Culture-adapted Plasmodium falciparum isolates from UK travellers: in vitro drug sensitivity, clonality and drug resistance markers.

BACKGROUND: The screening of lead compounds against in vitro parasite cultures is an essential step in the development of novel anti-malarial drugs, but currently relies on laboratory parasite lines established in vitro during the last century. This study sought to establish in continuous culture a series of recent Plasmodium falciparum isolates to represent the current parasite populations in Africa, all of which are now exposed to artemisinin combination therapy. METHODS: Pre-treatment P. falciparum isolates were obtained in EDTA, and placed into continuous culture after sampling of DNA. One post-treatment blood sample was also collected for each donor to monitor parasite clonality during clearance in vivo. IC₅₀ estimates were obtained for 11 anti-malarial compounds for each established parasite line, clonal multiplicity measured in vivo and in vitro, and polymorphic sites implicated in parasite sensitivity to drugs were investigated at the pfmdr1, pfcrt, pfdhfr, pfdhps and pfap2mu loci before and after treatment, and in the cultured lines. RESULTS: Plasmodium falciparum isolates from seven malaria patients with recent travel to three West African and two East African countries were successfully established in long-term culture. One of these, HL1211, was from a patient with recrudescent parasitaemia 14 days after a full course of artemether-lumefantrine. All established culture lines were shown to be polyclonal, reflecting the in vivo isolates from which they were derived, and at least two lines reliably produce gametocytes in vitro. Two lines displayed high chloroquine IC₅₀ estimates, and carried the CVIET haplotype at codons 72-76, whereas the remaining five lines carried the CVMNK haplotype and were sensitive in vitro. All were sensitive to the endoperoxides dihydroartemisinin and OZ277, but IC₅₀ estimates for lumefantrine varied, with the least sensitive parasites carrying pfmdr1 alleles encoding Asn at codon 86. CONCLUSIONS: This study describes the establishment in continuous culture, in vitro drug sensitivity testing and molecular characterization of a series of multiclonal P. falciparum isolates taken directly from UK malaria patients following recent travel to various malaria-endemic countries in Africa. These "HL" isolates are available as an open resource for studies of drug response, antigenic diversity and other aspects of parasite biology

Statistical Properties of Share Volume Traded in Financial Markets

We quantitatively investigate the ideas behind the often-expressed adage `it takes volume to move stock prices', and study the statistical properties of the number of shares traded $Q_{\Delta t}$ for a given stock in a fixed time interval $\Delta t$. We analyze transaction data for the largest 1000 stocks for the two-year period 1994-95, using a database that records every transaction for all securities in three major US stock markets. We find that the distribution $P(Q_{\Delta t})$ displays a power-law decay, and that the time correlations in $Q_{\Delta t}$ display long-range persistence. Further, we investigate the relation between $Q_{\Delta t}$ and the number of transactions $N_{\Delta t}$ in a time interval $\Delta t$, and find that the long-range correlations in $Q_{\Delta t}$ are largely due to those of $N_{\Delta t}$. Our results are consistent with the interpretation that the large equal-time correlation previously found between $Q_{\Delta t}$ and the absolute value of price change $| G_{\Delta t} |$ (related to volatility) are largely due to $N_{\Delta t}$.Comment: 4 pages, two-column format, four figure

Quantifying Stock Price Response to Demand Fluctuations

We address the question of how stock prices respond to changes in demand. We quantify the relations between price change $G$ over a time interval $\Delta t$ and two different measures of demand fluctuations: (a) $\Phi$, defined as the difference between the number of buyer-initiated and seller-initiated trades, and (b) $\Omega$, defined as the difference in number of shares traded in buyer and seller initiated trades. We find that the conditional expectations $<G >_{\Omega}$ and $_{\Phi}$ of price change for a given $\Omega$ or $\Phi$ are both concave. We find that large price fluctuations occur when demand is very small --- a fact which is reminiscent of large fluctuations that occur at critical points in spin systems, where the divergent nature of the response function leads to large fluctuations.Comment: 4 pages (multicol fomat, revtex

Analytical sensitivity of current best-in-class malaria rapid diagnostic tests

BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSION: 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

Development and clinical performance of high throughput loop-mediated isothermal amplification for detection of malaria.

BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: The HTP-LAMP system is a highly sensitive diagnostic test, with the potential to allow large scale population screening in malaria elimination campaigns

Economic Fluctuations and Diffusion

Stock price changes occur through transactions, just as diffusion in physical systems occurs through molecular collisions. We systematically explore this analogy and quantify the relation between trading activity - measured by the number of transactions $N_{\Delta t}$ - and the price change $G_{\Delta t}$, for a given stock, over a time interval $[t, t+\Delta t]$. To this end, we analyze a database documenting every transaction for 1000 US stocks over the two-year period 1994-1995. We find that price movements are equivalent to a complex variant of diffusion, where the diffusion coefficient fluctuates drastically in time. We relate the analog of the diffusion coefficient to two microscopic quantities: (i) the number of transactions $N_{\Delta t}$ in $\Delta t$, which is the analog of the number of collisions and (ii) the local variance $w^2_{\Delta t}$ of the price changes for all transactions in $\Delta t$, which is the analog of the local mean square displacement between collisions. We study the distributions of both $N_{\Delta t}$ and $w_{\Delta t}$, and find that they display power-law tails. Further, we find that $N_{\Delta t}$ displays long-range power-law correlations in time, whereas $w_{\Delta t}$ does not. Our results are consistent with the interpretation that the pronounced tails of the distribution of $G_{\Delta t} are due to$w_{\Delta t}$, and that the long-range correlations previously found for$| G_{\Delta t} |$are due to$N_{\Delta t}$.Comment: RevTex 2 column format. 6 pages, 36 references, 15 eps figure

MFGE8 does not influence chorio-retinal homeostasis or choroidal neovascularization in vivo

Purpose: Milk fat globule-epidermal growth factor-factor VIII (MFGE8) is necessary for diurnal outer segment phagocytosis and promotes VEGF-dependent neovascularization. The prevalence of two single nucleotide polymorphisms (SNP) in MFGE8 was studied in two exsudative or “wet” Age-related Macular Degeneration (AMD) groups and two corresponding control groups. We studied the effect of MFGE8 deficiency on retinal homeostasis with age and on choroidal neovascularization (CNV) in mice. Methods: The distribution of the SNP (rs4945 and rs1878326) of MFGE8 was analyzed in two groups of patients with “wet” AMD and their age-matched controls from Germany and France. MFGE8-expressing cells were identified in Mfge8+/− mice expressing ß-galactosidase. Aged Mfge8+/− and Mfge8−/− mice were studied by funduscopy, histology, electron microscopy, scanning electron microscopy of vascular corrosion casts of the choroid, and after laser-induced CNV. Results: rs1878326 was associated with AMD in the French and German group. The Mfge8 promoter is highly active in photoreceptors but not in retinal pigment epithelium cells. Mfge8−/− mice did not differ from controls in terms of fundus appearance, photoreceptor cell layers, choroidal architecture or laser-induced CNV. In contrast, the Bruch's membrane (BM) was slightly but significantly thicker in Mfge8−/− mice as compared to controls. Conclusions: Despite a reproducible minor increase of rs1878326 in AMD patients and a very modest increase in BM in Mfge8−/− mice, our data suggests that MFGE8 dysfunction does not play a critical role in the pathogenesis of AMD

Defining the next generation of Plasmodium vivax diagnostic tests for control and elimination: Target product profiles.

The global prevalence of malaria has decreased over the past fifteen years, but similar gains have not been realized against Plasmodium vivax because this species is less responsive to conventional malaria control interventions aimed principally at P. falciparum. Approximately half of all malaria cases outside of Africa are caused by P. vivax. This species places dormant forms in human liver that cause repeated clinical attacks without involving another mosquito bite. The diagnosis of acute patent P. vivax malaria relies primarily on light microscopy. Specific rapid diagnostic tests exist but typically perform relatively poorly compared to those for P. falciparum. Better diagnostic tests are needed for P. vivax. To guide their development, FIND, in collaboration with P. vivax experts, identified the specific diagnostic needs associated with this species and defined a series of three distinct target product profiles, each aimed at a particular diagnostic application: (i) point-of-care of acutely ill patients for clinical care purposes; (ii) point-of-care asymptomatic and otherwise sub-patent residents for public health purposes, e.g., mass screen and treat campaigns; and (iii) ultra-sensitive not point-of-care diagnosis for epidemiological research/surveillance purposes. This report presents and discusses the rationale for these P. vivax-specific diagnostic target product profiles. These contribute to the rational development of fit-for-purpose diagnostic tests suitable for the clinical management, control and elimination of P. vivax malaria

LAMP kit for diagnosis of non-falciparum malaria in Plasmodium ovale infected patients

Background: Microscopy and rapid diagnosis tests have a limited sensitivity in diagnosis of malaria by Plasmodium ovale. The LAMP kit (LoopAMP (R)) can be used in the field without special equipment and could have an important role in malaria control programmes in endemic areas and for malaria diagnosis in returned travellers. The performance of the Pan primer of the kit in detecting malaria by P. ovale was compared with the results of standard nPCR in samples of patients returning from P. ovale endemic areas. Methods: Plasmodium ovale positive samples (29, tested by PCR and/or microscopy) and malaria negative specimens (398, tested by microscopy and PCR) were collected in different hospitals of Europe from June 2014 to March 2016 and frozen at -20 degrees C. Boil and spin method was used to extract DNA from all samples and amplification was performed with LoopAMP (R) MALARIA kit (Eiken Chemical, Japan) in an automated turbidimeter (Eiken 500). The results of LAMP read by turbidimetry and with the naked eye were compared. Results: The kit showed a sensitivity of 100% and a specificity of 97.24% with positive and negative predictive values of 72.5 and 100%, respectively. Naked eyed readings were in accordance with turbidimetry readings (sensitivity, 92.5%, specificity, 98.96% and positive and negative predictive values, respectively, 90.24 and 99.22%). The limit of detection of LAMP assay for P. ovale was between 0.8 and 2 parasites/mu l. Conclusions: The Pan primer of the Malaria kit LoopAMP (R) can detect P. ovale at very low-levels and showed a predictive negative value of 100%. This tool can be useful in malaria control and elimination programmes and in returned travellers from P. ovale endemic areas. Naked eye readings are equivalent to automated turbidimeter readings in specimens obtained with EDTA.Peer reviewe