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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 events occurred; most were of mild severity with three being severe in the 3D7-MBE-008 study. Conclusion: The safety, growth and clearance profiles of the expanded 3D7-MBE-008 MCB closely resemble that of its parent, indicating its suitability for future studies. Trial Registration: Australian New Zealand Clinical Trials registry numbers: P3487 (3D7-V1): ACTRN12619001085167. P3491 (3D7-MBE-008): ACTRN12619001079134 [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Background: The COVID-19 pandemic has resulted in millions of infections, hundreds of thousands of deaths and major societal disruption due to lockdowns and other restrictions introduced to limit disease spread. Relatively little attention has been paid to understanding how the pandemic has affected treatment, prevention and control of malaria, which is a major cause of death and disease and predominantly affects people in less well-resourced settings.Main Body: Recent successes in malaria control and elimination have reduced the global malaria burden, but these gains are fragile and progress has stalled in the past 5 years. Withdrawing successful interventions often results in rapid malaria resurgence, primarily threatening vulnerable young children and pregnant women. Malaria programmes are being affected in many ways by COVID-19. For prevention of malaria, insecticide-treated nets need regular renewal, but distribution campaigns have been delayed or cancelled. For detection and treatment of malaria, individuals may stop attending health facilities, out of fear of exposure to COVID-19, or because they cannot afford transport, and health care workers require additional resources to protect themselves from COVID-19. Supplies of diagnostics and drugs are being interrupted, which is compounded by production of substandard and falsified medicines and diagnostics. These disruptions are predicted to double the number of young African children dying of malaria in the coming year and may impact efforts to control the spread of drug resistance. Using examples from successful malaria control and elimination campaigns, we propose strategies to re-establish malaria control activities and maintain elimination efforts in the context of the COVID-19 pandemic, which is likely to be a long-term challenge. All sectors of society, including governments, donors, private sector and civil society organisations, have crucial roles to play to prevent malaria resurgence. Sparse resources must be allocated efficiently to ensure integrated health care systems that can sustain control activities against COVID-19 as well as malaria and other priority infectious diseases.Conclusion: As we deal with the COVID-19 pandemic, it is crucial that other major killers such as malaria are not ignored. History tells us that if we do, the consequences will be dire, particularly in vulnerable populations. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Background: Timor-Leste has a high prevalence of soil-transmitted helminth (STH) infections. High proportions of the population have been reported as being anaemic, and extremely high proportions of children as stunted or wasted. There have been no published analyses of the contributions of STH to these morbidity outcomes in Timor-Leste. Methods: Using baseline cross-sectional data from 24 communities (18 communities enrolled in a cluster randomised controlled trial, and identically-collected data from six additional communities), analyses of the association between STH infections and community haemoglobin and child development indices were undertaken. Stool samples were assessed for STH using qPCR and participant haemoglobin, heights and weights were measured. Questionnaires were administered to collect demographic and socioeconomic data. Intensity of infection was categorised using correlational analysis between qPCR quantification cycle values and eggs per gram of faeces equivalents, with algorithms generated from seeding experiments. Mixed-effects logistic and multinomial regression were used to assess the association between STH infection intensity classes and anaemia, and child stunting, wasting and underweight. Results: Very high stunting (60%), underweight (60%), and wasting (20%) in children, but low anaemia prevalence (15%), were found in the study communities. STH were not significantly associated with morbidity outcomes. Male children and those in the poorest socioeconomic quintile were significantly more likely to be moderately and severely stunted. Male children were significantly more likely than female children to be severely underweight. Increasing age was also a risk factor for being underweight. Few risk factors emerged for wasting in these analyses. Conclusions: According to World Health Organization international reference standards, levels of child morbidity in this population constitute a public health emergency, although the international reference standards need to be critically evaluated for their applicability in Timor-Leste. Strategies to improve child development and morbidity outcomes, for example via nutrition and iron supplementation programmes, are recommended for these communities. Despite the apparent lack of an association from STH in driving anaemia, stunting, wasting and underweight, high endemicity suggests a need for STH control strategies. [ABSTRACT FROM AUTHOR]

Background: Previous studies from our group demonstrated the anti-inflammatory properties of statins on cardiopulmonary bypass (CPB), through inhibition of neutrophil transendothelial migration. We sought to determine the utility of preoperative statin on patients undergoing cardiac surgery, to investigate any moderating effects on the systemic inflammatory response (SIRS) with CPB, and to evaluate any clinical impact on our patients.Methods: This is a prospective, randomised controlled trial with national regulatory body approval. Eligible patients were already on oral statin therapy. They were then randomly assigned to either investigation arm (n = 15, atorvastatin 80 mg for 2 weeks before surgery) or control arm (n = 15, no change to current statin therapy). Blood and urine samples were collected at 3 timepoints. Postoperative clinical measures were documented.Results: Patients in the investigation arm have significantly lower troponin level (p = 0.016), and lower level of urine neutrophil gelatinase-associated lipocalin (NGAL; p = 0.002); thus demonstrating a lesser degree of cardiac and renal injury in these patients. Higher level of Interleukin-8 (IL-8) at baseline (p = 0.036) and 4 h post cross-clamp removal (p = 0.035) in the investiation arm. A similar trend is also observed in Matrix Metalloproteinase-9 (MMP-9; p > 0.05). There were however no differences in clinical outcomes.Conclusions: Maximizing the dose of statin in patients waiting for cardiac surgery has measurable biological effects. There is evidence of less cardiac and renal damage. The use of preoperative statins and in particular, high dose preoperative statin therapy, may prove a useful new tool for optimal preparation of patients for cardiac surgery.Trial Registration: EudraCT no. 2012-003396-20 . Registered 05 November 2012. [ABSTRACT FROM AUTHOR]

Background: Ferroquine (SSR97193) is a candidate anti-malarial currently undergoing clinical trials for malaria. To better understand its pharmacokinetic (PK) and pharmacodynamic (PD) parameters the compound was tested in the experimentally induced blood stage malaria infection model in volunteers. Methods: Male and non-pregnant female aged 18-50 years were screened for this phase II, controlled, single-centre clinical trial. Subjects were inoculated with ~1800 viable Plasmodium falciparum 3D7A-infected human erythrocytes, and treated with a single-dose of 800 mg ferroquine. Blood samples were taken at defined time-points to measure PK and PD parameters. The blood concentration of ferroquine and its active metabolite, SSR97213, were measured on dry blood spot samples by ultra-performance liquid chromatography with tandem mass spectrometry (LC-MS/MS). Parasitaemia and emergence of gametocytes were monitored by quantitative PCR. Safety was determined by recording adverse events and monitoring clinical laboratory assessments during the course of the study. Results: Eight subjects were enrolled into the study, inoculated with infected erythrocytes and treated with 800 mg ferroquine. Ferroquine was rapidly absorbed with maximal exposure after 4-8 and 4-12 h exposure for SSR97213. Non-compartmental PK analysis resulted in estimates for half-lives of 10.9 and 23.8 days for ferroquine and SSR97213, respectively. Parasite clearance as reported by parasite reduction ratio was 162.9 (95 % CI 141-188) corresponding to a parasite clearance half-life of 6.5 h (95 % CI: 6.4-6.7 h). PK/PD modelling resulted in a predicted minimal parasiticidal concentration of 20 ng/mL, and the single dosing tested in this study was predicted to maintain an exposure above this threshold for 454 h (37.8 days). Although ferroquine was overall well tolerated, transient elevated transaminase levels were observed in three subjects. Paracetamol was the only concomitant treatment among the two out of these three subjects that may have played a role in the elevated transaminases levels. No clinically significant ECG abnormalities were observed. Conclusions: The parameters and PK/PD model derived from this study pave the way to the further rational development of ferroquine as an anti-malarial partner drug. The safety of ferroquine has to be further explored in controlled human trials. [ABSTRACT FROM AUTHOR]

As malaria transmission continues to decrease, an increasing number of countries will enter pre-elimination and elimination. To interrupt transmission, changes in control strategies are likely to require more accurate identification of all carriers of Plasmodium parasites, both symptomatic and asymptomatic, using diagnostic tools that are highly sensitive, high throughput and with fast turnaround times preferably performed in local health service settings. Currently available immunochromatographic lateral low rapid diagnostic tests and field microscopy are unlikely to consistently detect infections at parasite densities less than 100 parasites/µL making them insufficiently sensitive for detecting all carriers. Molecular diagnostic platforms, such as PCR and LAMP, are currently available in reference laboratories, but at a cost both financially and in turnaround time. This review describes the recent progress in developing molecular diagnostic tools in terms of their capacity for high throughput and potential for performance in non-reference laboratories for malaria elimination. [ABSTRACT FROM AUTHOR]

Background: Angiostrongylus mackerrasae is a metastrongyloid nematode endemic to Australia, where it infects the native bush rat, Rattus fuscipes. This lungworm has an identical life cycle to that of Angiostrongylus cantonensis, a leading cause of eosinophilic meningitis in humans. The ability of A. mackerrasae to infect non-rodent hosts, specifically the black flying fox, raises concerns as to its zoonotic potential. To date, data on the taxonomy, epidemiology and population genetics of A. mackerrasae are unknown. Here, we describe the mitochondrial (mt) genome of A. mackerrasae with the aim of starting to address these knowledge gaps. Methods: The complete mitochondrial (mt) genome of A. mackerrasae was amplified from a single morphologically identified adult worm, by long-PCR in two overlapping amplicons (8 kb and 10 kb). The amplicons were sequenced using the MiSeq Illumina platform and annotated using an in-house pipeline. Amino acid sequences inferred from individual protein coding genes of the mt genomes were concatenated and then subjected to phylogenetic analysis using Bayesian inference. Results: The mt genome of A. mackerrasae is 13,640 bp in size and contains 12 protein coding genes (cox1-3, nad1-6, nad4L, atp6 and cob), and two ribosomal RNA (rRNA) and 22 transfer RNA (tRNA) genes. Conclusions: The mt genome of A. mackerrasae has similar characteristics to those of other Angiostrongylus species. Sequence comparisons reveal that A. mackerrasae is closely related to A. cantonensis and the two sibling species may have recently diverged compared with all other species in the genus with a highly specific host selection. This mt genome will provide a source of genetic markers for explorations of the epidemiology, biology and population genetics of A. mackerrasae. [ABSTRACT FROM AUTHOR]

Background: To detect all malaria infections in elimination settings sensitive, high throughput and field deployable diagnostic tools are required. Loop-mediated isothermal amplification (LAMP) represents a possible field-applicable molecular diagnostic tool. However, current LAMP platforms are limited by their capacity for high throughput. Methods: A high-throughput LAMP (HtLAMP) platform amplifying mitochondrial targets using a 96-well microtitre plate platform, processing 85 samples and 11 controls, using hydroxynaphtholblue as a colourimetric indicator was optimized for the detection of malaria parasites. Objective confirmation of visually detectable colour change results was made using a spectrophotometer. A dilution series of laboratory-cultured 3D7 Plasmodium falciparum parasites was used to determine the limit of detection of the HtLAMP assay, using P. falciparum (HtLAMP-Pf) and Plasmodium genus (HtLAMP-Pg) primers, on whole blood and filter paper, and using different DNA extraction protocols. The diagnostic accuracy of HtLAMP was validated using clinical samples from Papua New Guinea, Malaysia, Ghana and The Gambia and its field applicability was evaluated in Kota Marudu district hospital, Sabah, Malaysia. Results: The HtLAMP assay proved to be a simple method generating a visually-detectable blue and purple colour change that could be objectively confirmed in a spectrophotometer at a wavelength of 600 nm. When compared with PCR, overall HtLAMP-Pg had a sensitivity of 98 % (n = 260/266, 95 % Cl 95-99) and specificity 83 % (n = 15/18, 95 % Cl 59-96). HtLAMP-Pf had a sensitivity of 97 % (n = 124/128, 95 % Cl 92-99) and specificity of 96 % (n = 151/157, 95 % Cl 92-99). A validation study in a regional hospital laboratory demonstrated ease of performance and interpretation of the HtLAMP assay. HtLAMP-Pf performed in this field setting had a sensitivity of 100 % (n = 17/17, 95 % Cl 80-100) and specificity of 95 % (n = 123/128,95 % Cl 90-98) compared with multiplex PCR. HtLAMP-Pf also performed well on filter paper samples from asymptomatic Ghanaian children with a sensitivity of 88 % (n = 23/25, 95 % Cl 69-97). Conclusion: This colourimetric HtLAMP assay holds much promise as a field applicable molecular diagnostic tool for the purpose of malaria elimination. [ABSTRACT FROM AUTHOR]

Background: The ability to undertake controlled human malaria infection (CHMI) studies for preliminary evaluation of malaria vaccine candidates and anti-malaria drug efficacy has been limited by the need for access to sporozoite infected mosquitoes, aseptic, purified, cryopreserved sporozoites or blood-stage malaria parasites derived ex vivo from malaria infected individuals. Three different strategies are described for the manufacture of clinical grade cultured malaria cell banks suitable for use in CHMI studies. Methods: Good Manufacturing Practices (GMP)-grade NF54, clinically isolated 3D7, and Plasmodium falciparum research-grade 7G8 blood-stage malaria parasites were cultured separately in GMP-compliant facilities P. falciparum using screened blood components and then cryopreserved to produce three blood-stage malaria cell P. falciparum banks. These cell banks were evaluated according to specific criteria (parasitaemia, identity, viability, sterility, presence of endotoxin, presence of mycoplasma or other viral agents and anti-malarial drug sensitivity of the cell bank in vitro malaria parasites) to ensure they met the criteria to permit product release according to GMP requirements. Results: The NF54, 3D7 and 7G8 cell banks consisted of >78% ring stage parasites with a ring stage P. falciparum parasitaemia of >1.4%. Parasites were viable following thawing. The cell banks were free from contamination in vitro with bacteria, mycoplasma and a broad panel of viruses. The NF54, 3D7 and 7G8 parasites exhibited P. falciparum differential anti-malarial drug susceptibilities. The NF54 and 3D7 parasites were susceptible to all P. falciparum anti-malaria compounds tested, whereas the 7G8 parasites were resistant/had decreased susceptibility P. falciparum to four compounds. Following testing, all defined release criteria were met and the cell banks were P. falciparum deemed suitable for release. Ethical approval has been obtained for administration to human volunteers. Conclusions: The production of cultured blood-stage malaria cell banks represents a suitable approach P. falciparum for the generation of material suitable for CHMI studies. A key feature of this culture-based approach is the ability to take research-grade material through to a product suitable for administration in clinical trials. [ABSTRACT FROM AUTHOR]

Background Rapid diagnostic tests (RDTs) for detection of Plasmodium falciparum infection that target P. falciparum histidine-rich protein 2 (PfHRP2), a protein that circulates in the blood of patients infected with this species of malaria, are widely used to guide case management. Understanding determinants of PfHRP2 availability in circulation is therefore essential to understanding the performance of PfHRP2-detecting RDTs. Methods The possibility that pre-formed host anti-PfHRP2 antibodies may block target antigen detection, thereby causing false negative test results was investigated in this study. Results Anti-PfHRP2 antibodies were detected in 19/75 (25%) of plasma samples collected from patients with acute malaria from Cambodia, Nigeria and the Philippines, as well as in 3/28 (10.7%) asymptomatic Solomon Islands residents. Pre-incubation of plasma samples from subjects with high-titre anti-PfHRP2 antibodies with soluble PfHRP2 blocked the detection of the target antigen on two of the three brands of RDTs tested, leading to false negative results. Pre-incubation of the plasma with intact parasitized erythrocytes resulted in a reduction of band intensity at the highest parasite density, and a reduction of lower detection threshold by ten-fold on all three brands of RDTs tested. Conclusions These observations indicate possible reduced sensitivity for diagnosis of P. falciparum malaria using PfHRP2-detecting RDTs among people with high levels of specific antibodies and low density infection, as well as possible interference with tests configured to detect soluble PfHRP2 in saliva or urine samples. Further investigations are required to assess the impact of pre-formed anti-PfHRP2 antibodies on RDT performance in different transmission settings. [ABSTRACT FROM AUTHOR]

Malaria rapid diagnostic tests (RDTs) play a critical role in malaria case management, surveillance and case investigations. Test performance is largely determined by design and quality characteristics, such as detection sensitivity, specificity, and thermal stability. However, parasite characteristics such as variable or absent expression of antigens targeted by RDTs can also affect RDT performance. Plasmodium falciparum parasites lacking the PfHRP2 protein, the most common target antigen for detection of P. falciparum, have been reported in some regions. Therefore, accurately mapping the presence and prevalence of P. falciparum parasites lacking pfhrp2 would be an important step so that RDTs targeting alternative antigens, or microscopy, can be preferentially selected for use in such regions. Herein the available evidence and molecular basis for identifying malaria parasites lacking PfHRP2 is reviewed, and a set of recommended procedures to apply for future investigations for parasites lacking PfHRP2, is proposed. [ABSTRACT FROM AUTHOR]

Background: Early and accurate diagnosis of Plasmodium falciparum infection is important for providing appropriate treatment to patients with malaria. However, technical limitations of currently available diagnostic tests limit their use in control programs. One possible explanation for the vulnerability of current antibodies used in RDTs is their propensity to degrade at high ambient temperatures. Isolation of new antibodies with better thermal stability represents an appealing approach to improve the performance of RDTs. Methods: In this study, phage display technology was deployed to isolate novel binders by screening a human naïve scFv antibody library against recombinant Plasmodium falciparum histidine rich protein 2 (rPfHRP2). The isolated scFv clones were reformatted to whole IgG and the recombinant mAbs were produced in a mammalian CHO cell expression system. To verify the biological activity of these purified recombinant mAbs, range of functional assays were characterized. Results: Two unique clones (D2 and F9) were isolated after five rounds of biopanning. The reformatted and expressed antibodies demonstrated high binding specificity to malaria recombinant PfHRP2 and native proteins. When 5 μg/mL of mAbs applied, mAb C1-13 had the highest sensitivity, with an OD value of 1, the detection achieved 5 ng/mL of rPfHRP2, followed by mAbs D2 and F9 at 10 ng/mL and 100 ng/mL of rPfHRP2, respectively. Although the sensitivity of mAbs D2 and F9 was lower than the control, these recombinant human mAbs have shown better stability compared to mouse mAb C1-13 at various temperatures in DSC and blot assays. In view of epitope mapping, the predominant motif of rPfHRP2 recognized by mAb D2 was AHHAADAHHA, whereas mAb F9 was one amino acid shorter, resulting in AHHAADAHH. mAb F9 had the strongest binding affinity to rPfHRP2 protein, with a KD value of 4.27 × 10−11 M, followed by control mAb C1-13 at 1.03 × 10−10 M and mAb D2 at 3.05 × 10−10 M. Conclusions: Overall, the performance of these mAbs showed comparability to currently available PfHRP2-specific mouse mAb C1-13. The stability of these novel binders indicate that they merit further work to evaluate their utility in the development of new generation point of care diagnosis of malaria. [ABSTRACT FROM AUTHOR]

Background: Prognostic multibiomarker signatures in prostate cancer (PCa) may improve patient management and provide a bridge for developing novel therapeutics and imaging methods. Our objective was to evaluate the association between expression of 33 candidate protein biomarkers and time to biochemical failure (BF) after prostatectomy. Methods: PCa tissue microarrays were constructed representing 160 patients for whom clinicopathologic features and follow-up data after surgery were available. Immunohistochemistry for each of 33 proteins was quantified using automated digital pathology techniques. Relationships between clinicopathologic features, staining intensity, and time to BF were assessed. Predictive modeling using multiple imputed datasets was performed to identify the top biomarker candidates. Results: In univariate analyses, lymph node positivity, surgical margin positivity, non-localized tumor, age at prostatectomy, and biomarkers CCND1, HMMR, IGF1, MKI67, SIAH2, and SMAD4 in malignant epithelium were significantly associated with time to BF. HMMR, IGF1, and SMAD4 remained significantly associated with BF after adjusting for clinicopathologic features while additional associations were observed for HOXC6 and MAP4K4 following adjustment. In multibiomarker predictive models, 3 proteins including HMMR, SIAH2, and SMAD4 were consistently represented among the top 2, 3, 4, and 5 most predictive biomarkers, and a signature comprised of these proteins best predicted BF at 3 and 5 years. Conclusions: This study provides rationale for investigation of HMMR, HOXC6, IGF1, MAP4K4, SIAH2, and SMAD4 as biomarkers of PCa aggressiveness in larger cohorts. [ABSTRACT FROM AUTHOR]

The diagnosis and management of glucose-6-phosphate dehydrogenase (G6PD) deficiency is a crucial aspect in the current phases of malaria control and elimination, which will require the wider use of 8-aminoquinolines for both reducing Plasmodium falciparum transmission and achieving the radical cure of Plasmodium vivax. 8-aminoquinolines, such as primaquine, can induce severe haemolysis in G6PD-deficient individuals, potentially creating significant morbidity and undermining confidence in 8-aminoquinoline prescription. On the other hand, erring on the side of safety and excluding large numbers of people with unconfirmed G6PD deficiency from treatment with 8-aminoquinolines will diminish the impact of these drugs. Estimating the remaining G6PD enzyme activity is the most direct, accessible, and reliable assessment of the phenotype and remains the gold standard for the diagnosis of patients who could be harmed by the administration of primaquine. Genotyping seems an unambiguous technique, but its use is limited by cost and the large range of recognized G6PD genotypes. A number of enzyme activity assays diagnose G6PD deficiency, but they require a cold chain, specialized equipment, and laboratory skills. These assays are impractical for care delivery where most patients with malaria live. Improvements to the diagnosis of G6PD deficiency are required for the broader and safer use of 8-aminoquinolines to kill hypnozoites, while lower doses of primaquine may be safely used to kill gametocytes without testing. The discussions and conclusions of a workshop conducted in Incheon, Korea in May 2012 to review key knowledge gaps in G6PD deficiency are reported here. [ABSTRACT FROM AUTHOR]

Background: Relapsing Plasmodium vivax infection results in significant morbidity for the individual and is a key factor in transmission. Primaquine remains the only licensed drug for prevention of relapse. To minimize relapse rates, treatment guidelines have recently been revised to recommend an increased primaquine dose, aiming to achieve a cumulative dose of ≥6 mg/kg, i.e. ≥420 mg in a 70 kg patient. The aims of this study were to characterize the epidemiology of P. vivax infection imported into Queensland Australia, to determine the rates of relapse, to investigate the use of primaquine therapy, and its efficacy in the prevention of relapse. Methods: A retrospective study was undertaken of laboratory confirmed P. vivax infection presenting to the two major tertiary hospitals in Queensland, Australia between January 1999 and January 2011. Primaquine dosing was classified as no dose, low dose (<420 mg), high dose (≥420 mg), or unknown. The dose of primaquine prescribed to patients who subsequently relapsed that prescribed to patients who did not relapse. Results: Twenty relapses occurred following 151 primary episodes of P. vivax infection (13.2%). Relapses were confirmed among 3/21 (14.2%), 9/50 (18.0%), 1/54 (1.9%) and 7/18 (38.9%) of patients administered no dose, low dose, high dose and unknown primaquine dose respectively. High dose primaquine therapy was associated with a significantly lower rate of relapse compared to patients who were prescribed low dose therapy (OR 11.6, 95% CI 1.5-519, p = 0.005). Conclusions: Relapse of P. vivax infection is more likely in patients who received low dose primaquine therapy. This study supports the recommendations that high dose primaquine therapy is necessary to minimize relapse of P. vivax malaria. [ABSTRACT FROM AUTHOR]

Background: Obtaining single parasite clones is required for many techniques in malaria research. Cloning by limiting dilution using microscopy-based assessment for parasite growth is an arduous and labor-intensive process. An alternative method for the detection of parasite growth in limiting dilution assays is using a commercial ELISA histidine-rich protein II (HRP2) detection kit. Methods: Detection of parasite growth was undertaken using HRP2 ELISA and compared to thick film microscopy. An HRP2 protein standard was used to determine the detection threshold of the HRP2 ELISA assay, and a HRP2 release model was used to extrapolate the amount of parasite growth required for a positive result. Results: The HRP2 ELISA was more sensitive than microscopy for detecting parasite growth. The minimum level of HRP2 protein detection of the ELISA was 0.11ng/ml. Modeling of HRP2 release determined that 2,116 parasites are required to complete a full erythrocytic cycle to produce sufficient HRP2 to be detected by the ELISA. Under standard culture conditions this number of parasites is likely to be reached between 8 to 14 days of culture. Conclusions: This method provides an accurate and simple way for the detection of parasite growth in limiting dilution assays, reducing time and resources required in traditional methods. Furthermore the method uses spent culture media instead of the parasite-infected red blood cells, enabling culture to continue. [ABSTRACT FROM AUTHOR]

Background: Glucose-6-phosphate dehydrogenase deficiency poses a significant impediment to primaquine use for the elimination of liver stage infection with Plasmodium vivax and for gametocyte clearance, because of the risk of life-threatening haemolytic anaemia that can occur in G6PD deficient patients. Although a range of methods for screening G6PD deficiency have been described, almost all require skilled personnel, expensive laboratory equipment, freshly collected blood, and are time consuming; factors that render them unsuitable for massscreening purposes. Methods: A published WST8/1-methoxy PMS method was adapted to assay G6PD activity in a 96-well format using dried blood spots, and used it to undertake population screening within a malaria survey undertaken in Isabel Province, Solomon Islands. The assay results were compared to a biochemical test and a recently marketed rapid diagnostic test. Results: Comparative testing with biochemical and rapid diagnostic test indicated that results obtained by filter paper assay were accurate providing that blood spots were assayed within 5 days when stored at ambient temperature and 10 days when stored at 4 degrees. Screening of 8541 people from 41 villages in Isabel Province, Solomon Islands revealed the prevalence of G6PD deficiency as defined by enzyme activity < 30% of normal control was 20.3% and a prevalence of severe deficiency that would predispose to primaquine-induced hemolysis (WHO Class I-II) of 6.9%. Conclusions: The assay enabled simple and quick semi-quantitative population screening in a malaria-endemic region. The study indicated a high prevalence of G6PD deficiency in Isabel Province and highlights the critical need to consider G6PD deficiency in the context of P. vivax malaria elimination strategies in Solomon Islands, particularly in light of the potential role of primaquine mass drug administration. [ABSTRACT FROM AUTHOR]

Background: Recent evidence suggests that Sarcoptes scabiei var. hominis mites collected from scabies endemic communities in northern Australia show increasing tolerance to 5% permethrin and oral ivermectin. Previous findings have implicated detoxification pathways in developing resistance to these acaricides. We investigated the contribution of Glutathione S-transferase (GST) enzymes to permethrin and ivermectin tolerance in scabies mites using biochemical and molecular approaches. Results: Increased in vitro survival following permethrin exposure was observed in S. scabiei var. hominis compared to acaricide naïve mites (p < 0.0001). The addition of the GST inhibitor diethyl maleate restored in vitro permethrin susceptibility, confirming GST involvement in permethrin detoxification. Assay of GST enzymatic activity in mites demonstrated that S. scabiei var. hominis mites showed a two-fold increase in activity compared to naïve mites (p < 0.0001). Increased transcription of three different GST molecules was observed in permethrin resistant S. scabiei var. canis- mu 1 (p < 0.0001), delta 1 (p < 0.001), and delta 3 (p < 0.0001). mRNA levels of GST mu 1, delta 3 and Pglycoprotein also significantly increased in S. scabiei var. hominis mites collected from a recurrent crusted scabies patient over the course of ivermectin treatment. Conclusions: These findings provide further support for the hypothesis that increased drug metabolism and efflux mediate permethrin and ivermectin resistance in scabies mites and highlight the threat of emerging acaricide resistance to the treatment of scabies worldwide. This is one of the first attempts to define specific genes involved in GST mediated acaricide resistance at the transcriptional level, and the first application of such studies to S. scabiei, a historically challenging ectoparasite. [ABSTRACT FROM AUTHOR]

Background: Distraction osteogenesis is the standard treatment for the management of lower limb length discrepancy of more than 3 cm and bone loss secondary to congenital anomalies, trauma or infection. This technique consists of an osteotomy of the bone to be lengthened, application of an external fixator, followed by gradual and controlled distraction of the bone ends. Although limb lengthening using the Ilizarov distraction osteogenesis principle yields excellent results in most cases, the technique has numerous problems and is not well tolerated by many children. The objective of the current study is to determine if Botulinum Toxin A (BTX-A), which is known to possess both analgesic and paralytic actions, can be used to alleviate post-operative pain and improve the functional outcome of children undergoing distraction osteogenesis. Methods/Design: The study design consists of a multi centre, randomized, double-blinded, placebo-controlled trial. Patients between ages 5-21 years requiring limb lengthening or deformity correction using distraction will be recruited from 6 different sites (Shriners Hospital for Children in Montreal, Honolulu, Philadelphia and Portland as well as DuPont Hospital for Children in Wilmington, Delaware and Hospital for Sick Children in Toronto, Ont). Approximately 150 subjects will be recruited over 2 years and will be randomized to either receive 10 units per Kg of BTX-A or normal saline (control group) intraoperatively following the surgery. Functional outcome effects will be assessed using pain scores, medication dosages, range of motion, flexibility, strength, mobility function and quality of life of the patient. IRB approval was obtained from all sites and adverse reactions will be monitored vigorously and reported to IRB, FDA and Health Canada. Discussion: BTX-A injection has been widely used world wide with no major side effects reported. However, to the best of our knowledge, this is the first time BTX-A is being used under the context of limb lengthening and deformity correction. Trial Registration: NCT00412035 [ABSTRACT FROM AUTHOR]

Background: The tetraspan protein CD63, originally described as a stage-specific melanoma antigen but also present in a number of normal cells, regulates melanoma cell growth in nude mice, motility in serum containing media, and adhesion to several extracellular matrix proteins. CD63 has been reported to associate with β1 and β2 integrins, but the mechanism of signal transduction by CD63 is not clear. This study examined whether CD63 is associated with protein kinase and can transmit signals in melanoma cells. Methods: Immunoprecipitation and radiolabeling were used to test for association of protein kinase activity with CD63. Adhesion of cells to monoclonal antibodies immobilized to microtiter plates was used to examine the ability of CD63 to transmit signals. Results: CD63 was capable of transmitting a signal in melanoma cells that required extracellular calcium. In the absence of extracellular calcium at the time of binding to the CD63 mAb, the cell was no longer responsive to stimulation by CD63. Immunoprecipitation studies demonstrated protein kinase activity associated with CD63, and phosphoamino acid analysis revealed that most of this protein kinase activity was due to serine kinase activity. Conclusion: The current study suggests that serine protein kinase activity associated with CD63 may play a role in signaling by CD63 in melanoma cells. [ABSTRACT FROM AUTHOR]

Background: Ov-CHI-1 is a chitinase specifically expressed in the infective stage larvae of the human filarial parasite Onchocerca volvulus. Evidence has show that it could be a vaccine candidate, however, there is no data available regarding the immunological status of people naturally exposed to infective stage larvae and thus provoked by this antigen. Method: We analysed the Ov-CHI-1-specific immune response present in four endemic foci of human onchocerciasis (Ecuador, Nigeria, Togo and Cameroon) by enzyme-linked immunosorbent assays and T-cell proliferation assays. Results: In these foci of infection, antibodies to Ov-CHI-1 were found to be present in only 22% of individuals from Ecuador, but were detected in 42-62% of infected individuals in the three foci from West Africa (Nigeria, Togo and Cameroon). There was found to be no relationship between antibody level and age, gender, or infection intensity as indicated by microfilarial density and numbers of skin nodules. The isotype response to Ov-CHI-1 was dominated by the presence of IgG3, IgG1 was present to a lesser extent. Our results show a positive correlation between N- and C-termini of Ov-CHI-1 in their ability to provoke humoral and cellular immune responses in the human. Peripheral blood mononuclear cell (PBMC) proliferative responses to Ov-CHI-1 when assayed, were found to be significantly higher in the individuals from endemic areas and there was a statistically elevated response to Ov-CHI-1 in the infected individuals when compared to putative immune individuals. Conclusion: Ov-CHI-1 is an antigen that we have found strongly induces both humoral and cellular immune responses in humans. [ABSTRACT FROM AUTHOR]

Background: Malaria rapid diagnostic tests (RDTs) represent an important antibody based immunoassay platform. Unfortunately, conventional monoclonal antibodies are subject to degradation shortening shelf lives of RDTs. The variable region of the receptor (VNAR) from shark has a potential as alternative to monoclonal antibodies in RDTs due to high thermal stability. Methods: In this study, new binders derived from shark VNAR domains library were investigated. Following immunization of a wobbegong shark (Orectolobus ornatus) with three recombinant malaria biomarker proteins (PfHRP2, PfpLDH and Pvaldolase), a single domain antibody (sdAb) library was constructed from splenocytes. Target-specific VNAR phage were isolated by panning. One specific clone was selected for expression in Escherichia coli expression system, and study of binding reactivity undertaken. Results: The primary VNAR domain library possessed a titre of 1.16 × 106 pfu/mL. DNA sequence analysis showed 82.5% of isolated fragments appearing to contain an in-frame sequence. After multiple rounds of biopanning, a highly dominant clone specific to PfHRP2 was identified and selected for protein production in an E. coli expression system. Biological characterization showed the recombinant protein expressed in periplasmic has better detection sensitivity than that of cytoplasmic proteins. Assays of binding activity indicated that its reactivity was inferior to the positive control mAb C1–13. Conclusions: Target-specific bacteriophage VNARs were successfully isolated after a series of immunization, demonstrating that phage display technology is a useful tool for selection of antigen binders. Generation of new binding reagents such as VNAR antibodies that specifically recognize the malaria biomarkers represents an appealing approach to improve the performance of RDTs. [ABSTRACT FROM AUTHOR]

Background: Evaluating the efficacy of transmission-blocking interventions relies on mosquito-feeding assays, with transmission typically assessed by microscopic identification of oocysts in mosquito midguts; however, microscopy has limited throughput, sensitivity and specificity. Where low prevalence and intensity mosquito infections occur, as observed during controlled human malaria infection studies or natural transmission, a reliable method for detection and quantification of low-level midgut infection is required. Here, a semi-automated, Taqman quantitative PCR (qPCR) assay sufficiently sensitive to detect a single-oocyst midgut infection is described. Results: Extraction of genomic DNA from Anopheles stephensi midguts using a semi-automated extraction process was shown to have equivalent extraction efficiency to manual DNA extraction. An 18S Plasmodium falciparum qPCR assay was adapted for quantitative detection of P. falciparum midgut oocyst infection using synthetic DNA standards. The assay was validated for sensitivity and specificity, and the limit of detection was 0.7 genomes/µL (95% CI 0.4–1.6 genomes/µL). All microscopy-confirmed oocyst infected midgut samples were detected by qPCR, including all single-oocyst positive midguts. The genome number per oocyst was assessed 8–9 days after feeding assay using both qPCR and droplet digital PCR and was 3722 (IQR: 2951–5453) and 3490 (IQR: 2720–4182), respectively. Conclusions: This semi-automated qPCR method enables accurate detection of low-level P. falciparum oocyst infections in mosquito midguts, and may improve the sensitivity, specificity and throughput of assays used to evaluate candidate transmission-blocking interventions. [ABSTRACT FROM AUTHOR]

The recent article by Mohammed et al. demonstrates an impressive effect of ivermectin mass drug administration for lymphatic filariasis on the burden of scabies. Partnering scabies research within the evaluation and monitoring of Neglected Tropical Disease programmes could potentially increase our understanding of the epidemiology and control of scabies and its important bacterial complications. [ABSTRACT FROM AUTHOR]

An abstract of the article "Elimination challenges in the Pacific: vivax and submicroscopic parasitemia," by James McCarthy is presented.