first description of a fully validated method for the measurement of TQ and 5,6-OQTQ quantification in urine.

BACKGROUND: Plasmodium falciparum malaria increases plasma levels of the cytokine Fms-like tyrosine kinase 3 ligand (Flt3L), a haematopoietic factor associated with dendritic cell (DC) expansion. It is unknown if the zoonotic parasite Plasmodium knowlesi impacts Flt3L or DC in human malaria. This study investigated circulating DC and Flt3L associations in adult malaria and in submicroscopic experimental infection. METHODS: Plasma Flt3L concentration and blood CD141+ DC, CD1c+ DC and plasmacytoid DC (pDC) numbers were assessed in (i) volunteers experimentally infected with P. falciparum and in Malaysian patients with uncomplicated (ii) P. falciparum or (iii) P. knowlesi malaria. RESULTS: Plasmodium knowlesi caused a decline in all circulating DC subsets in adults with malaria. Plasma Flt3L was elevated in acute P. falciparum and P. knowlesi malaria with no increase in a subclinical experimental infection. Circulating CD141+ DCs, CD1c+ DCs and pDCs declined in all adults tested, for the first time extending the finding of DC subset decline in acute malaria to the zoonotic parasite P. knowlesi. CONCLUSIONS: In adults, submicroscopic Plasmodium infection causes no change in plasma Flt3L but does reduce circulating DCs. Plasma Flt3L concentrations increase in acute malaria, yet this increase is insufficient to restore or expand circulating CD141+ DCs, CD1c+ DCs or pDCs. These data imply that haematopoietic factors, yet to be identified and not Flt3L, involved in the sensing/maintenance of circulating DC are impacted by malaria and a submicroscopic infection. The zoonotic P. knowlesi is similar to other Plasmodium spp in compromising DC in adult malaria.

BACKGROUND: The suboptimal sensitivity and specificity of available diagnostic methods for scabies hampers clinical management, trials of new therapies and epidemiologic studies. Additionally, parasitologic diagnosis by microscopic examination of skin scrapings requires sample collection with a sharp scalpel blade, causing discomfort to patients and difficulty in children. Polymerase chain reaction (PCR)-based diagnostic assays, combined with non-invasive sampling methods, represent an attractive approach. In this study, we aimed to develop a real-time probe-based PCR test for scabies, test a non-invasive sampling method and evaluate its diagnostic performance in two clinical settings. METHODOLOGY/PRINCIPAL FINDINGS: High copy-number repetitive DNA elements were identified in draft Sarcoptes scabiei genome sequences and used as assay targets for diagnostic PCR. Two suitable repetitive DNA sequences, a 375 base pair microsatellite (SSR5) and a 606 base pair long tandem repeat (SSR6), were identified. Diagnostic sensitivity and specificity were tested using relevant positive and negative control materials and compared to a published assay targeting the mitochondrial cox1 gene. Both assays were positive at a 1:100 dilution of DNA from a single mite; no amplification was observed in DNA from samples from 19 patients with other skin conditions nor from house dust, sheep or dog mites, head and body lice or from six common skin bacterial and fungal species. Moderate sensitivity of the assays was achieved in a pilot study, detecting 5/7 (71.4% [95% CI: 29.0% - 96.3%]) of clinically diagnosed untreated scabies patients). Greater sensitivity was observed in samples collected by FLOQ swabs compared to skin scrapings. CONCLUSIONS/SIGNIFICANCE: This newly developed qPCR assay, combined with the use of an alternative non-invasive swab sampling technique offers the possibility of enhanced diagnosis of scabies. Further studies will be required to better define the diagnostic perfor

BACKGROUND: Malaria-associated anaemia, arising from symptomatic, asymptomatic and submicroscopic infections, is a significant cause of morbidity worldwide. Induced blood stage malaria volunteer infection studies (IBSM-VIS) provide a unique opportunity to evaluate the haematological response to early Plasmodium falciparum and Plasmodium vivax infection. METHODS: This study was an analysis of the haemoglobin, red cell counts, and parasitaemia data from 315 participants enrolled in IBSM-VIS between 2012 and 2019, including 269 participants inoculated with the 3D7 strain of P. falciparum (Pf3D7), 15 with an artemisinin-resistant P. falciparum strain (PfK13) and 46 with P. vivax. Factors associated with the fractional fall in haemoglobin (Hb-FF) were evaluated, and the malaria-attributable erythrocyte loss after accounting for phlebotomy-related losses was estimated. The relative contribution of parasitized erythrocytes to the malaria-attributable erythrocyte loss was also estimated. RESULTS: The median peak parasitaemia prior to treatment was 10,277 parasites/ml (IQR 3566-27,815), 71,427 parasites/ml [IQR 33,236-180,213], and 34,840 parasites/ml (IQR 13,302-77,064) in participants inoculated with Pf3D7, PfK13, and P. vivax, respectively. The median Hb-FF was 10.3% (IQR 7.8-13.3), 14.8% (IQR 11.8-15.9) and 11.7% (IQR 8.9-14.5) in those inoculated with Pf3D7, PfK13 and P. vivax, respectively, with the haemoglobin nadir occurring a median 12 (IQR 5-21), 15 (IQR 7-22), and 8 (IQR 7-15) days following inoculation. In participants inoculated with P. falciparum, recrudescence was associated with a greater Hb-FF, while in those with P. vivax, the Hb-FF was associated with a higher pre-treatment parasitaemia and later day of anti-malarial treatment. After accounting for phlebotomy-related blood losses, the estimated Hb-FF was 4.1% (IQR 3.1-5.3), 7.2% (IQR 5.8-7.8), and 4.9% (IQR 3.7-6.1) in participants inoculated with Pf3D7, PfK13, and P. vivax, respectively. Parasitized eryth

The spiroindolone cipargamin, a new antimalarial compound that inhibits Plasmodium ATP4, is currently in clinical development. This study aimed to characterize the antimalarial activity of cipargamin in healthy volunteers experimentally infected with blood-stage Plasmodium falciparum Eight subjects were intravenously inoculated with parasite-infected erythrocytes and received a single oral dose of 10 mg cipargamin 7 days later. Blood samples were collected to monitor the development and clearance of parasitemia and plasma cipargamin concentrations. Parasite regrowth was treated with piperaquine monotherapy to clear asexual parasites, while allowing gametocyte transmissibility to mosquitoes to be investigated. An initial rapid decrease in parasitemia occurred in all participants following cipargamin dosing, with a parasite clearance half-life of 3.99 h. As anticipated from the dose selected, parasite regrowth occurred in all 8 subjects 3 to 8 days after dosing and allowed the pharmacokinetic/pharmacodynamic relationship to be determined. Based on the limited data from the single subtherapeutic dose cohort, a MIC of 11.6 ng/ml and minimum parasiticidal concentration that achieves 90% of maximum effect of 23.5 ng/ml were estimated, and a single 95-mg dose (95% confidence interval [CI], 50 to 270) was predicted to clear 109 parasites/ml. Low gametocyte densities were detected in all subjects following piperaquine treatment, which did not transmit to mosquitoes. Serious adverse liver function changes were observed in three subjects, which led to premature study termination. The antimalarial activity characterized in this study supports the further clinical development of cipargamin as a new treatment for P. falciparum malaria, although the hepatic safety profile of the compound warrants further evaluation. (This study has been registered at ClinicalTrials.gov under identifier NCT02543086.).

Primaquine, an 8-aminoquinoline, is the only medication approved by the World Health Organization to treat the hypnozoite stage of Plasmodium vivax and P. ovale malaria. Relapse, triggered by activation of dormant hypnozoites in the liver, can occur weeks to years after primary infection, and provides the predominant source of transmission in endemic settings. Hence, primaquine is essential for individual treatment and P. vivax elimination efforts. However, primaquine use is limited by the risk of life-threatening acute hemolytic anemia in glucose-6-phosphate dehydrogenase (G6PD) deficient individuals. More recently, studies have demonstrated decreased efficacy of primaquine due to cytochrome P450 2D6 (CYP2D6) polymorphisms conferring an impaired metabolizer phenotype. Failure of standard primaquine therapy has occurred in individuals with decreased or absent CYP2D6 activity. Both G6PD and CYP2D6 are highly polymorphic genes, with considerable geographic and interethnic variability, adding complexity to primaquine use. Innovative strategies are required to overcome the dual challenge of G6PD deficiency and impaired primaquine metabolism. Further understanding of the pharmacogenetics of primaquine is key to utilizing its full potential. Accurate CYP2D6 genotype-phenotype translation may optimize primaquine dosing strategies for impaired metabolizers and expand its use in a safe, efficacious manner. At an individual level the current challenges with G6PD diagnostics and CYP2D6 testing limit clinical implementation of pharmacogenetics. However, further characterisation of the overlap and spectrum of G6PD and CYP2D6 activity may optimize primaquine use at a population level and facilitate region-specific dosing strategies for mass drug administration. This precision public health approach merits further investigation for P. vivax elimination.

BACKGROUND: New anti-malarial therapeutics are required to counter the threat of increasing drug resistance. Malaria volunteer infection studies (VIS), particularly the induced blood stage malaria (IBSM) model, play a key role in accelerating anti-malarial drug development. Supply of the reference 3D7-V2 Plasmodium falciparum malaria cell bank (MCB) is limited. This study aimed to develop a new MCB, and compare the safety and infectivity of this MCB with the existing 3D7-V2 MCB, in a VIS. A second bank (3D7-V1) developed in 1995 was also evaluated. METHODS: The 3D7-V2 MCB was expanded in vitro using a bioreactor to produce a new MCB designated 3D7-MBE-008. This bank and 3D7-V1 were then evaluated using the IBSM model, where healthy participants were intravenously inoculated with blood-stage parasites. Participants were treated with artemether-lumefantrine when parasitaemia or clinical thresholds were reached. Safety, infectivity and parasite growth and clearance were evaluated. RESULTS: The in vitro expansion of 3D7-V2 produced 200 vials of the 3D7-MBE-008 MCB, with a parasitaemia of 4.3%. This compares to 0.1% in the existing 3D7-V2 MCB, and < 0.01% in the 3D7-V1 MCB. All four participants (two per MCB) developed detectable P. falciparum infection after inoculation with approximately 2800 parasites. For the 3D7-MBE-008 MCB, the parasite multiplication rate of 48 h (PMR48) using non-linear mixed effects modelling was 34.6 (95% CI 18.5-64.6), similar to the parental 3D7-V2 line; parasitaemia in both participants exceeded 10,000/mL by day 8. Growth of the 3D7-V1 was slower (PMR48 of 11.5 [95% CI 8.5-15.6]), with parasitaemia exceeding 10,000 parasites/mL on days 10 and 8.5. Rapid parasite clearance followed artemether-lumefantrine treatment in all four participants, with clearance half-lives of 4.01 and 4.06 (weighted mean 4.04 [95% CI 3.61-4.57]) hours for 3D7-MBE-008 and 4.11 and 4.52 (weighted mean 4.31 [95% CI 4.16-4.47]) hours for 3D7-V1. A total of 59 adverse ev

The use of quantitative qRT-PCR assays for detection and quantification of late gametocyte stages has revealed the high transmission capacity of the human malaria parasite, Plasmodium falciparum. To understand how the parasite adjusts its transmission in response to in-host environmental conditions including antimalarials requires simultaneous quantification of early and late gametocytes. Here, we describe qRT-PCR assays that specifically detect and quantify early-stage P. falciparum gametocytes. The assays are based on expression of known early and late gametocyte genes and were developed using purified stage II and stage V gametocytes and tested in natural and controlled human infections. Genes pfpeg4 and pfg27 are specifically expressed at significant levels in early gametocytes with a limit of quantification of 190 and 390 gametocytes/mL, respectively. In infected volunteers, transcripts of pfpeg4 and pfg27 were detected shortly after the onset of blood stage infection. In natural infections, both early (pfpeg4/pfg27) and late gametocyte transcripts (pfs25) were detected in 71.2% of individuals, only early gametocyte transcripts in 12.6%, and only late gametocyte transcripts in 15.2%. The pfpeg4/pfg27 qRT-PCR assays are sensitive and specific for quantification of circulating sexually committed ring stages/early gametocytes and can be used to increase our understanding of epidemiological processes that modulate P. falciparum transmission.

BACKGROUND: Volunteer infection studies have become a standard model for evaluating drug efficacy against Plasmodium infections. Molecular techniques such as qPCR are used in these studies due to their ability to provide robust and accurate estimates of parasitaemia at increased sensitivity compared to microscopy. The validity and reliability of assays need to be ensured when used to evaluate the efficacy of candidate drugs in clinical trials. METHODS: A previously described 18S rRNA gene qPCR assay for quantifying Plasmodium falciparum in blood samples was evaluated. Assay performance characteristics including analytical sensitivity, reportable range, precision, accuracy and specificity were assessed using experimental data and data compiled from phase 1 volunteer infection studies conducted between 2013 and 2019. Guidelines for validation of laboratory-developed molecular assays were followed. RESULTS: The reportable range was 1.50 to 6.50 log10 parasites/mL with a limit of detection of 2.045 log10 parasites/mL of whole blood based on a parasite diluted standard series over this range. The assay was highly reproducible with minimal intra-assay (SD = 0.456 quantification cycle (Cq) units [0.137 log10 parasites/mL] over 21 replicates) and inter-assay (SD = 0.604 Cq units [0.182 log10 parasites/mL] over 786 qPCR runs) variability. Through an external quality assurance program, the QIMR assay was shown to generate accurate results (quantitative bias + 0.019 log10 parasites/mL against nominal values). Specificity was 100% after assessing 164 parasite-free human blood samples. CONCLUSIONS: The 18S rRNA gene qPCR assay is specific and highly reproducible and can provide reliable and accurate parasite quantification. The assay is considered fit for use in evaluating drug efficacy in malaria clinical trials.

BACKGROUND: Streptococcus pyogenes is a leading cause of infection-related morbidity and mortality. A reinvigorated vaccine development effort calls for new clinically relevant human S pyogenes experimental infection models to support proof of concept evaluation of candidate vaccines. We describe the initial Controlled Human Infection for Vaccination Against S pyogenes (CHIVAS-M75) study, in which we aimed to identify a dose of emm75 S pyogenes that causes acute pharyngitis in at least 60% of volunteers when applied to the pharynx by swab. METHODS: This observational, dose-finding study was done in a clinical trials facility in Melbourne (VIC, Australia). Groups of healthy volunteers aged 18-40 years, at low risk of complicated S pyogenes disease, and without high type-specific anti-emm75 IgG antibodies against the challenge strain were challenged and closely monitored as inpatients for up to 6 days, and then as outpatients for 6 months. Antibiotics were started upon diagnosis (clinical signs and symptoms of pharyngitis and a positive rapid molecular test) or after 5 days in those without pharyngitis. Rapid test results were confirmed by standard bacterial culture. After a sentinel participant, cohorts of five and then ten participants were challenged, with protocol-directed dose-escalation or de-escalation for subsequent cohorts. The primary outcome was the proportion of participants at each dose level with pharyngitis by day 5 after challenge. The study is registered with ClinicalTrials.gov, NCT03361163. FINDINGS: Between July 10, 2018, and Sept 23, 2019, 25 healthy adults were challenged with emm75 S pyogenes and included in analyses. Pharyngitis was diagnosed in 17 (85%; 95% CI 62-97) of 20 participants at the starting dose level (1-3 × 105 colony-forming units [CFU]/mL). This high proportion prompted dose de-escalation. At the lower dose level (1-3 × 104 CFU/mL), pharyngitis was diagnosed in one of five participants. Immunological, biochemical, and microbiologi

In 1896, a serendipitous laboratory accident led to the understanding that hookworms propagate infection by penetrating skin, a theory that was then confirmed with the first experimental human infection, reported in 1901. Experimental human infections undertaken in the 20th century enabled understanding of the natural history of infection and the immune response. More recently, experimental hookworm infection has been performed to investigate the immunomodulatory potential of hookworm infection and for the evaluation of hookworm vaccines and chemotherapeutic interventions. Experimental human hookworm infection has been proven to be safe, with no deaths observed in over 500 participants (although early reports predate systematic adverse event reporting) and no serious adverse events described in over 200 participants enrolled in contemporary clinical trials. While experimental human hookworm infection holds significant promise, as both a challenge model for testing anti-hookworm therapies and for treating various diseases of modernity, there are many challenges that present. These challenges include preparation and storage of larvae, which has not significantly changed since Harada and Mori first described their coproculture method in 1955. In vitro methods of hookworm larval culture, storage, and the development of meaningful potency or release assays are required. Surrogate markers of intestinal infection intensity are required because faecal egg counts or hookworm faecal DNA intensity lack the fidelity required for exploration of hookworm infection as a vaccine/drug testing platform or as a regulated therapy.

BACKGROUND: M5717 is the first plasmodium translation elongation factor 2 inhibitor to reach clinical development as an antimalarial. We aimed to characterise the safety, pharmacokinetics, and antimalarial activity of M5717 in healthy volunteers. METHODS: This first-in-human study was a two-part, single-centre clinical trial done in Brisbane, QLD, Australia. Part one was a double-blind, randomised, placebo-controlled, single ascending dose study in which participants were enrolled into one of nine dose cohorts (50, 100, 200, 400, 600, 1000, 1250, 1800, or 2100 mg) and randomly assigned (3:1) to M5717 or placebo. A sentinel dosing strategy was used for each dose cohort whereby two participants (one assigned to M5717 and one assigned to placebo) were initially randomised and dosed. Randomisation schedules were generated electronically by independent, unblinded statisticians. Part two was an open-label, non-randomised volunteer infection study using the Plasmodium falciparum induced blood-stage malaria model in which participants were enrolled into three dose cohorts. Healthy men and women of non-childbearing potential aged 18-55 years were eligible for inclusion; individuals in the volunteer infection study were required to be malaria naive. Safety and tolerability (primary outcome of the single ascending dose study and secondary outcome of the volunteer infection study) were assessed by frequency and severity of adverse events. The pharmacokinetic profile of M5717 was also characterised (primary outcome of the volunteer infection study and secondary outcome of the single ascending dose study). Parasite clearance kinetics (primary outcome of the volunteer infection study) were assessed by the parasite reduction ratio and the corresponding parasite clearance half-life; the incidence of recrudescence up to day 28 was determined (secondary outcome of the volunteer infection study). Recrudescent parasites were tested for genetic mutations (exploratory outcome). The trial

Dihydroartemisinin-piperaquine is a recommended first-line artemisinin combination therapy for Plasmodium falciparum malaria. Piperaquine is also under consideration for other antimalarial combination therapies. The aim of this study was to develop a pharmacokinetic-pharmacodynamic model that might be useful when optimizing the use of piperaquine in new antimalarial combination therapies. The pharmacokinetic-pharmacodynamic model was developed using data from a previously reported dose-ranging study where 24 healthy volunteers were inoculated with 1,800 blood-stage Plasmodium falciparum parasites. All volunteers received a single oral dose of piperaquine (960 mg, 640 mg, or 480 mg) on day 7 or day 8 after parasite inoculation in separate cohorts. Parasite densities were measured by quantitative PCR (qPCR), and piperaquine levels were measured in plasma samples. We used nonlinear mixed-effect modeling to characterize the pharmacokinetic properties of piperaquine and the parasite dynamics associated with piperaquine exposure. The pharmacokinetics of piperaquine was described by a three-compartment disposition model. A semimechanistic parasite dynamics model was developed to explain the maturation of parasites, sequestration of mature parasites, synchronicity of infections, and multiplication of parasites, as seen in natural clinical infections with P. falciparum malaria. Piperaquine-associated parasite killing was estimated using a maximum effect (Emax) function. Treatment simulations (i.e., 3-day oral dosing of dihydroartemisinin-piperaquine) indicated that to be able to combat multidrug-resistant infections, an ideal additional drug in a new antimalarial triple-combination therapy should have a parasite reduction ratio of ≥102 per life cycle (38.8 h) with a duration of action of ≥2 weeks. The semimechanistic pharmacokinetic-pharmacodynamic model described here offers the potential to be a valuable tool for assessing and optimizing current and new antimalarial drug c

The COVID-19 pandemic has highlighted the importance of understanding the immune response to seasonal human coronavirus (HCoV) infections such as HCoV-NL63, how existing neutralising antibodies to HCoV may modulate responses to SARS-CoV-2 infection, and the utility of seasonal HCoV as human challenge models. Therefore, in this study we quantified HCoV-NL63 neutralising antibody titres in a healthy adult population using plasma from 100 blood donors in Australia. A microneutralisation assay was performed with plasma diluted from 1:10 to 1:160 and tested with the HCoV-NL63 Amsterdam-1 strain. Neutralising antibodies were detected in 71% of the plasma samples, with a median geometric mean titre of 14. This titre was similar to those reported in convalescent sera taken from individuals 3-7 months following asymptomatic SARS-CoV-2 infection, and 2-3 years post-infection from symptomatic SARS-CoV-1 patients. HCoV-NL63 neutralising antibody titres decreased with increasing age (R2 = 0.042, p = 0.038), but did not differ by sex. Overall, this study demonstrates that neutralising antibody to HCoV-NL63 is detectable in approximately 71% of the healthy adult population of Australia. Similar titres did not impede the use of another seasonal human coronavirus (HCoV-229E) in a human challenge model, thus, HCoV-NL63 may be useful as a human challenge model for more pathogenic coronaviruses.

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 represen

Soil-transmitted helminths (STH) infect up to one-quarter of the global population, with a significant associated disease burden. The main human STH are: Ancylostoma spp. and Necator americanus (hookworms); Ascaris lumbricoides, Trichuris trichiura, and Strongyloides stercoralis. The aim of this study was to establish a scalable system for stool STH multiplex quantitative real-time polymerase chain reactions (qPCR). Stool samples collected in Fiji and preserved in potassium dichromate were transferred to Melbourne at ambient temperature. Samples were washed to remove potassium dichromate and DNA was extracted with the Mini-Beadbeater-24 and a column-based kit. A SYBR green qPCR to detect the vertebrate mitochondrial gene was used as a DNA extraction control. Samples were tested using a probe-based multiplex qPCR targeting A. lumbricoides, T. trichiura and S. stercoralis, and in a second multiplex reaction to detect hookworms to the species level (A. duodenale, A. ceylanicum, N. americanus). An internal amplification control in both multiplex assays was included to prevent false-negative results due to PCR inhibitors. Samples were homogenised for a single cycle of 40 seconds to release STH DNA and washed stool was stored for up to 15 weeks at -30°C without compromising DNA. Our multiplex qPCR detected multiple species of STH without reduced sensitivity compared to singleplex. qPCR data from 40 stools was validated against STH-positive stools determined by microscopy. We have developed and validated an efficient and staged system for detecting six clinically important STH affecting humans that could be easily implemented without advanced automation in any qPCR-capable laboratory.

BACKGROUND: Plasmodium vivax has been proposed to infect and replicate in the human spleen and bone marrow. Compared to Plasmodium falciparum, which is known to undergo microvascular tissue sequestration, little is known about the behavior of P. vivax outside of the circulating compartment. This may be due in part to difficulties in studying parasite location and activity in life. METHODS AND FINDINGS: To identify organ-specific changes during the early stages of P. vivax infection, we performed 18-F fluorodeoxyglucose (FDG) positron emission tomography/magnetic resonance imaging (PET/MRI) at baseline and just prior to onset of clinical illness in P. vivax experimentally induced blood-stage malaria (IBSM) and compared findings to P. falciparum IBSM. Seven healthy, malaria-naive participants were enrolled from 3 IBSM trials: NCT02867059, ACTRN12616000174482, and ACTRN12619001085167. Imaging took place between 2016 and 2019 at the Herston Imaging Research Facility, Australia. Postinoculation imaging was performed after a median of 9 days in both species (n = 3 P. vivax; n = 4 P. falciparum). All participants were aged between 19 and 23 years, and 6/7 were male. Splenic volume (P. vivax: +28.8% [confidence interval (CI) +10.3% to +57.3%], P. falciparum: +22.9 [CI -15.3% to +61.1%]) and radiotracer uptake (P. vivax: +15.5% [CI -0.7% to +31.7%], P. falciparum: +5.5% [CI +1.4% to +9.6%]) increased following infection with each species, but more so in P. vivax infection (volume: p = 0.72, radiotracer uptake: p = 0.036). There was no change in FDG uptake in the bone marrow (P. vivax: +4.6% [CI -15.9% to +25.0%], P. falciparum: +3.2% [CI -3.2% to +9.6%]) or liver (P. vivax: +6.2% [CI -8.7% to +21.1%], P. falciparum: -1.4% [CI -4.6% to +1.8%]) following infection with either species. In participants with P. vivax, hemoglobin, hematocrit, and platelet count decreased from baseline at the time of postinoculation imaging. Decrements in hemoglobin and hematocrit were significantl

Plasmodium parasite–specific antibodies are critical for protection against malaria, yet the development of long-lived and effective humoral immunity against Plasmodium takes many years and multiple rounds of infection and cure. Here, we report that the rapid development of short-lived plasmablasts during experimental malaria unexpectedly hindered parasite control by impeding germinal center responses. Metabolic hyperactivity of plasmablasts resulted in nutrient deprivation of the germinal center reaction, limiting the generation of memory B cell and long-lived plasma cell responses. Therapeutic administration of a single amino acid to experimentally infected mice was sufficient to overcome the metabolic constraints imposed by plasmablasts and enhanced parasite clearance and the formation of protective humoral immune memory responses. Thus, our studies not only challenge the current model describing the role and function of blood-stage Plasmodium-induced plasmablasts but they also reveal new targets and strategies to improve anti-Plasmodium humoral immunity.

INTRODUCTION: Celiac disease is an autoimmune disorder where intestinal immunopathology arises after gluten consumption. Previous studies suggested that hookworm infection restores gluten tolerance; however, these studies were small (n = 12) and not placebo controlled. METHODS: We undertook a randomized, placebo-controlled trial of hookworm infection in 54 people with celiac disease. The 94-week study involved treatment with either 20 or 40 Necator americanus third-stage larvae (L3-20 or L3-40) or placebo, followed by escalating gluten consumption (50 mg/d for 12 weeks, 1 g intermittent twice weekly for 12 weeks, 2 g/d sustained for 6 weeks, liberal diet for 1 year). RESULTS: Successful study completion rates at week 42 (primary outcome) were similar in each group (placebo: 57%, L3-20: 37%, and L3-40: 44%; P = 0.61), however gluten-related adverse events were significantly reduced in hookworm-treated participants: Median (range) adverse events/participant were as follows: placebo, 4 (1–9); L3-20, 1 (0–9); and L3-40, 0 (0–3) (P = 0.019). Duodenal villous height:crypt depth deteriorated similarly compared with their enrolment values in each group (mean change [95% confidence interval]: placebo, −0.6 [−1.3 to 0.2]; L3-20, −0.5 [−0.8 to 0.2]; and L3-40, −1.1 [−1.8 to 0.4]; P = 0.12). A retrospective analysis revealed that 9 of the 40 L3-treated participants failed to establish hookworm infections. Although week 42 completion rates were similar in hookworm-positive vs hookworm-negative participants (48% vs 44%, P = 0.43), quality of life symptom scores were lower in hookworm-positive participants after intermittent gluten challenge (mean [95% confidence interval]: 38.9 [33.9–44] vs 45.9 [39.2–52.6]). DISCUSSION: Hookworm infection does not restore tolerance to sustained moderate consumption of gluten (2 g/d) but was associated with improved symptom scores after intermittent consumption of lower, intermittent gluten doses.

Endothelial activation and microvascular dysfunction are key pathogenic processes in severe malaria. We evaluated the early role of these processes in experimentally induced Plasmodium falciparum and P. vivax infection. Participants were enrolled in induced blood-stage malaria clinical trials. Plasma osteoprotegerin, angiopoietin-2, and von Willebrand Factor (vWF) levels were measured as biomarkers of endothelial activation. Microvascular function was assessed using peripheral arterial tonometry and near-infrared spectroscopy, and the endothelial glycocalyx was assessed by sublingual videomicroscopy and measurement of biomarkers of degradation. Forty-five healthy, malaria-naive participants were recruited from 5 studies. Osteoprotegerin and vWF levels increased in participants following inoculation with P. vivax (n = 16) or P. falciparum (n = 15), with the angiopoietin-2 level also increasing in participants following inoculation with P. falciparum. For both species, the most pronounced increase was seen in osteoprotegerin. This was particularly marked in participants inoculated with P. vivax, where the osteoprotegerin level correlated with the levels of parasitemia and the malaria clinical score. There were no changes in measures of endothelial glycocalyx or microvascular function. Plasma biomarkers of endothelial activation increased in early P. falciparum and P. vivax infection and preceded changes in the endothelial glycocalyx or microvascular function. The more pronounced increase in osteoprotegerin suggests that this biomarker may play a role in disease pathogenesis.

Liver transaminase elevations after treatment in malaria volunteer infection studies (VISs) have raised safety concerns. We investigated transaminase elevations from two human Plasmodium vivax VISs where subjects were treated with chloroquine (n = 24) or artefenomel (n = 8) and compared them with studies in Thailand (n = 41) and Malaysia (n = 76). In the VISs, alanine transaminase (ALT) increased to ≥ 2.5 × upper limit of normal (ULN) in 11/32 (34%) volunteers, peaking 5-8 days post-treatment. Transaminase elevations were asymptomatic, were not associated with elevated bilirubin, and resolved by day 42. The risk of an ALT ≥ 2.5 × ULN increased more than 4-fold (odds ratio [OR] 4.28; 95% CI: 1.26-14.59; P = 0.02) for every log10 increase in the parasite clearance burden (PCB), defined as the log-fold reduction in parasitemia 24 hours post-treatment. Although an elevated ALT ≥ 2.5 × ULN was more common after artefenomel than after chloroquine (5/8 [63%] versus 6/24 [25%]; OR 5.0; 95% CI: 0.91-27.47; P = 0.06), this risk disappeared when corrected for PCB. Peak ALT also correlated with peak C-reactive protein (R = 0.44; P = 0.012). Elevations in ALT (≥ 2.5 × ULN) were less common in malaria-endemic settings, occurring in 1/41 (2.5%) Thai patients treated with artefenomel, and in none of 76 Malaysians treated with chloroquine or artemisinin combination therapy. Post-treatment transaminase elevations are common in experimental P. vivax infection but do not appear to impact on participant safety. Although the mechanism of these changes remains uncertain, host inflammatory response to parasite clearance may be contributory.

Artefenomel and DSM265 are two new compounds that have been shown to be well tolerated and effective when administered as monotherapy malaria treatment. This study aimed to determine the safety, pharmacokinetics, and pharmacodynamics of artefenomel and DSM265 administered in combination to healthy subjects in a volunteer infection study using the Plasmodium falciparum-induced blood-stage malaria model. Thirteen subjects were inoculated with parasite-infected erythrocytes on day 0 and received a single oral dose of artefenomel and DSM265 on day 7. Cohort 1 (n = 8) received 200 mg artefenomel plus 100 mg DSM265, and cohort 2 (n = 5) received 200 mg artefenomel plus 50 mg DSM265. Blood samples were collected to measure parasitemia, gametocytemia, and artefenomel-DSM265 plasma concentrations. There were no treatment-related adverse events. The pharmacokinetic profiles of artefenomel and DSM265 were similar to those of the compounds when administered as monotherapy, suggesting no pharmacokinetic interactions. A reduction in parasitemia occurred in all subjects following treatment (log10 parasite reduction ratios over 48 h [PRR48] of 2.80 for cohort 1 and 2.71 for cohort 2; parasite clearance half-lives of 5.17 h for cohort 1 and 5.33 h for cohort 2). Recrudescence occurred in 5/8 subjects in cohort 1 between days 19 and 28 and in 5/5 subjects in cohort 2 between days 15 and 22. Low-level gametocytemia (1 to 330 female gametocytes/ml) was detected in all subjects from day 14. The results of this single-dosing combination study support the further clinical development of the use of artefenomel and DSM265 in combination as a treatment for falciparum malaria. (This study has been registered at ClinicalTrials.gov under identifier NCT02389348.).

Background Growth rate of malaria parasites in the blood of infected subjects is an important measure of efficacy of drugs and vaccines. Methods We used log-linear and sine-wave models to estimate the parasite growth rate of the 3D7 strain of Plasmodium falciparum using data from 177 subjects from 14 induced blood stage malaria (IBSM) studies conducted at QIMR Berghofer. We estimated parasite multiplication rate per 48 hours (PMR48), PMR per life-cycle (PMRLC), and parasite life-cycle duration. We compared these parameters to those from studies conducted elsewhere with infections induced by IBSM (n = 66), sporozoites via mosquito bite (n = 336), or injection (n = 51). Results The parasite growth rate of 3D7 in QIMR Berghofer studies was 0.75/day (95% confidence interval [CI], .73–.77/day), PMR48 was 31.9 (95% CI, 28.7–35.4), PMRLC was 16.4 (95% CI, 15.1–17.8), and parasite life-cycle was 38.8 hours (95% CI, 38.3–39.2 hours). These parameters were similar to estimates from IBSM studies elsewhere (0.71/day, 95% CI, .67–.75/day; PMR48 26.6, 95% CI, 22.2–31.8) but significantly higher (P < .001) than in sporozoite studies (0.47/day, 95% CI, .43–.50/day; PMR48 8.6, 95% CI, 7.3–10.1). Conclusions Parasite growth rates were similar across different IBSM studies and higher than infections induced by sporozoite.

Soil-transmitted helminth (STH) infections are endemic in Indonesia. However, prevalence data for many parts of the country are incomplete. The aim of this study was to determine human STH prevalence and knowledge and practices relating to STH risk behaviour, to provide a current view of the status of STH infection in rural communities in Central Java. A cross-sectional survey of 16 villages was conducted in Semarang, Central Java in 2015. Demographic and household data together with information about knowledge and practices relating to STH and hygiene were elicited through face-to-face interviews. Stool samples were collected and examined using the flotation method. Children (aged 2–12 years) also had their haemoglobin (Hb) levels, height and weight data collected, and BMI estimated. Data were analysed using univariate logistic regression analysis. A total of 6,466 individuals with a mean age of 33.5 years (range: 2–93) from 2,195 households were interviewed. The overall prevalence of STH was 33.8% with Ascaris lumbricoides (roundworm) the predominant nematode identified (prevalence = 26.0%). Hookworm and Trichuris trichiura (whipworm) were found in 7.9% and 1.8% of participants, respectively. Females were at increased odds of infection with A. lumbricoides (adjusted OR 1.14, 95% CI [1.02–1.29], p = 0.02). Adults in age groups 51–60 and over 60 years had the highest odds of being infected with hookworm (adjusted OR 3.01, 95% CI [1.84–4.91], p<0.001 and adjusted OR 3.79, 95% CI [2.30–6.26], p<0.001, respectively) compared to 6–12 year olds. Farmers also had higher odds of being infected with hookworm (adjusted OR 2.36, 95% CI [1.17–4.76], p = 0.02) compared to other occupation categories. Poverty (OR 2.14, 95% CI [1.77–2.58], p<0.001), overcrowding (OR 1.35, 95% CI [1.27–1.44], p<0.001), goat ownership (OR 1.61, 95% CI [1.10–2.41], p = 0.02) and the presence of dry floor space in the home (OR 0.73, 95% CI [0.58–0.91], p = 0.01) were all household factors significant

MMV390048 is a novel antimalarial compound that inhibits Plasmodium phosphatidylinositol-4-kinase. The safety, tolerability, pharmacokinetic profile, and antimalarial activity of MMV390048 were determined in healthy volunteers in three separate studies. A first-in-human, double-blind, randomized, placebo-controlled, single-ascending-dose study was performed. Additionally, a volunteer infection study investigated the antimalarial activity of MMV390048 using the Plasmodium falciparum induced blood-stage malaria (IBSM) model. Due to the high pharmacokinetic variability with the powder-in-bottle formulation used in both of these studies, a third study was undertaken to select a tablet formulation of MMV390048 to take forward into future studies. MMV390048 was generally well tolerated when administered as a single oral dose up to 120 mg, with rapid absorption and a long elimination half-life. Twelve adverse events were considered to be potentially related to MMV390048 in the first-in-human study but with no obvious correlation between these and MMV390048 dose or exposure. Although antimalarial activity was evident in the IBSM study, rapid recrudescence occurred in most subjects after treatment with 20 mg MMV390048, a dose expected to be subtherapeutic. Reformulation of MMV390048 into two tablet formulations (tartaric acid and Syloid) resulted in significantly reduced intersubject pharmacokinetic variability. Overall, the results of this study suggest that MMV390048 is well tolerated in humans, and the pharmacokinetic properties of the compound indicate that it has the potential to be used for antimalarial prophylaxis or inclusion in a single-dose cure. MMV390048 is currently being tested in a phase 2a study in Ethiopian adults with acute, uncomplicated falciparum or vivax malaria monoinfection. (The three clinical trials described here were each registered with ClinicalTrials.gov as follows: first-in-human study, registration no. NCT02230579; IBSM study, registration no.

Scabies is a neglected tropical disease of global significance. Our understanding of host-parasite interactions has been limited, particularly in crusted scabies (CS), a severe clinical manifestation involving hyper-infestation of Sarcoptes scabiei mites. Susceptibility to CS may be associated with immunosuppressive conditions but CS has also been seen in cases with no identifiable risk factor or immune deficit. Due to ethical and logistical difficulties with undertaking research on clinical patients with CS, we adopted a porcine model which parallels human clinical manifestations. Transcriptomic analysis using microarrays was used to explore scabies pathogenesis, and to identify early events differentiating pigs with ordinary (OS) and crusted scabies. Pigs with OS (n = 4), CS (n = 4) and non-infested controls (n = 4) were compared at pre-infestation, weeks 1, 2, 4 and 8 post-infestation. In CS relative to OS, there were numerous differentially expressed genes including pro-inflammatory cytokines (IL17A, IL8, IL19, IL20 and OSM) and chemokines involved in immune cell activation and recruitment (CCL20, CCL27 and CXCL6). The influence of genes associated with immune regulation (CD274/PD-L1 and IL27), immune signalling (TLR2, TLR8) and antigen presentation (RFX5, HLA-5 and HLA-DOB) were highlighted in the early host response to CS. We observed similarities with gene expression profiles associated with psoriasis and atopic dermatitis and confirmed previous observations of Th2/17 pronounced responses in CS. This is the first comprehensive study describing transcriptional changes associated with the development of CS and significantly, the distinction between OS and CS. This provides a basis for clinical follow-up studies, potentially identifying new control strategies for this severely debilitating disease.

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 pa

Chloroquine has been used for the treatment of malaria for > 70 years; however, chloroquine pharmacokinetic (PK) and pharmacodynamic (PD) profile in Plasmodium vivax malaria is poorly understood. The objective of this study was to describe the PK/PD relationship of chloroquine and its major metabolite, desethylchloroquine, in a P. vivax volunteer infection study. We analyzed data from 24 healthy subjects who were inoculated with blood-stage P. vivax malaria and administered a standard treatment course of chloroquine. The PK of chloroquine and desethylchloroquine was described by a two-compartment model with first-order absorption and elimination. The relationship between plasma and whole blood concentrations of chloroquine and P. vivax parasitemia was characterized by a PK/PD delayed response model, where the equilibration half-lives were 32.7 hours (95% confidence interval (CI) 27.4-40.5) for plasma data and 24.1 hours (95% CI 19.0-32.7) for whole blood data. The estimated parasite multiplication rate was 17 folds per 48 hours (95% CI 14-20) and maximum parasite killing rate by chloroquine was 0.213 hour-1 (95% CI 0.196-0.230), translating to a parasite clearance half-life of 4.5 hours (95% CI 4.1-5.0) and a parasite reduction ratio of 400 every 48 hours (95% CI 320-500). This is the first study that characterized the PK/PD relationship between chloroquine plasma and whole blood concentrations and P. vivax clearance using a semimechanistic population PK/PD modeling. This PK/PD model can be used to optimize dosing scenarios and to identify optimal dosing regimens for chloroquine where resistance to chloroquine is increasing.

Malaria is spread by the transmission of sexual stage parasites, called gametocytes. However, with Plasmodium falciparum, gametocytes can only be detected in peripheral blood when they are mature and transmissible to a mosquito, which complicates control efforts. Here, we identify the set of genes overexpressed in patient blood samples with high levels of gametocyte-committed ring stage parasites. Expression of all 18 genes is regulated by transcription factor AP2-G, which is required for gametocytogenesis. We select three genes, not expressed in mature gametocytes, to develop as biomarkers. All three biomarkers we validate in vitro using 6 different parasite lines and develop an algorithm that predicts gametocyte production in ex vivo samples and volunteer infection studies. The biomarkers are also sensitive enough to monitor gametocyte production in asymptomatic P. falciparum carriers allowing early detection and treatment of infectious reservoirs, as well as the in vivo analysis of factors that modulate sexual conversion.

CD4+ T follicular helper cells (Tfh) are key drivers of antibody development. During Plasmodium falciparum malaria in children, the activation of Tfh is restricted to the Th1 subset and not associated with antibody levels. To identify Tfh subsets that are associated with antibody development in malaria, we assess Tfh and antibodies longitudinally in human volunteers with experimental P. falciparum infection. Tfh cells activate during infection, with distinct dynamics in different Tfh subsets. Th2-Tfh cells activate early, during peak infection, while Th1-Tfh cells activate 1 week after peak infection and treatment. Th2-Tfh cell activation is associated with the functional breadth and magnitude of parasite antibodies. In contrast, Th1-Tfh activation is not associated with antibody development but instead with plasma cells, which have previously been shown to play a detrimental role in the development of long-lived immunity. Thus, our study identifies the contrasting roles of Th2 and Th1-Tfh cells during experimental P. falciparum malaria.

Background Malaria transmission from humans to Anopheles mosquitoes requires the presence of gametocytes in human peripheral circulation, and the dynamics of transmission are determined largely by the density and sex ratio of the gametocytes. Molecular methods are thus employed to measure gametocyte densities, particularly when assessing transmission epidemiology and the efficacy of transmission-blocking interventions. However, accurate quantification of male and female gametocytes with molecular methods requires pure male and female gametocytes as reference standards, which are not widely available. Methods qRT-PCR assays were used to quantify levels of sex-specific mRNA transcripts in Plasmodium falciparum female and male gametocytes (pfs25 and pfMGET, respectively) using synthetic complimentary RNA standards and in vitro cultured gametocytes. Assays were validated and assay performance was investigated in blood samples of clinical trial participants using these standards and compared to absolute quantification by droplet digital PCR (ddPCR). Results The number of transcript copies per gametocyte were determined to be 279.3 (95% CI 253.5–307.6) for the female-specific transcript pfs25, and 12.5 (95% CI 10.6–14.9) for the male-specific transcript pfMGET. These numbers can be used to convert from transcript copies/mL to gametocyte/mL. The reportable range was determined to be 5.71 × 106 to 5.71 female gametocytes/mL for pfs25, and 1.73 × 107 to 1.73 × 101 male gametocytes/mL for pfMGET. The limit of detection was 3.9 (95% CI 2.5–8.2) female gametocytes/mL for pfs25, and 26.9 (95% CI 19.3–51.7) male gametocytes/mL for PfMGET. Both assays showed minimal intra-assay and inter-assay variability with coefficient of variation < 3%. No cross-reactivity was observed in both assays in uninfected human blood samples. Comparison of results from ddPCR to qRT-PCR assays on clinical blood samples indicated a high-level agreement (ICC = 0.998 for pfs25 and 0.995 for pfMGET). Concl

Background MMV390048 is the first Plasmodium phosphatidylinositol 4-kinase inhibitor to reach clinical development as a new antimalarial. We aimed to characterize the safety, pharmacokinetics, and antimalarial activity of a tablet formulation of MMV390048. Methods A 2-part, phase 1 trial was conducted in healthy adults. Part 1 was a double-blind, randomized, placebo-controlled, single ascending dose study consisting of 3 cohorts (40, 80, 120 mg MMV390048). Part 2 was an open-label volunteer infection study using the Plasmodium falciparum induced blood-stage malaria model consisting of 2 cohorts (40 mg and 80 mg MMV390048). Results Twenty four subjects were enrolled in part 1 (n = 8 per cohort, randomized 3:1 MMV390048:placebo) and 15 subjects were enrolled in part 2 (40 mg [n = 7] and 80 mg [n = 8] cohorts). One subject was withdrawn from part 2 (80 mg cohort) before dosing and was not included in analyses. No serious or severe adverse events were attributed to MMV390048. The rate of parasite clearance was greater in subjects administered 80 mg compared to those administered 40 mg (clearance half-life 5.5 hours [95% confidence interval {CI}, 5.2–6.0 hours] vs 6.4 hours [95% CI, 6.0–6.9 hours]; P = .005). Pharmacokinetic/pharmacodynamic modeling estimated a minimum inhibitory concentration of 83 ng/mL and a minimal parasiticidal concentration that would achieve 90% of the maximum effect of 238 ng/mL, and predicted that a single 120-mg dose would achieve an adequate clinical and parasitological response with 92% certainty. Conclusions The safety, pharmacokinetics, and pharmacodynamics of MMV390048 support its further development as a partner drug of a single-dose combination therapy for malaria. Clinical Trials Registration NCT02783820 (part 1); NCT02783833 (part 2).

BACKGROUND. Interventions that interrupt Plasmodium vivax transmission or eliminate dormant P. vivax liver-stage parasites will be essential for malaria elimination. Development of these interventions has been hindered by the lack of P. vivax in vitro culture and could be accelerated by a safe and reproducible clinical model in malaria-naive individuals. METHODS. Healthy, malaria-naive adults were enrolled in 2 studies to assess the safety, infectivity, and transmissibility of a new P. vivax isolate. Participants (Study 1, n = 2; Study 2, n = 24) were inoculated with P. vivax–infected red blood cells to initiate infection, and were treated with artemether-lumefantrine (Study 1) or chloroquine (Study 2). Primary endpoints were safety and infectivity of the new isolate. In Study 2, transmission to mosquitoes was also evaluated using mosquito feeding assays, and sporozoite viability was assessed using in vitro cultured hepatocytes. RESULTS. Parasitemia and gametocytemia developed in all participants and was cleared by antimalarial treatment. Adverse events were mostly mild or moderate and none were serious. Sixty-nine percent of participants (11/16) were infectious to Anopheles mosquitoes at peak gametocytemia. Mosquito infection rates reached 97% following membrane feeding with gametocyte-enriched blood, and sporozoites developed into liver-stage schizonts in culture. CONCLUSION. We have demonstrated the safe, reproducible, and efficient transmission of P. vivax gametocytes from humans to mosquitoes, and have established an experimental model that will accelerate the development of interventions targeting multiple stages of the P. vivax life cycle. TRIAL REGISTRATION. ACTRN12614000930684 and ACTRN12616000174482. FUNDING. (Australian) National Health and Medical Research Council Program Grant 1132975 (Study 1). Bill and Melinda Gates Foundation (OPP1111147) (Study 2).

The World Health Organization (WHO) has defined moderate-to-heavy intensity (M&HI) infections with soil-transmitted helminths (Ascaris lumbricoides, Trichuris trichiura and the two hookworms, Ancylostoma duodenale and Necator americanus) based on specific values of eggs per gram of stool, as measured by the Kato-Katz method. There are a variety of novel microscopy and DNA-based methods but it remains unclear whether applying current WHO thresholds on to these methods allows for a reliable classification of M&HI infections. We evaluated both WHO and method-specific thresholds for classifying the M&HI infections for novel microscopic (FECPAKG2, McMaster and Mini-FLOTAC) and DNA-based (qPCR) diagnostic methods. For this, we determined method-specific thresholds that best classified M&HI infections (defined by Kato-Katz and WHO thresholds; reference method) in two multi-country drug efficacy studies. Subsequently, we verified whether applying these method-specific thresholds improved the agreement in classifying M&HI infections compared to the reference method. When we applied the WHO thresholds, the new microscopic methods mainly misclassified M&HI as low intensity, and to a lesser extent low intensity infection as M&HI. For FECPAKG2, applying the method-specific thresholds significantly improved the agreement for Ascaris (moderate → substantial), Trichuris and hookworms (fair → moderate). For Mini-FLOTAC, a significantly improved agreement was observed for hookworms only (fair → moderate). For the other STHs, the agreement was almost perfect and remained unchanged. For McMaster, the method-specific thresholds revealed a fair to a substantial agreement but did not significantly improve the agreement. For qPCR, the method-specific thresholds based on genome equivalents per ml of DNA moderately agreed with the reference method for hookworm and Trichuris infections. For Ascaris, there was a substantial agreement. We defined method-specific thresholds that improved the cla