Variability of voriconazole plasma levels measured by new high-performance liquid chromatography and bioassay methods.

Voriconazole (VRC) is a broad-spectrum antifungal triazole with nonlinear pharmacokinetics. The utility of measurement of voriconazole blood levels for optimizing therapy is a matter of debate. Available high-performance liquid chromatography (HPLC) and bioassay methods are technically complex, time-consuming, or have a narrow analytical range. Objectives of the present study were to develop new, simple analytical methods and to assess variability of voriconazole blood levels in patients with invasive mycoses. Acetonitrile precipitation, reverse-phase separation, and UV detection were used for HPLC. A voriconazole-hypersusceptible Candida albicans mutant lacking multidrug efflux transporters (cdr1Delta/cdr1Delta, cdr2Delta/cdr2Delta, flu1Delta/flu1Delta, and mdr1Delta/mdr1Delta) and calcineurin subunit A (cnaDelta/cnaDelta) was used for bioassay. Mean intra-/interrun accuracies over the VRC concentration range from 0.25 to 16 mg/liter were 93.7% +/- 5.0%/96.5% +/- 2.4% (HPLC) and 94.9% +/- 6.1%/94.7% +/- 3.3% (bioassay). Mean intra-/interrun coefficients of variation were 5.2% +/- 1.5%/5.4% +/- 0.9% and 6.5% +/- 2.5%/4.0% +/- 1.6% for HPLC and bioassay, respectively. The coefficient of concordance between HPLC and bioassay was 0.96. Sequential measurements in 10 patients with invasive mycoses showed important inter- and intraindividual variations of estimated voriconazole area under the concentration-time curve (AUC): median, 43.9 mg x h/liter (range, 12.9 to 71.1) on the first and 27.4 mg x h/liter (range, 2.9 to 93.1) on the last day of therapy. During therapy, AUC decreased in five patients, increased in three, and remained unchanged in two. A toxic encephalopathy probably related to the increase of the VRC AUC (from 71.1 to 93.1 mg x h/liter) was observed. The VRC AUC decreased (from 12.9 to 2.9 mg x h/liter) in a patient with persistent signs of invasive aspergillosis. These preliminary observations suggest that voriconazole over- or underexposure resulting from variability of blood levels might have clinical implications. Simple HPLC and bioassay methods offer new tools for monitoring voriconazole therapy.

Variability of voriconazole plasma levels measured by new high-performance liquid chromatography and bioassay methods, [and] Voriconazole therapeutic drug monitoring in patients with invasive mycoses improves efficacy and safety outcomes

Rapport de synthèse1. Partie de laboratoireCette première étude décrit le développement et la validation, selon les standards internationaux, de deux techniques de mesure des concentrations sanguines de voriconazole, un nouvel agent antifongique à large spectre: 1) la chromatographic en phase liquide à haute pression et 2) le bio-essai utilisant une souche mutante de Candida hypersensible au voriconazole. Ce travail a aussi permis de mettre en évidence une importante et imprévisible variabilité inter- et intra-individuelle des concentrations sanguines de voriconazole malgré l'utilisation des doses recommandées par le fabriquant. Ce travail a été publié dans un journal avec "peer-review": "Variability of voriconazole plasma levels measured by new high- performance liquid chromatography and bioassay methods" by A. Pascual, V. Nieth, T. Calandra, J. Bille, S. Bolay, L.A. Decosterd, T. Buclin, P.A. Majcherczyk, D. Sanglard, 0. Marchetti. Antimicrobial Agents Chemotherapy, 2007; 51:137-432. Partie CliniqueCette deuxième étude a évalué de façon prospective l'impact clinique des concentrations sanguines de voriconazole sur l'efficacité et sécurité thérapeutique chez des patients atteints d'infections fongiques. Des concentrations sanguines élevées étaient significativement associés à la survenue d'une toxicité neurologique (encéphalopathie avec confusion, hallucinations et myoclonies) et des concentrations sanguines basses à une réponse insuffisante au traitement antifongique (persistance ou progression des signes cliniques et radiologiques de l'infection). Dans la majorité des cas, un ajustement de la dose de voriconazole, sur la base des concentrations mesurées, a abouti à une récupération neurologique complète ou à une résolution de l'infection, respectivement. Ce travail a été publié dans un journal avec "peer-review": " Voriconazole Therapeutic Drug Monitoring in Patients with Invasive Mycoses Improves Efficacy and Safety Outcomes" by A. Pascual, T. Calandra, S. Bolay, T. Buclin, J. Bille, and O. Marchetti. Clinical Infectious Diseases, 2008 January 15; 46(2): 201-11.Ces deux études, financées de façon conjointe par un "grant" international de la Société suisse d'infectiologie et la Société internationale de maladies infectieuses et par la Fondation pour le progrès en microbiologie médicale et maladies infectieuses (FAMMID, Lausanne), ont été réalisées au sein du Service des Maladies Infectieuses, Département de Médecine, au CHUV, en étroite collaboration avec la Division de Pharmacologie Clinique, Département de Médecine, au CHUV et l'Institut de Microbiologie du CHUV et de l'Université de Lausanne.

Comparison of parasitological, immunological and molecular methods for the diagnosis of leishmaniasis in dogs with different clinical signs

Aiming to improve the diagnosis of canine leishmaniasis (CanL) in an endemic area of the Northwest region of São Paulo State, Brazil, the efficacy of parasitological, immunological and molecular diagnostic methods were studied. Dogs with and without clinical sips of the disease and positive for Leishmania, by direct parasite identification on lymph node smears and/or specific antibody detection by ELISA, were selected for the study. According to the clinical signs, 89 dogs attending the Veterinary Hospital of UNESP in Aracatuba (SP, Brazil) were divided into three groups: symptomatic (36%), oligosymptomatic (22%) and asymptomatic (22%). Twenty-six dogs from an area non-endemic for CanL were used as negative controls (20%). Fine-needle aspiration biopsies (FNA) of popliteal lymph nodes were collected and Diff-Quick (R)-stained for optical microscopy. Direct immumofluorescence, immunocytochemistry and parasite DNA amplification by PCR were also performed. After euthanasia, fragments of popliteal lymph nodes, spleen, bone marrow and liver were collected and processed for HE and immunohistochemistry. Parasite detection by both HE and immunohistochemistry was specifically more effective in lymph nodes, when compared with the other organs. Immunolabeling provided higher sensitivity for parasite detection in the tissues. In the symptomatic group, assay sensitivity was 75.61% for direct parasite search on Diff-Quick (R)-stained FNAs, 92.68% for direct immunofluorescence, 92.68% for immunocytochemistry and 100% for PCR; the corresponding values in the other clinical groups were: 32, 60, 76 and 96% (oligosymptomatic), and 39.13, 73.91, 100 and 95.65% (asymptomatic). Results of the control animals from the CanL non-endemic area were all negative, indicating that the methods used were 100% specific. (C) 2006 Elsevier B.V. All rights reserved.

Ultra-performance liquid chromatography mass spectrometry and sensitive bioassay methods for quantification of posaconazole plasma concentrations after oral dosing.

Posaconazole (POS) is a new antifungal agent for prevention and therapy of mycoses in immunocompromised patients. Variable POS pharmacokinetics after oral dosing may influence efficacy: a trough threshold of 0.5 ?g/ml has been recently proposed. Measurement of POS plasma concentrations by complex chromatographic techniques may thus contribute to optimize prevention and management of life-threatening infections. No microbiological analytical method is available. The objective of this study was to develop and validate a new simplified ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) method and a sensitive bioassay for quantification of POS over the clinical plasma concentration range. The UPLC-MS/MS equipment consisted of a triple quadrupole mass spectrometer, an electrospray ionization (ESI) source, and a C(18) analytical column. The Candida albicans POS-hypersusceptible mutant (MIC of 0.002 ?g/ml) ?cdr1 ?cdr2 ?flu ?mdr1 ?can constructed by targeted deletion of multidrug efflux transporters and calcineurin genes was used for the bioassay. POS was extracted from plasma by protein precipitation with acetonitrile-methanol (75%/25%, vol/vol). Reproducible standard curves were obtained over the range 0.014 to 12 (UPLC-MS/MS) and 0.028 to 12 ?g/ml (bioassay). Intra- and interrun accuracy levels were 106% ± 2% and 103% ± 4% for UPLC-MS/MS and 102% ± 8% and 104% ± 1% for bioassay, respectively. The intra- and interrun coefficients of variation were 7% ± 4% and 7% ± 3% for UPLC-MS/MS and 5% ± 3% and 4% ± 2% for bioassay, respectively. An excellent correlation between POS plasma concentrations measured by UPLC-MS/MS and bioassay was found (concordance, 0.96). In 26 hemato-oncological patients receiving oral POS, 27/69 (39%) trough plasma concentrations were lower than 0.5 ?g/ml. The UPLC-MS/MS method and sensitive bioassay offer alternative tools for accurate and precise quantification of the plasma concentrations in patients receiving oral posaconazole.

Leaching of Atrazine in Commercial and Xerogel Formulations in Oxisol Using Bioassay and Chromatographic Methods

Mobility of atrazine in soil has contributed to the detection of levels above the legal limit in surface water and groundwater in Europe and the United States. The use of new formulations can reduce or minimize the impacts caused by the intensive use of this herbicide in Brazil, mainly in regions with higher agricultural intensification. The objective of this study was to compare the leaching of a commercial formulation of atrazine (WG) with a controlled release formulation (xerogel) using bioassay and chromatographic methods of analysis. The experiment was a split plot randomized block design with four replications, in a (2 x 6) + 1 arrangement. The main formulations of atrazine (WG and xerogel) were allocated in the plots, and the herbicide concentrations (0, 3200, 3600, 4200, 5400 and 8000 g ha-1), in the subplots. Leaching was determined comparatively by using bioassays with oat and chromatographic analysis. The results showed a greater concentration of the herbicide in the topsoil (0-4 cm) in the treatment with the xerogel formulation in comparison with the commercial formulation, which contradicts the results obtained with bioassays, probably because the amount of herbicide available for uptake by plants in the xerogel formulation is less than that available in the WG formulation.

Stability study of fluconazole applying validated bioassay and stability-indicating LC methods

Chemical degradation of drugs may result in altered therapeutic efficacy and even toxic effects. Therefore, understanding the factors that change the stability of pharmaceuticals and identifying ways to guarantee their stability are important. In this work stability-indicating Liquid Chromatographic (LC) and bioassay methods were validated and employed in the fluconazole stability studies. The correlation of sample results from both methods was evaluated. Fluconazole raw material stability was investigated in aqueous, acid (0.1 M HCl), alkaline (0.1 M NaOH) and oxidative (3% v/v H2O2) reflux for 6 hours, by LC method. Fluconazole capsules were exposed to UVC (254 nm, 66 and 180 days), climatic chamber (40°C, 75% RH, 90 days) and oven (60°C, 60 days), these samples were analyzed by LC and bioassay methods It was found that the drug is degraded (10% decrease) with arising of a possible degradation product in an oxidative medium and UVC exposure, in all the others conditions fluconazole remained chemically stable (higher than 98%) when analyzed by LC. However when the capsules stressed samples were evaluated through bioassay very low antifungal activity was found (about 30%). Fluconazole showed to be an unstable drug and it indicates that special care must be taken during the handling, storage and quality control using appropriated methods to analyze this therapeutic agent. This work suggests monitoring the fluconazole stability by bioassay and the stability-indicating LC methods.

Emergent and re-emergent parasites in HIV-infected children: immunological and socio-environmental conditions that are involved in the transmission of Giardia spp. and Cryptosporidium spp.

ABSTRACT INTRODUCTION: Emergent and re-emergent waterborne protozoans have become a worldwide public health problem, especially among vulnerable groups. METHODS: This cross-sectional study evaluated 17 HIV-infected children and their families. RESULTS: A high (76.5%) percentage of parasite-infected children was observed, even among children with CD4+ T-cell counts of >200 cells/mm3. Giardia spp., Cryptosporidium spp. and Cyclospora spp. were observed in 41.2% of these children Low income, poor hygiene practices, and co-infection in domestic, peridomestic and scholastic environments were significant sources of these intestinal infections. CONCLUSIONS: Early diagnosis, timely treatment, and socio-educational interventions may improve the health conditions of this vulnerable population.

Immunological and clinical consequences of treating a patient with natalizumab.

BACKGROUND: Long-term therapy with natalizumab increases the risk of progressive multifocal leukoencephalopathy (PML). OBJECTIVES: We present a patient study through therapy, the diagnosis of PML (after 29 infusions), plasma exchange (PE) and development of immune reconstitution inflammatory syndrome (IRIS). METHODS: Routine diagnostics, magnetic resonance imaging (MRI), immunological status (flow cytometry, T-cell migration assays and T-cell repertoire analysis), and brain biopsy with immunohistological analysis. RESULTS: CD49d decreased after 12 months of treatment. At PML diagnosis, CD49d expression and migratory capacity of T cells was low and peripheral T-cell receptor (TCR) complexity showed severe perturbations. The distribution of peripheral monocytes changed from CCR5+ to CCR7+. After PE some changes reverted: CD49d increased and overshot earliest levels, migratory capacities of T cells recovered and peripheral TCR complexity increased. With no clinical, routine laboratory or cerebrospinal fluid (CSF) changes, MRI 2 months after PE demonstrated progressive lesion development. Brain histopathology confirmed the presence of infiltrates indicative of IRIS without clinical signs, immunologically accompanied by CCR7/CCR5 recovery of peripheral monocytes. CONCLUSION: Natalizumab-associated immunological changes accompanying PML were reversible after PE; IRIS can occur very late, remain asymptomatic and be elusive to CSF analysis. Our study may provide insights into the changes under treatment with natalizumab associated with JC virus control.

Performance characteristics of high performance liquid chromatography, first order derivative UV spectrophotometry and bioassay for fluconazole determination in capsules

The bioassay, first order derivative UV spectrophotometry and chromatographic methods for assaying fluconazole capsules were compared. They have shown great advantages over the earlier published methods. Using the first order derivative, the UV spectrophotometry method does not suffer interference of excipients. Validation parameters such as linearity, precision, accuracy, limit of detection and limit of quantitation were determined. All methods were linear and reliable within acceptable limits for antibiotic pharmaceutical preparations being accurate, precise and reproducible. The application of each method as a routine analysis should be investigated considering cost, simplicity, equipment, solvents, speed, and application to large or small workloads.

Detection of sandal spike phytoplasma using immunological and molecular techniques

Spike disease in sandal is generally diagnosed by the manifestation of external symptoms. Attempts have been made to detect the diseased plants by determining the length/breadth ratio of leaves (lyengar, 1961) and histochemical tests using Mann's stain (Parthasarathi et al., 1966), Dienes' stain (Ananthapadmanabha et a/., 1973) aniline blue and Hoechst 33258 (Ghosh et a/., 1985, Rangaswamy, 1995). But most of these techniques are insensitive, indirect detection methods leading to misinterpretation of results. Moreover, to identify disease resistant sandal trees, highly sensitive techniques are needed to detect the presence of the pathogen. In sandal forests, several host plants of sandal like Zizyphus oenop/ea (Fig. 1.3) also exhibit the yellows type disease symptoms. Immunological and molecular assays have to be developed to confirm the presence of sandal spike phytoplasma in such hosts. The major objectives of the present work includes:In situ detection of sandal spike phytoplasma by epifluorescence microscopy and scanning electron microscopy.,Purification of sandal spike phytoplasma and production of polyclonal antibodies.,Amino acid and total protein estimation of sandal spike phytoplasma.,Immunological detection of sandal spike phytoplasma., Molecular detection of sandal spike phytoplasma.,Screening for phytoplasma in host plants of spike disease affected sandal using immunological and molecular techniques.

Performance characteristics of high performance liquid chromatography, first order derivative UV spectrophotometry and bioassay for fluconazole determination in capsules

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Diabetes induced immunological and biochemical changes in human colostrum

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Immunological and biochemical techniques in the analysis of tear proteins

This study is concerned with the analysis of tear proteins, paying particular attention to the state of the tears (e.g. non-stimulated, reflex, closed), created during sampling, and to assess their interactions with hydrogel contact lenses. The work has involved the use of a variety of biochemical and immunological analytical techniques for the measurement of proteins, (a), in tears, (b), on the contact lens, and (c), in the eluate of extracted lenses. Although a diverse range of tear components may contribute to contact lens spoilation, proteins were of particular interest in this study because of their theoretical potential for producing immunological reactions. Although normal host proteins in their natural state are generally not treated as dangerous or non-self, those which undergo denaturation or suffer a conformational change may provoke an excessive and unnecessary immune response. A novel on-lens cell based assay has been developed and exploited in order to study the role of the ubiquitous cell adhesion glycoprotein, vitronectin, in tears and contact lens wear under various parameters. Vitronectin, whose levels are known to increase in the closed eye environment and shown here to increase during contact lens wear, is an important immunoregulatory protein and may be a prominent marker of inflammatory activity. Immunodiffusion assays were developed and optimised for use in tear analysis, and in a series of subsequent studies used for example in the measurement of albumin, lactoferrin, IgA and IgG. The immunodiffusion assays were then applied in the estimation of the closed eye environment; an environment which has been described as sustaining a state of sub-clinical inflammation. The role and presence of a lesser understood and investigated protein, kininogen, was also estimated, in particular, in relation to contact lens wear. Difficulties arise when attempting to extract proteins from the contact lens in order to examine the individual nature of the proteins involved. These problems were partly alleviated with the use of the on-lens cell assay and a UV spectrophotometry assay, which can analyse the lens surface and bulk respectively, the latter yielding only total protein values. Various lens extraction methods were investigated to remove protein from the lens and the most efficient was employed in the analysis of lens extracts. Counter immunoelectrophoresis, an immunodiffusion assay, was then applied to the analysis of albumin, lactoferrin, IgA and IgG in the resultant eluates.