Characterization of the commercially-available fluorescent chloroquine-BODIPY conjugate, LynxTag-CQGREEN, as a marker for chloroquine resistance and uptake in a 96-well plate assay

Chloroquine was a cheap, extremely effective drug against Plasmodium falciparum until resistance arose. One approach to reversing resistance is the inhibition of chloroquine efflux from its site of action, the parasite digestive vacuole. Chloroquine accumulation studies have traditionally relied on radiolabelled chloroquine, which poses several challenges. There is a need for development of a safe and biologically relevant substitute. We report here a commercially-available green fluorescent chloroquine-BODIPY conjugate, LynxTag-CQGREEN, as a proxy for chloroquine accumulation. This compound localized to the digestive vacuole of the parasite as observed under confocal microscopy, and inhibited growth of chloroquine-sensitive strain 3D7 more extensively than in the resistant strains 7G8 and K1. Microplate reader measurements indicated suppression of LynxTag-CQGREEN efflux after pretreatment of parasites with known reversal agents. Microsomes carrying either sensitive or resistant-type PfCRT were assayed for uptake; resistant-type PfCRT exhibited increased accumulation of LynxTag-CQGREEN, which was suppressed by pretreatment with known chemosensitizers. Eight laboratory strains and twelve clinical isolates were sequenced for PfCRT and Pgh1 haplotypes previously reported to contribute to drug resistance, and pfmdr1 copy number and chloroquine IC50s were determined. These data were compared with LynxTag-CQGREEN uptake/fluorescence by multiple linear regression to identify genetic correlates of uptake. Uptake of the compound correlated with the logIC50 of chloroquine and, more weakly, a mutation in Pgh1, F1226Y

The dominant Anopheles vectors of human malaria in the Asia-Pacific region: occurrence data, distribution maps and bionomic précis

<p>Abstract</p> <p>Background</p> <p>The final article in a series of three publications examining the global distribution of 41 dominant vector species (DVS) of malaria is presented here. The first publication examined the DVS from the Americas, with the second covering those species present in Africa, Europe and the Middle East. Here we discuss the 19 DVS of the Asian-Pacific region. This region experiences a high diversity of vector species, many occurring sympatrically, which, combined with the occurrence of a high number of species complexes and suspected species complexes, and behavioural plasticity of many of these major vectors, adds a level of entomological complexity not comparable elsewhere globally. To try and untangle the intricacy of the vectors of this region and to increase the effectiveness of vector control interventions, an understanding of the contemporary distribution of each species, combined with a synthesis of the current knowledge of their behaviour and ecology is needed.</p> <p>Results</p> <p>Expert opinion (EO) range maps, created with the most up-to-date expert knowledge of each DVS distribution, were combined with a contemporary database of occurrence data and a suite of open access, environmental and climatic variables. Using the Boosted Regression Tree (BRT) modelling method, distribution maps of each DVS were produced. The occurrence data were abstracted from the formal, published literature, plus other relevant sources, resulting in the collation of DVS occurrence at 10116 locations across 31 countries, of which 8853 were successfully geo-referenced and 7430 were resolved to spatial areas that could be included in the BRT model. A detailed summary of the information on the bionomics of each species and species complex is also presented.</p> <p>Conclusions</p> <p>This article concludes a project aimed to establish the contemporary global distribution of the DVS of malaria. The three articles produced are intended as a detailed reference for scientists continuing research into the aspects of taxonomy, biology and ecology relevant to species-specific vector control. This research is particularly relevant to help unravel the complicated taxonomic status, ecology and epidemiology of the vectors of the Asia-Pacific region. All the occurrence data, predictive maps and EO-shape files generated during the production of these publications will be made available in the public domain. We hope that this will encourage data sharing to improve future iterations of the distribution maps.</p

Fluid prescription practices of anesthesiologists managing patients undergoing elective colonoscopy: an observational study.

BACKGROUND: Routine fluid prescription is common practice amongst anesthesiologists caring for patients undergoing colonoscopy. However there is limited information about routine procedural fluid prescription practices of anesthesiologists in this setting. Routine fluid administration may also have important pharmaco-economic implications for the health care budget. Therefore we performed a prospective observational study assessing the fluid prescription practices of anesthesiologists caring for patients undergoing elective colonoscopy. METHODS: With Institutional Review Board approval, adult patients receiving procedural fluid intervention during elective colonoscopy were included. DATA COLLECTED: size of intravenous cannula inserted, volumes of fluid administered, adverse events, procedure duration, and pharmaco-economic costs associated with fluid prescription. Anesthesiologists and gastroenterologists were blinded to the study. RESULTS: We collected data on 289 patients who received fluid prescription by their attending anesthesiologist. Median patient age: 48 yrs (range 18-83), gender: 174 (60%) female; median duration of procedure: 24 minutes (range 12-48). Cannula size: 181 (63%) patients received a 22G cannula or smaller. Median volume of fluid administered during the colonoscopy was 325 ml (range 0 to 1000 ml). Median duration of the procedure: 25 minutes (range 12 to 48 minutes). Median volume of fluid administered in the post anaesthesia recovery unit: 450 ml (range 0 to 1000 ml). Fifteen patients (5%) became hypotensive during the procedure and two patients (<1%) developed hypotension in the PACU. There was no difference in the median fluid requirements between patients with hypotension and those without. Fluid volumes were strongly associated with increasing cannula diameter (p = 0.0001), however there was no association between fluid volumes administered and vasopressor use, peri-procedural adverse events, or procedure duration. At our institution fluid therapy currently cost about AUD$4.90 per patient: 1 L crystalloid$1.18 and fluid delivery set $3.77 Our institution performs over 9000 endoscopic procedures annually with fluid therapy costing about$45,000/year. CONCLUSIONS: Routine fluid prescription by anesthesiologists managing patients undergoing colonoscopy was ineffective with low actual fluid volumes delivered during the procedure. There was no association between volumes of fluid delivered and procedural hypotension, adverse events, or procedure duration. Anesthesiologists should question the clinical and pharmaco-economic value of routine fluid administration for patients undergoing elective endoscopy

Contrasting ex vivo efficacies of "reversed chloroquine" compounds in chloroquine-resistant Plasmodium falciparum and P. vivax isolates.

Chloroquine (CQ) has been the mainstay of malaria treatment for more than 60 years. However, the emergence and spread of CQ resistance now restrict its use to only a few areas where malaria is endemic. The aim of the present study was to investigate whether a novel combination of a CQ-like moiety and an imipramine-like pharmacophore can reverse CQ resistance ex vivo. Between March to October 2011 and January to September 2013, two "reversed chloroquine" (RCQ) compounds (PL69 and PL106) were tested against multidrug-resistant field isolates of Plasmodium falciparum (n = 41) and Plasmodium vivax (n = 45) in Papua, Indonesia, using a modified ex vivo schizont maturation assay. The RCQ compounds showed high efficacy against both CQ-resistant P. falciparum and P. vivax field isolates. For P. falciparum, the median 50% inhibitory concentrations (IC50s) were 23.2 nM for PL69 and 26.6 nM for PL106, compared to 79.4 nM for unmodified CQ (P &lt; 0.001 and P = 0.036, respectively). The corresponding values for P. vivax were 19.0, 60.0, and 60.9 nM (P &lt; 0.001 and P = 0.018, respectively). There was a significant correlation between IC50s of CQ and PL69 (Spearman's rank correlation coefficient [r s] = 0.727, P &lt; 0.001) and PL106 (rs = 0.830, P &lt; 0.001) in P. vivax but not in P. falciparum. Both RCQs were equally active against the ring and trophozoite stages of P. falciparum, but in P. vivax, PL69 and PL106 showed less potent activity against trophozoite stages (median IC50s, 130.2 and 172.5 nM) compared to ring stages (median IC50s, 17.6 and 91.3 nM). RCQ compounds have enhanced ex vivo activity against CQ-resistant clinical isolates of P. falciparum and P. vivax, suggesting the potential use of reversal agents in antimalarial drug development. Interspecies differences in RCQ compound activity may indicate differences in CQ pharmacokinetics between the two Plasmodium species