Chemotherapy is still the cornerstone for malaria control. Developing drugs against Plasmodium parasites and monitoring their efficacy requires methods to accurately determine the parasite killing ...rate in response to treatment. Commonly used techniques essentially measure metabolic activity as a proxy for parasite viability. However, these approaches are susceptible to artefacts, as viability and metabolism are two parameters that are coupled during the parasite life cycle but can be differentially affected in response to drug actions. Moreover, traditional techniques do not allow to measure the speed-of-action of compounds on parasite viability, which is an essential efficacy determinant. We present here a comprehensive methodology to measure in vitro the direct effect of antimalarial compounds over the parasite viability, which is based on limiting serial dilution of treated parasites and re-growth monitoring. This methodology allows to precisely determine the killing rate of antimalarial compounds, which can be quantified by the parasite reduction ratio and parasite clearance time, which are key mode-of-action parameters. Importantly, we demonstrate that this technique readily permits to determine compound killing activities that might be otherwise missed by traditional, metabolism-based techniques. The analysis of a large set of antimalarial drugs reveals that this viability-based assay allows to discriminate compounds based on their antimalarial mode-of-action. This approach has been adapted to perform medium throughput screening, facilitating the identification of fast-acting antimalarial compounds, which are crucially needed for the control and possibly the eradication of malaria.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
MicroRNAs (miRNAs) repress target genes through a poorly defined antisense mechanism. Cell‐free and cell‐based assays have supported the idea that miRNAs repress their target mRNAs by blocking ...initiation of translation, whereas studies in animal models argued against this possibility. We examined endogenous targets of the let‐7 miRNA, an important regulator of stem cell fates. We report that let‐7 represses translation initiation in Caenorhabditis elegans, demonstrating this mode of action for the first time in an organism. Unexpectedly, although the lin‐4 miRNA was previously reported to repress its targets at a step downstream of translation initiation, we also observe repression of translation initiation for this miRNA. This repressive mechanism, which frequently but not always coincides with transcript degradation, requires the GW182 proteins AIN‐1 and AIN‐2, and acts on several mRNAs targeted by different miRNAs. Our analysis of an expanded set of endogenous miRNA targets therefore indicates widespread repression of translation initiation under physiological conditions and establishes C. elegans as a genetic system for dissection of the underlying mechanisms.
Accurate and efficient detection of sub-microscopic malaria infections is crucial for enabling rapid treatment and interruption of transmission. Commercially available malaria LAMP kits have ...excellent diagnostic performance, though throughput is limited by the need to prepare samples individually. Here, we evaluate the clinical performance of a newly developed high throughput (HTP) sample processing system for use in conjunction with the Eiken malaria LAMP kit.
The HTP system utilised dried blood spots (DBS) and liquid whole blood (WB), with parallel sample processing of 94 samples per run. The system was evaluated using 699 samples of known infection status pre-determined by gold standard nested PCR.
The sensitivity and specificity of WB-HTP-LAMP was 98.6% (95% CI, 95.7-100), and 99.7% (95% CI, 99.2-100); sensitivity of DBS-HTP-LAMP was 97.1% (95% CI, 93.1-100), and specificity 100% against PCR. At parasite densities greater or equal to 2 parasites/μL, WB and DBS HTP-LAMP showed 100% sensitivity and specificity against PCR. At densities less than 2 p/μL, WB-HTP-LAMP sensitivity was 88.9% (95% CI, 77.1-100) and specificity was 99.7% (95% CI, 99.2-100); sensitivity and specificity of DBS-HTP-LAMP was 77.8% (95% CI, 54.3-99.5) and 100% respectively.
The HTP-LAMP system is a highly sensitive diagnostic test, with the potential to allow large scale population screening in malaria elimination campaigns.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
A major gap in the Plasmodium vivax elimination toolkit is the identification of individuals carrying clinically silent and undetectable liver-stage parasites, called hypnozoites. This study ...developed a panel of serological exposure markers capable of classifying individuals with recent P. vivax infections who have a high likelihood of harboring hypnozoites. We measured IgG antibody responses to 342 P. vivax proteins in longitudinal clinical cohorts conducted in Thailand and Brazil and identified candidate serological markers of exposure. Candidate markers were validated using samples from year-long observational cohorts conducted in Thailand, Brazil and the Solomon Islands and antibody responses to eight P. vivax proteins classified P. vivax infections in the previous 9 months with 80% sensitivity and specificity. Mathematical models demonstrate that a serological testing and treatment strategy could reduce P. vivax prevalence by 59-69%. These eight antibody responses can serve as a biomarker, identifying individuals who should be targeted with anti-hypnozoite therapy.
Regulated by histone acetyltransferases and deacetylases (HDACs), histone acetylation is a key epigenetic mechanism controlling chromatin structure, DNA accessibility, and gene expression. HDAC ...inhibitors induce growth arrest, differentiation, and apoptosis of tumor cells and are used as anticancer agents. Here we describe the effects of HDAC inhibitors on microbial sensing by macrophages and dendritic cells in vitro and host defenses against infection in vivo. HDAC inhibitors down-regulated the expression of numerous host defense genes, including pattern recognition receptors, kinases, transcription regulators, cytokines, chemokines, growth factors, and costimulatory molecules as assessed by genome-wide microarray analyses or innate immune responses of macrophages and dendritic cells stimulated with Toll-like receptor agonists. HDAC inhibitors induced the expression of Mi-2β and enhanced the DNA-binding activity of the Mi-2/NuRD complex that acts as a transcriptional repressor of macrophage cytokine production. In vivo, HDAC inhibitors increased the susceptibility to bacterial and fungal infections but conferred protection against toxic and septic shock. Thus, these data identify an essential role for HDAC inhibitors in the regulation of the expression of innate immune genes and host defenses against microbial pathogens.
Rapid diagnostic tests (RDTs) are today the most widely used method for malaria diagnosis and are recommended, alongside microscopy, for the confirmation of suspected cases before the administration ...of anti-malarial treatment. The diagnostic performance of RDTs, as compared to microscopy or PCR is well described but the actual analytical sensitivity of current best-in-class tests is poorly documented. This value is however a key performance indicator and a benchmark value needed to developed new RDTs of improved sensitivity.
Thirteen RDTs detecting either the Plasmodium falciparum histidine rich protein 2 (HRP2) or the plasmodial lactate dehydrogenase (pLDH) antigens were selected from the best performing RDTs according to the WHO-FIND product testing programme. The analytical sensitivity of these products was evaluated using a range of reference materials including P. falciparum and Plasmodium vivax whole parasite samples as well as recombinant proteins.
The best performing HRP2-based RDTs could detect all P. falciparum cultured samples at concentrations as low as 0.8 ng/mL of HRP2. The limit of detection of the best performing pLDH-based RDT specifically detecting P. vivax was 25 ng/mL of pLDH.
The analytical sensitivity of P. vivax and Pan pLDH-based RDTs appears to vary considerably from product to product, and improvement of the limit-of-detection for P. vivax detecting RDTs is needed to match the performance of HRP2 and Pf pLDH-based RDTs for P. falciparum. Different assays using different reference materials produce different values for antigen concentration in a given specimen, highlighting the need to establish universal reference assays.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Loop-mediated isothermal DNA amplification (LAMP) methodology offers an opportunity for point-of-care (POC) molecular detection of asymptomatic malaria infections. However, there is still little ...evidence on the feasibility of implementing this technique for population screenings in isolated field settings.
Overall, we recruited 1167 individuals from terrestrial ('road') and hydric ('riverine') communities of the Peruvian Amazon for a cross-sectional survey to detect asymptomatic malaria infections. The technical performance of LAMP was evaluated in a subgroup of 503 samples, using real-time Polymerase Chain Reaction (qPCR) as reference standard. The operational feasibility of introducing LAMP testing in the mobile screening campaigns was assessed based on field-suitability parameters, along with a pilot POC-LAMP assay in a riverine community without laboratory infrastructure.
LAMP had a sensitivity of 91.8% (87.7-94.9) and specificity of 91.9% (87.8-95.0), and the overall accuracy was significantly better among samples collected during road screenings than riverine communities (p≤0.004). LAMP-based diagnostic strategy was successfully implemented within the field-team logistics and the POC-LAMP pilot in the riverine community allowed for a reduction in the turnaround time for case management, from 12-24 hours to less than 5 hours. Specimens with haemolytic appearance were regularly observed in riverine screenings and could help explaining the hindered performance/interpretation of the LAMP reaction in these communities.
LAMP-based molecular malaria diagnosis can be deployed outside of reference laboratories, providing similar performance as qPCR. However, scale-up in remote field settings such as riverine communities needs to consider a number of logistical challenges (e.g. environmental conditions, labour-intensiveness in large population screenings) that can influence its optimal implementation.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Antimalarial drug resistance is a major constraint for malaria control and elimination efforts. Artemisinin-based combination therapy is now the mainstay for malaria treatment. However, delayed ...parasite clearance following treatment with artemisinin derivatives has now spread in the Greater Mekong Sub region and may emerge or spread to other malaria endemic regions. This spread is of great concern for malaria control programmes, as no alternatives to artemisinin-based combination therapies are expected to be available in the near future. There is a need to strengthen surveillance systems for early detection and response to the antimalarial drug resistance threat. Current surveillance is mainly done through therapeutic efficacy studies; however these studies are complex and both time- and resource-intensive. For multiple common antimalarials, parasite drug resistance has been correlated with specific genetic mutations, and the molecular markers associated with antimalarial drug resistance offer a simple and powerful tool to monitor the emergence and spread of resistant parasites. Different techniques to analyse molecular markers associated with antimalarial drug resistance are available, each with advantages and disadvantages. However, procedures are not adequately harmonized to facilitate comparisons between sites. Here we describe the target product profiles for tests to analyse molecular markers associated with antimalarial drug resistance, discuss how use of current techniques can be standardised, and identify the requirements for an ideal product that would allow malaria endemic countries to provide useful spatial and temporal information on the spread of resistance.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Abstract
Background
The diagnosis of malaria, using microscopy or rapid diagnostic tests (RDTs), requires the collection of capillary blood. This procedure is relatively simple to perform but ...invasive and poses potential risks to patients and health workers, arising from the manipulation of potentially infectious bodily fluids. Less or non-invasive diagnostic tests, based on urine, saliva or requiring no sampling, have the potential to generate less discomfort for the patient and to offer simpler and less risky testing procedures that could be safely performed by untrained staff or even self-performed. To explore the potential acceptance and perceived value of such non-invasive tests, an online, international survey was conducted to gather feedback from National Malaria Control Programme (NMCP) representatives.
Methods
An online survey comprising nineteen questions, available in English, French or Spanish, was emailed to 300 individuals who work with NMCPs in malaria-endemic countries. Answers were collected between November and December 2017; responses were qualitatively analysed to identify key themes and trends and quantitatively analysed to determine average values stratified by region.
Results
Responses were received from 70 individuals, from 33 countries. Approximately half of the respondents (52 %) considered current blood-based tests for malaria to be minimally invasive and non-problematic in their setting. For these participants, non-invasive tests would only be of interest if they brought additional performance improvements, as compared with the performance of microscopy and RDTs. Most respondents were of the view that saliva-based (80 %) and urine-based (66 %) tests would be more readily acceptable among children than blood-based tests. Potential use-case scenarios of interest for both saliva- and urine-based tests were ease-of-testing by community health workers, additional surveillance, self-testing, and outbreak investigation. Many respondents (41 %) thought that if saliva-based tests retailed at <$0.50 per unit they could largely replace conventional RDTs, whereas only 25 % of respondents thought a similarly priced urine-based test would do so.
Conclusions
Although limited to NMCP stakeholders, this survey indicated that current tests for malaria, based on capillary blood, are generally perceived to be minimally invasive and non-problematic. Non-invasive tests, especially if saliva-based, would be welcome if they could match or out-perform the price and performance of current blood-based tests.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Resistance is a constant challenge for anti-infective drug development. Since they kill sensitive organisms, anti-infective agents are bound to exert an evolutionary pressure toward the emergence and ...spread of resistance mechanisms, if such resistance can arise by stochastic mutation events. New classes of medicines under development must be designed or selected to stay ahead in this vicious circle of resistance control. This involves both circumventing existing resistance mechanisms and selecting molecules which are resilient against the development and spread of resistance. Cell-based screening methods have led to a renaissance of new classes of anti-malarial medicines, offering us the potential to select and modify molecules based on their resistance potential. To that end, a standardized in vitro methodology to assess quantitatively these characteristics in Plasmodium falciparum during the early phases of the drug development process has been developed and is presented here. It allows the identification of anti-malarial compounds with overt resistance risks and the prioritization of the most robust ones. The integration of this strategy in later stages of development, registration, and deployment is also discussed.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
PDF
