Melbourne Medical School Collected Works - Research Publications
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ItemSoil-Transmitted Helminths in Children in a Remote Aboriginal Community in the Northern Territory: Hookworm is Rare but Strongyloides stercoralis and Trichuris trichiura Persist(MDPI, 2017-12)(1) Background: soil-transmitted helminths are a problem worldwide, largely affecting disadvantaged populations. The little data available indicates high rates of infection in some remote Aboriginal communities in Australia. Studies of helminths were carried out in the same remote community in the Northern Territory in 1994⁻1996 and 2010⁻2011; (2) Methods: fecal samples were collected from children aged <10 years and examined for helminths by direct smear microscopy. In the 2010⁻2011 study, some fecal samples were also analyzed by agar plate culture and PCR for Strongyloides stercoralis DNA. Serological analysis of fingerprick dried blood spots using a S. stercoralis NIE antigen was also conducted; (3) Results and Conclusions: a reduction in fecal samples positive for S. stercoralis, hookworm and Trichuris trichiura was seen between the studies in 1994⁻1996 and 2010⁻2011, likely reflecting public health measures undertaken in the region to reduce intestinal helminths. Comparison of methods to detect S. stercoralis showed that PCR of fecal samples and serological testing of dried blood spots was at least as sensitive as direct smear microscopy and agar plate culture. These methods have advantages for use in remote field studies.
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ItemPerformance of a High-Sensitivity Rapid Diagnostic Test for Plasmodium falciparum Malaria in Asymptomatic Individuals from Uganda and Myanmar and Naive Human Challenge Infections(AMER SOC TROP MED & HYGIENE, 2017)Sensitive field-deployable diagnostic tests can assist malaria programs in achieving elimination. The performance of a new Alere™ Malaria Ag P.f Ultra Sensitive rapid diagnostic test (uRDT) was compared with the currently available SD Bioline Malaria Ag P.f RDT in blood specimens from asymptomatic individuals in Nagongera, Uganda, and in a Karen Village, Myanmar, representative of high- and low-transmission areas, respectively, as well as in pretreatment specimens from study participants from four Plasmodium falciparum-induced blood-stage malaria (IBSM) studies. A quantitative reverse transcription PCR (qRT-PCR) and a highly sensitive enzyme-linked immunosorbent assay (ELISA) test for histidine-rich protein II (HRP2) were used as reference assays. The uRDT showed a greater than 10-fold lower limit of detection for HRP2 compared with the RDT. The sensitivity of the uRDT was 84% and 44% against qRT-PCR in Uganda and Myanmar, respectively, and that of the RDT was 62% and 0% for the same two sites. The specificities of the uRDT were 92% and 99.8% against qRT-PCR for Uganda and Myanmar, respectively, and 99% and 99.8% against the HRP2 reference ELISA. The RDT had specificities of 95% and 100% against qRT-PCR for Uganda and Myanmar, respectively, and 96% and 100% against the HRP2 reference ELISA. The uRDT detected new infections in IBSM study participants 1.5 days sooner than the RDT. The uRDT has the same workflow as currently available RDTs, but improved performance characteristics to identify asymptomatic malaria infections. The uRDT may be a useful tool for malaria elimination strategies.
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ItemSingle Domain Antibodies as New Biomarker Detectors(MDPI, 2017-12)Biomarkers are defined as indicators of biological processes, pathogenic processes, or pharmacological responses to a therapeutic intervention. Biomarkers have been widely used for early detection, prediction of response after treatment, and for monitoring the progression of diseases. Antibodies represent promising tools for recognition of biomarkers, and are widely deployed as analytical tools in clinical settings. For immunodiagnostics, antibodies are now exploited as binders for antigens of interest across a range of platforms. More recently, the discovery of antibody surface display and combinatorial chemistry techniques has allowed the exploration of new binders from a range of animals, for instance variable domains of new antigen receptors (VNAR) from shark and variable heavy chain domains (VHH) or nanobodies from camelids. These single domain antibodies (sdAbs) have some advantages over conventional murine immunoglobulin owing to the lack of a light chain, making them the smallest natural biomarker binders thus far identified. In this review, we will discuss several biomarkers used as a means to validate diseases progress. The potential functionality of modern singe domain antigen binders derived from phylogenetically early animals as new biomarker detectors for current diagnostic and research platforms development will be described.
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ItemComparison of statistical models to estimate parasite growth rate in the induced blood stage malaria model(BMC, 2017-08-25)BACKGROUND: The efficacy of vaccines aimed at inhibiting the growth of malaria parasites in the blood can be assessed by comparing the growth rate of parasitaemia in the blood of subjects treated with a test vaccine compared to controls. In studies using induced blood stage malaria (IBSM), a type of controlled human malaria infection, parasite growth rate has been measured using models with the intercept on the y-axis fixed to the inoculum size. A set of statistical models was evaluated to determine an optimal methodology to estimate parasite growth rate in IBSM studies. METHODS: Parasite growth rates were estimated using data from 40 subjects published in three IBSM studies. Data was fitted using 12 statistical models: log-linear, sine-wave with the period either fixed to 48 h or not fixed; these models were fitted with the intercept either fixed to the inoculum size or not fixed. All models were fitted by individual, and overall by study using a mixed effects model with a random effect for the individual. RESULTS: Log-linear models and sine-wave models, with the period fixed or not fixed, resulted in similar parasite growth rate estimates (within 0.05 log10 parasites per mL/day). Average parasite growth rate estimates for models fitted by individual with the intercept fixed to the inoculum size were substantially lower by an average of 0.17 log10 parasites per mL/day (range 0.06-0.24) compared with non-fixed intercept models. Variability of parasite growth rate estimates across the three studies analysed was substantially higher (3.5 times) for fixed-intercept models compared with non-fixed intercept models. The same tendency was observed in models fitted overall by study. Modelling data by individual or overall by study had minimal effect on parasite growth estimates. CONCLUSIONS: The analyses presented in this report confirm that fixing the intercept to the inoculum size influences parasite growth estimates. The most appropriate statistical model to estimate the growth rate of blood-stage parasites in IBSM studies appears to be a log-linear model fitted by individual and with the intercept estimated in the log-linear regression. Future studies should use this model to estimate parasite growth rates.
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ItemHuman vaccination against Plasmodium vivax Duffy-binding protein induces strain-transcending antibodies(AMER SOC CLINICAL INVESTIGATION INC, 2017-06-15)BACKGROUND: Plasmodium vivax is the most widespread human malaria geographically; however, no effective vaccine exists. Red blood cell invasion by the P. vivax merozoite depends on an interaction between the Duffy antigen receptor for chemokines (DARC) and region II of the parasite's Duffy-binding protein (PvDBP_RII). Naturally acquired binding-inhibitory antibodies against this interaction associate with clinical immunity, but it is unknown whether these responses can be induced by human vaccination. METHODS: Safety and immunogenicity of replication-deficient chimpanzee adenovirus serotype 63 (ChAd63) and modified vaccinia virus Ankara (MVA) viral vectored vaccines targeting PvDBP_RII (Salvador I strain) were assessed in an open-label dose-escalation phase Ia study in 24 healthy UK adults. Vaccines were delivered by the intramuscular route in a ChAd63-MVA heterologous prime-boost regimen using an 8-week interval. RESULTS: Both vaccines were well tolerated and demonstrated a favorable safety profile in malaria-naive adults. PvDBP_RII-specific ex-vivo IFN-γ T cell, antibody-secreting cell, memory B cell, and serum IgG responses were observed after the MVA boost immunization. Vaccine-induced antibodies inhibited the binding of vaccine homologous and heterologous variants of recombinant PvDBP_RII to the DARC receptor, with median 50% binding-inhibition titers greater than 1:100. CONCLUSION: We have demonstrated for the first time to our knowledge that strain-transcending antibodies can be induced against the PvDBP_RII antigen by vaccination in humans. These vaccine candidates warrant further clinical evaluation of efficacy against the blood-stage P. vivax parasite. TRIAL REGISTRATION: Clinicaltrials.gov NCT01816113. FUNDING: Support was provided by the UK Medical Research Council, UK National Institute of Health Research Oxford Biomedical Research Centre, and the Wellcome Trust.
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ItemPlasmacytoid dendritic cells appear inactive during sub-microscopic Plasmodium falciparum blood-stage infection, yet retain their ability to respond to TLR stimulation(NATURE PORTFOLIO, 2017-06-01)Plasmacytoid dendritic cells (pDC) are activators of innate and adaptive immune responses that express HLA-DR, toll-like receptor (TLR) 7, TLR9 and produce type I interferons. The role of human pDC in malaria remains poorly characterised. pDC activation and cytokine production were assessed in 59 malaria-naive volunteers during experimental infection with 150 or 1,800 P. falciparum-parasitized red blood cells. Using RNA sequencing, longitudinal changes in pDC gene expression were examined in five adults before and at peak-infection. pDC responsiveness to TLR7 and TLR9 stimulation was assessed in-vitro. Circulating pDC remained transcriptionally stable with gene expression altered for 8 genes (FDR < 0.07). There was no upregulation of co-stimulatory molecules CD86, CD80, CD40, and reduced surface expression of HLA-DR and CD123 (IL-3R-α). pDC loss from the circulation was associated with active caspase-3, suggesting pDC apoptosis during primary infection. pDC remained responsive to TLR stimulation, producing IFN-α and upregulating HLA-DR, CD86, CD123 at peak-infection. In clinical malaria, pDC retained HLA-DR but reduced CD123 expression compared to convalescence. These data demonstrate pDC retain function during a first blood-stage P. falciparum exposure despite sub-microscopic parasitaemia downregulating HLA-DR. The lack of evident pDC activation in both early infection and malaria suggests little response of circulating pDC to infection.
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ItemSafety, tolerability, pharmacokinetics, and activity of the novel long-acting antimalarial DSM265: a two-part first-in-human phase 1a/1b randomised study(ELSEVIER SCI LTD, 2017-06)BACKGROUND: DSM265 is a novel antimalarial that inhibits plasmodial dihydroorotate dehydrogenase, an enzyme essential for pyrimidine biosynthesis. We investigated the safety, tolerability, and pharmacokinetics of DSM265, and tested its antimalarial activity. METHODS: Healthy participants aged 18-55 years were enrolled in a two-part study: part 1, a single ascending dose (25-1200 mg), double-blind, randomised, placebo-controlled study, and part 2, an open-label, randomised, active-comparator controlled study, in which participants were inoculated with Plasmodium falciparum induced blood-stage malaria (IBSM) and treated with DSM265 (150 mg) or mefloquine (10 mg/kg). Primary endpoints were DSM265 safety, tolerability, and pharmacokinetics. Randomisation lists were created using a validated, automated system. Both parts were registered with the Australian New Zealand Clinical Trials Registry, number ACTRN12613000522718 (part 1) and number ACTRN12613000527763 (part 2). FINDINGS: In part 1, 73 participants were enrolled between April 12, 2013, and July 14, 2015 (DSM265, n=55; placebo, n=18). In part 2, nine participants were enrolled between Sept 30 and Nov 25, 2013 (150 mg DSM265, n=7; 10 mg/kg mefloquine, n=2). In part 1, 117 adverse events were reported; no drug-related serious or severe events were reported. The most common drug-related adverse event was headache. The mean DSM265 peak plasma concentration (Cmax) ranged between 1310 ng/mL and 34 800 ng/mL and was reached in a median time (tmax) between 1·5 h and 4 h, with a mean elimination half-life between 86 h and 118 h. In part 2, the log10 parasite reduction ratio at 48 h in the DSM265 (150 mg) group was 1·55 (95% CI 1·42-1·67) and in the mefloquine (10 mg/kg) group was 2·34 (2·17-2·52), corresponding to a parasite clearance half-life of 9·4 h (8·7-10·2) and 6·2 h (5·7-6·7), respectively. The median minimum inhibitory concentration of DSM265 in blood was estimated as 1040 ng/mL (range 552-1500), resulting in a predicted single efficacious dose of 340 mg. Parasite clearance was significantly faster in participants who received mefloquine than in participants who received DSM265 (p<0·0001). INTERPRETATION: The good safety profile, long elimination half-life, and antimalarial effect of DSM265 supports its development as a partner drug in a single-dose antimalarial combination treatment. FUNDING: Wellcome Trust, UK Department for International Development, Global Health Innovative Technology Fund, Bill & Melinda Gates Foundation.
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ItemAn environmental assessment and risk map of Ascaris lumbricoides and Necator americanus distributions in Manufahi District, Timor-Leste(PUBLIC LIBRARY SCIENCE, 2017-05)BACKGROUND: In Timor-Leste there have been intermittent and ineffective soil-transmitted helminth (STH) deworming programs since 2004. In a resource-constrained setting, having information on the geographic distribution of STH can aid in prioritising high risk communities for intervention. This study aimed to quantify the environmental risk factors for STH infection and to produce a risk map of STH in Manufahi district, Timor-Leste. METHODOLOGY/PRINCIPAL FINDINGS: Georeferenced cross-sectional data and stool samples were obtained from 2,194 participants in 606 households in 24 villages in the Manufahi District as part of cross sectional surveys done in the context of the "WASH for Worms" randomised controlled trial. Infection status was determined for Ascaris lumbricoides and Necator americanus using real-time quantitative polymerase chain reaction. Baseline infection data were linked to environmental data obtained for each household. Univariable and multivariable multilevel mixed-effects logistic regression analysis with random effects at the village and household level were conducted, with all models adjusted for age and sex. For A. lumbricoides, being a school-aged child increased the odds of infection, whilst higher temperatures in the coolest quarter of the year, alkaline soils, clay loam/loam soils and woody savannas around households were associated with decreased infection odds. For N. americanus, greater precipitation in the driest month, higher average enhanced vegetation index, age and sandy loam soils increased infection odds, whereas being female and living at higher elevations decreased the odds of infection. Predictive risk maps generated for Manufahi based upon these final models highlight the high predicted risk of N. americanus infection across the district and the more focal nature of A. lumbricoides infection. The predicted risk of any STH infection is high across the entire district. CONCLUSIONS/SIGNIFICANCE: The widespread predicted risk of any STH infection in 6 to 18 year olds provides strong evidence to support strategies for control across the entire geographical area. As few studies include soil texture and pH in their analysis, this study adds to a growing body of evidence suggesting these factors influence STH infection distribution. This study also further supports that A. lumbricoides prefers acidic soils, highlighting a potential relatively unexplored avenue for control. TRIAL REGISTRATION: ClinicalTrials.gov ACTRN12614000680662.
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ItemPlasmodium malariae and P. ovale genomes provide insights into malaria parasite evolution(NATURE PORTFOLIO, 2017-02-02)Elucidation of the evolutionary history and interrelatedness of Plasmodium species that infect humans has been hampered by a lack of genetic information for three human-infective species: P. malariae and two P. ovale species (P. o. curtisi and P. o. wallikeri). These species are prevalent across most regions in which malaria is endemic and are often undetectable by light microscopy, rendering their study in human populations difficult. The exact evolutionary relationship of these species to the other human-infective species has been contested. Using a new reference genome for P. malariae and a manually curated draft P. o. curtisi genome, we are now able to accurately place these species within the Plasmodium phylogeny. Sequencing of a P. malariae relative that infects chimpanzees reveals similar signatures of selection in the P. malariae lineage to another Plasmodium lineage shown to be capable of colonization of both human and chimpanzee hosts. Molecular dating suggests that these host adaptations occurred over similar evolutionary timescales. In addition to the core genome that is conserved between species, differences in gene content can be linked to their specific biology. The genome suggests that P. malariae expresses a family of heterodimeric proteins on its surface that have structural similarities to a protein crucial for invasion of red blood cells. The data presented here provide insight into the evolution of the Plasmodium genus as a whole.
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ItemGriseofulvin impairs intraerythrocytic growth of Plasmodium falciparum through ferrochelatase inhibition but lacks activity in an experimental human infection study(NATURE PORTFOLIO, 2017-02-08)Griseofulvin, an orally active antifungal drug used to treat dermatophyte infections, has a secondary effect of inducing cytochrome P450-mediated production of N-methyl protoporphyrin IX (N-MPP). N-MPP is a potent competitive inhibitor of the heme biosynthetic-enzyme ferrochelatase, and inhibits the growth of cultured erythrocyte stage Plasmodium falciparum. Novel drugs against Plasmodium are needed to achieve malaria elimination. Thus, we investigated whether griseofulvin shows anti-plasmodial activity. We observed that the intraerythrocytic growth of P. falciparum is inhibited in red blood cells pretreated with griseofulvin in vitro. Treatment with 100 μM griseofulvin was sufficient to prevent parasite growth and induce the production of N-MPP. Inclusion of the ferrochelatase substrate PPIX blocked the inhibitory activity of griseofulvin, suggesting that griseofulvin exerts its activity through the N-MPP-dependent inhibition of ferrochelatase. In an ex-vivo study, red blood cells from griseofulvin-treated subjects were refractory to the growth of cultured P. falciparum. However, in a clinical trial griseofulvin failed to show either therapeutic or prophylactic effect in subjects infected with blood stage P. falciparum. Although the development of griseofulvin as an antimalarial is not warranted, it represents a novel inhibitor of P. falciparum growth and acts via the N-MPP-dependent inhibition of ferrochelatase.