Validated HPLC-UV method for amphotericin B quantification in a critical patient receiving AmBisome and treated with extracorporeal replacement therapies.

Amphotericin B (AMB) is a polyene macrolide antifungal agent used for treating invasive fungal infections. Liposomal AMB is a lipid dosage form, available as AmBisome, which reduces the toxicity of the drug. A simple HPLC-UV method was developed for the determination of AMB in plasma to study its pharmacokinetic profile in a critical patient receiving AmBisome and treated with extracorporeal replacement therapies. Sample preparation was performed using plasma deproteinization and drug release from liposome by the addition of acetonitrile (ACN)/zinc sulfate and ultrasonication. Chromatographic separation was performed using a C18 column and a mobile phase consisting of phosphate buffer (pH 3.0)/ACN (65/35, v/v). The UV detector was set at 407 nm. The total run time analysis was 23 min. The method was validated according to the standard guidelines and applied to study the pharmacokinetics of AMB in a critical patient. The total run time analysis obtained was shorter than that of the previously reported methods, being useful for therapeutic drug monitoring or pharmacokinetic profile research.

Chalcogenide nanoparticles and organic photosensitizers for synergetic antimicrobial photodynamic therapy.

Synergistic antimicrobial effects were observed for copper sulfide (CuS) nanoparticles together with indocyanine green (ICG) in the elimination of wild type pathogenic bacteria (Staphylococcus aureus ATCC 29213 and Pseudomonas aeruginosa ATCC 27853) and also opportunistic fungal infective yeast (Candida albicans ATCC 10231). Furthermore, large antibacterial effects were observed for clinical isolates of Methicillin-resistant S. aureus (MRSA) PFGE strain-type USA300. This efficient antimicrobial action was attributed to the combined extra- and intracellular generation of reactive oxygen species upon light irradiation. Instead of the use of visible-light for the activation of common photosensitizers, both ICG and CuS nanoparticles can be activated in the near infrared (NIR)-region of the electromagnetic spectrum and therefore, superior tissue penetration would be expected in a potential elimination of pathogenic microorganisms not only on the skin but also in the soft tissue. In the different bacteria studied a 3-log reduction in the bacterial counts was achieved after only 6 min of NIR irradiation and treatment with ICG or CuS alone at concentrations of 40 and 160 µg mL-1, respectively. A maximum bactericidal effect against S. aureus and USA300 strains was obtained for the combination of both photosensitizers at the same concentration. Regarding P. aeruginosa, a 4-log reduction in the CFU was observed for the combination of CuS and ICG at various concentrations. In Candida albicans the combination of both ICG and CuS and light irradiation showed an antimicrobial dose-dependent effect with the reduction of at least 3-log in the cell counts for the combination of ICG + CuS at reduced concentrations. The observed antimicrobial effect was solely attributed to a photodynamic effect and any photothermal effect was avoided to discard any potential thermal injury in a potential clinical application. The generation of reactive oxygen species upon near infrared-light irradiation for those photosensitizers used was measured either alone or in combination. The cytocompatibility of the proposed materials at the doses used in photodynamic therapy was also demonstrated in human dermal fibroblasts and keratinocytes by cell culturing and flow cytometry studies.

Speciation across mountains: Phylogenomics, species delimitation and taxonomy of the Liolaemus leopardinus clade (Squamata, Liolaemidae).

Organisms rapidly diversifying across unstable environments such as mountain tops provide substantial challenges for resolving evolutionary histories and delimiting species. The Liolaemus leopardinus clade is a group of five species of lizards adapted to high altitudes in central Chile, with most species found in the Andes, but one species, L. frassinettii is found in the independent Costa Cordillera. Despite their allopatric distributions, they display shallow mitochondrial divergences, making phylogenetics and species delimitation of this clade hard to resolve. We use an integrative approach to delimit species by considering morphological data (linear and landmark-based), mitochondrial DNA (mtDNA), and nuclear DNA (Sequences and SNPs collected with ddRADseq). We find strong conflicting signals between phylogenetic analyses of the nuclear and mtDNA data. While mtDNA places L. frassinettii as sister to the rest of the clade, the SNPs support a south to north order of divergences, with southernmost species (new taxon described here) as sister to the rest of the clade. Moreover, species delimitation using mtDNA only supports two species (one in the Costa and one in the Andes), whereas combined analyses using the nuclear data and morphology support multiple Andean taxa, including a new one we describe here. Based on these results, population structure analyses and our knowledge of the geological and climatic history of the Andes, we argue that this mito-nuclear discordance is explained by past introgression among the Andean taxa, likely during glacial periods that forced these lizards to lower altitudes where they would hybridize. The complete isolation between the Costa and Andes cordilleras has prevented any further contact between taxa on either mountain chain. Our study highlights the importance of using multiple lines of evidence to resolve evolutionary histories, and the potential misleading results from relying solely on mtDNA.

Pharmacokinetic/Pharmacodynamic Analysis of Voriconazole Against Candida spp. and Aspergillus spp. in Allogeneic Stem Cell Transplant Recipients.

BACKGROUND: To evaluate the adequacy of different dosing regimens of voriconazole for the prophylaxis of invasive candidiasis and aspergillosis in adult allogeneic stem cell transplant recipients by means of population pharmacokinetic (PK) modelling and simulation. METHODS: Allogeneic stem cell transplant recipients receiving voriconazole were included in this observational study. A population PK model was developed. Three oral voriconazole-dosing regimens were simulated: 200, 300, and 400 mg twice daily. The pharmacodynamic target was defined as fAUC0-24/0.7. A probability of target attainment ≥90% was considered optimal. The cumulative fraction of response was defined as the fraction of patients achieving the pharmacodynamic target when a population of simulated patients is matched with a simulated population of different Candida spp. and Aspergillus spp. The percentage of patients with trough plasma concentrations at steady state (Ctrough) within the reference range (1-5.5 mg/L) was also calculated. RESULTS: A 2-compartment PK model was developed using data from 40 patients, which contributed 237 voriconazole plasma samples, including trough and maximum concentrations. Voriconazole 200, 300, and 400 mg twice daily achieved probability of target attainment ≥90% for minimal inhibitory concentration values ≤0.25, ≤0.38, and ≤0.50 mg/L, respectively. The cumulative fraction of response for A. niger, A. versicolor, and A. flavus increased >10% when increasing voriconazole dose from 200 to 400 mg twice daily (from 72.5% to 89.5% for A. niger; from 77.7% to 88.7% for A. versicolor; and from 82.4% to 94.9% for A flavus). The percentage of patients with Ctrough within the reference range increased 15% when voriconazole dose was increased from 200 to 300 mg twice daily. CONCLUSIONS: The PK simulations in this study suggest that transplant recipients on voriconazole prophylaxis against invasive candidiasis or aspergillosis are likely to achieve the target concentrations associated with the desired treatment outcomes if the maintenance dose is 200 mg twice daily. However, Aspergillus spp. with high minimal inhibitory concentrations could require higher maintenance doses.

Stability of mycophenolate mofetil in polypropylene 5% dextrose infusion bags and chemical compatibility associated with the use of the Equashield® closed-system transfer device.

Stability studies are necessary in healthcare settings as they facilitate fast, cost-effective and efficient work related to batch manufacturing and availability of supplies. We studied the stability of 1-10 mg/mL mycophenolate mofetil (MMF) in polypropylene 5% dextrose infusion bags prepared from Cellcept® and with a generic brand name (Micofenolato de Mofetilo Accord) at different storage temperatures. To ensure chemical compatibility during preparation, we also tested MMF sorption to the Equashield® closed-system drug transfer device used in this step. For this, a validated stability-indicating high-performance liquid chromatography method was developed for the quantification and identification of MMF in the infusion bags. The analytical selectivity of the assay was determined by subjecting an MMF sample to extreme values of pH, oxidative stress and heat conditions to force degradation. Protected from light, 1-10 mg/mL MMF in infusion polypropylene bags prepared from reconstituted Cellcept® 500 mg or Accord 500 mg in 5% dextrose was stable for at least 35 days when stored at 2-8°C or between -15 and -25°C, and for 14 days when stored at 25°C. MMF loss owing to chemical sorption to the Equashield® closed-system drug transfer device set was negligible.

A systematic review of parasites and micropredators of non-avian reptiles (Reptilia=Sauropsida) in Chile.

Knowledge about the identity of parasites in vertebrates is relevant because of their influence on ecological processes and health of their hosts. This is particularly important for groups of animals currently facing conservation issues, such as reptiles. The diversity of species and supra-specific taxa of microparasites and macroparasites (such as helminths and arthropods) present in non-avian reptiles in Chile was analyzed through a systematic review. A total of 49 scientific documents (thesis projects, abstracts in congresses, book chapters and peer-reviewed articles) concerning parasites, taxonomic descriptions and parasite ecology were included in this review. The suborder Iguania was among the most studied group with 35 native species recognized as hosts of parasites and 39 species of parasites reported up to the category of species or genus. Liolaemus tenuis was the species with the most taxa of recorded parasites (11 taxa, and only three to species level), but Liolaemus chillanensis had the highest number of identified species of parasites. In addition, only one native species of Serpentes, one of Gymnophthalmoidea, and two of Gekkota, as well as some reports of exotic species, were recorded as hosts. Among parasites, Trombidiformes was the richest order with 10 species from the superfamily Pterygosomatoidea and 16 species from Trombiculoidea. Current knowledge about the richness of helminths is very limited and there were only a few records of microparasites. In general, there is an urgent need for the development of collaborative works between specialists in reptile taxonomy and epidemiology in parasitology destined to evaluate the consequences that reptiles and their parasites will suffer due to the ongoing processes of habitat loss, climate change and the still present taxonomic issues of the native reptiles.

Validated HPLC-UV detection method for the simultaneous determination of ceftolozane and tazobactam in human plasma.

AIM: A simple, rapid, economical and sensitive HPLC-UV method was developed for the simultaneous quantification of ceftolozane and tazobactam in plasma samples. METHODOLOGY: After deproteinization followed by a liquid-liquid back-extraction, the compounds were separated on a C18 column (150 mm × 4.6 mm, 5 µm) with UV-visible detection at 220 nm. The mobile phase consisted of acetonitrile and potassium dihydrogenphosphate buffer at pH 3.0 (8:92, v/v), delivered isocratically at a flow rate of 1.0 ml/min and at a column oven temperature of 30°C. Cefepime was used as an internal standard. RESULTS: Linearity was achieved in the concentration range of 0.50-100.00 µg/ml for ceftolozane and 0.25-50.00 µg/ml for tazobactam. The intra- and interday precision showed good reproducibility with coefficients of variation of less than 9.26% for ceftolozane and 9.62% for tazobactam. CONCLUSION: The sample preparation procedure avoids expensive or time-consuming steps used by other previously published methods. The methodology was validated according to standard guidelines and was used for quantification of ceftolozane and tazobactam in plasma samples from critically ill patients.

Dosing of caspofungin based on a pharmacokinetic/pharmacodynamic index for the treatment of invasive fungal infections in critically ill patients on continuous venovenous haemodiafiltration.

INTRODUCTION: The study objective was to evaluate the efficacy of different dosages of caspofungin in the treatment of invasive candidiasis and aspergillosis, in relation to the probability of pharmacokinetic/pharmacodynamic (PK/PD) target attainment, using modelling and Monte Carlo simulations in critically ill adult patients on continuous haemodiafiltration. METHODS: Critically ill adult patients on continuous venovenous haemodiafiltration treated with caspofungin were analysed. A population PK model was developed. Four caspofungin dosing regimens were simulated: the licensed regimen, 70 mg/day, 100 mg/day or 200 mg/day. A PK/PD target was defined as the ratio between the area under the caspofungin concentration-time curve over 24 hours and the minimal inhibitory concentration (AUC/MIC) for candidiasis or the minimal effective concentrations (AUC/MEC) for Aspergillus spp. Target attainment based on preclinical target for Candida and Aspergillus was assessed for different MIC or MEC, respectively. RESULTS: Concentration-time data were described by a two-compartment model. Body-weight and protein concentration were the only covariates identified by the model. Goodness-of-fit plots and bootstrap analysis proved the model had a satisfactory performance. As expected, a higher maintenance dose resulted in a higher exposure. Target attainment was >90% for candidiasis (MIC≤0.06 mg/L) and aspergillosis (MEC≤0.5 mg/L), irrespective of the dosing regimen, but not for C. parapsilosis. Standard regimen was insufficient to reach the target for C. albicans and C. parapsilosis with MIC≥0.1 mg/L. CONCLUSION: The licensed regimen of caspofungin is insufficient to achieve the PK/PD targets in critically ill patients on haemodiafiltration. The determination of MICs will enable dose scheme selection.

Stability of tacrolimus ophthalmic solution.

PURPOSE: The stability of 0.3-mg/mL tacrolimus ophthalmic solution at different storage temperatures was studied. METHODS: A sterile ophthalmic solution of 0.3 mg/mL tacrolimus was prepared in triplicate under aseptic conditions by diluting tacrolimus in eye drops. Three aliquots of this solution were transferred into polypropylene bottles and stored at 25, 2-8, or -15 to -25 °C. Samples were collected immediately after preparation and at selected time points and assayed in triplicate using high-performance liquid chromatography (HPLC). Samples were also visually examined for macroscopic changes. The 0.3-mg/mL tacrolimus solution was also exposed to acidic treatment and heat to force its degradation and to evaluate the selectivity of the analytic method. The tacrolimus ophthalmic solution was considered stable if at least 90% of the mean initial concentration remained when analyzed by HPLC. RESULTS: When stored at 2-8 °C and between -15 and -25 °C, at least 90% of the initial tacrolimus concentration remained throughout the 85-day study period. There were no significant differences in tacrolimus concentrations between the starting and ending points (p > 0.05). However, when tacrolimus solution was stored at 25 °C, the percentage of the initial tacrolimus concentration remaining had decreased to less than 90% on day 28. CONCLUSION: Tacrolimus diluted to 0.3 mg/mL in eye drop solution was stable for 20 days when stored at 25 °C and for at least 85 days when stored at 2-8 °C or between -15 and -25 °C in polypropylene bottles and protected from light.

The missing leopard lizard: Liolaemus ubaghsi sp. nov., a new species of the leopardinus clade (Reptilia: Squamata: Liolaemidae) from the Andes of the O'Higgins Region in Chile.

Liolaemus is an extremely species rich genus of iguanid lizards from southern South America. Most of the diversity though is found in the Andes Cordillera, between Argentina and Chile. Here we describe Liolaemus ubaghsi sp. nov., from El Teniente Mine, in the Andean mountains of the O'Higgins Region in Chile. This species presents scalation and pattern traits that belong to the leopardinus clade, a group of viviparous, high altitude lizards that inhabit the mountain ranges surrounding Santiago City. The species of this clade in turn belong to the Andean and Patagonian elongatus-kriegi complex. Liolaemus ubaghsi sp. nov. has been previously recognized as L. leopardinus and L. elongatus, nevertheless we present diagnostic traits that allow us to describe it as a new species. It mainly differs from the rest of the leopardinus clade (L. leopardinus, L. ramonensis, L. valdesianus and L. frassinettii) by having the following unique combination of traits: ochre background coloration, a wide dark occipital stripe, dark flanks, white dots dispersed on the dorsum, absence of leopard-like spots and enlarged infralabial scales. 

On the identity of Liolaemus nigromaculatus Wiegmann, 1834 (Iguania, Liolaemidae) and correction of its type locality.

In the current study, we review the taxonomic status of Liolaemus nigromaculatus. Despite being the nominal species of the nigromaculatus group and being the second species of the genus Liolaemus that was described, this species is of uncertain type locality and its true identification is a matter of discussion. After carefully analyzing several digital pictures of the holotype (juvenile male), reviewing all of the literature concerning the issue, examining specimens of nearly all recognized species of the nigromaculatus group, and determining the locations visited by the specimen collector, we are able to point out the following: 1) Liolaemus nigromaculatus was collected between Puerto Viejo and Copiapó of the Atacama region in Chile, and not in Huasco 2) Liolaemus bisignatus is a nomen nudum, and populations attributed to Liolaemus bisignatus should be referred to as Liolaemus nigromaculatus. 3) There is agreement that Liolaemus copiapoensis is indistinguishable from populations currently referred to as Liolaemus bisignatus (= Liolaemus nigromaculatus), 4) Populations found in Huasco (currently considered the type locality of Liolaemus nigromaculatus) are very similar to those found in Caldera (currently considered Liolaemus bisignatus) and should be designated as Liolaemus nigromaculatus, and 5) Liolaemus oxycephalus and Liolaemus inconspicuus are not synonymous with Liolaemus nigromaculatus, although their true identities are difficult to determine. We also detail several characteristic based on the holotype of Liolaemus nigromaculatus, in addition to drawing diagnostic comparisons between this species and others belonging to the nigromaculatus group.

Deep Divergences within Liolaemus nigroviridis (Squamata, Liolaemidae) Lineages Associated with Sky Islands in Central Chile.

Evolution of montane species may be strongly influenced by climate oscillations, particularly species distributed in isolated high-elevation areas (sky islands). Chilean topography is exemplified by montane environments including the Andes and Coastal Mountains. To test hypotheses related to genetic divergence associated with sky islands, we explored population genetics and phylogenetic signatures in the montane lizard Liolaemus nigroviridis Müller and Hellmich 1932. We sequenced the mitochondrial cytochrome b for samples collected from six montane areas in central Chile. We found high genetic divergence among populations, congruent with well-supported clades from phylogeny reconstructions. The most recent common ancestor of all samples of L. nigroviridis was dated around the limit of Pliocene-Pleistocene (2.7 Mya), congruent with early vicariance of Andean and coastal populations. Deep lineage divergences suggest that allopatric populations accumulated high nucleotide differences and maintained long periods without gene exchange. We discuss potential taxonomic revisions considering relative genetic divergence.