A single LC-tandem mass spectrometry method for the simultaneous determination of 14 antimalarial drugs and their metabolites in human plasma.

Among the various determinants of treatment response, the achievement of sufficient blood levels is essential for curing malaria. For helping us at improving our current understanding of antimalarial drugs pharmacokinetics, efficacy and toxicity, we have developed a liquid chromatography-tandem mass spectrometry method (LC-MS/MS) requiring 200mul of plasma for the simultaneous determination of 14 antimalarial drugs and their metabolites which are the components of the current first-line combination treatments for malaria (artemether, artesunate, dihydroartemisinin, amodiaquine, N-desethyl-amodiaquine, lumefantrine, desbutyl-lumefantrine, piperaquine, pyronaridine, mefloquine, chloroquine, quinine, pyrimethamine and sulfadoxine). Plasma is purified by a combination of protein precipitation, evaporation and reconstitution in methanol/ammonium formate 20mM (pH 4.0) 1:1. Reverse-phase chromatographic separation of antimalarial drugs is obtained using a gradient elution of 20mM ammonium formate and acetonitrile both containing 0.5% formic acid, followed by rinsing and re-equilibration to the initial solvent composition up to 21min. Analyte quantification, using matrix-matched calibration samples, is performed by electro-spray ionization-triple quadrupole mass spectrometry by selected reaction monitoring detection in the positive mode. The method was validated according to FDA recommendations, including assessment of extraction yield, matrix effect variability, overall process efficiency, standard addition experiments as well as antimalarials short- and long-term stability in plasma. The reactivity of endoperoxide-containing antimalarials in the presence of hemolysis was tested both in vitro and on malaria patients samples. With this method, signal intensity of artemisinin decreased by about 20% in the presence of 0.2% hemolysed red-blood cells in plasma, whereas its derivatives were essentially not affected. The method is precise (inter-day CV%: 3.1-12.6%) and sensitive (lower limits of quantification 0.15-3.0 and 0.75-5ng/ml for basic/neutral antimalarials and artemisinin derivatives, respectively). This is the first broad-range LC-MS/MS assay covering the currently in-use antimalarials. It is an improvement over previous methods in terms of convenience (a single extraction procedure for 14 major antimalarials and metabolites reducing significantly the analytical time), sensitivity, selectivity and throughput. While its main limitation is investment costs for the equipment, plasma samples can be collected in the field and kept at 4 degrees C for up to 48h before storage at -80 degrees C. It is suited to detecting the presence of drug in subjects for screening purposes and quantifying drug exposure after treatment. It may contribute to filling the current knowledge gaps in the pharmacokinetics/pharmacodynamics relationships of antimalarials and better define the therapeutic dose ranges in different patient populations.

Acquired epileptic dysgraphia: a longitudinal study.

A male presenting with benign partial epilepsy with rolandic spikes from the age of 7 years was evaluated at age 11 years for worsening of his epilepsy associated with a specific regression of graphomotor skills. A longitudinal study over nearly 2 years showed an improvement in handwriting to an almost normal level under modified antiepileptic therapy. A detailed analysis with a computer-monitored graphics table showed at first a rapid improvement of skills followed by protracted slower progress. We argue that the initial rapid recovery of skills was directly linked to the improvement of his epilepsy. The slower late acquisition of motor programmes that had never been fully established was due to long-standing interference by his epilepsy. The specificity of the deficit within the graphomotor system and its possible neurobiological basis are also discussed. The analytical method and approach used in a single patient might provide an example for other patients in whom epilepsy can interfere in the acquisition, progress, and maintenance of new skills and can be responsible for selective deficits.

Missing value imputation in longitudinal measures of alcohol consumption

Attrition in longitudinal studies can lead to biased results. The study is motivated by the unexpected observation that alcohol consumption decreased despite increased availability, which may be due to sample attrition of heavy drinkers. Several imputation methods have been proposed, but rarely compared in longitudinal studies of alcohol consumption. The imputation of consumption level measurements is computationally particularly challenging due to alcohol consumption being a semi-continuous variable (dichotomous drinking status and continuous volume among drinkers), and the non-normality of data in the continuous part. Data come from a longitudinal study in Denmark with four waves (2003-2006) and 1771 individuals at baseline. Five techniques for missing data are compared: Last value carried forward (LVCF) was used as a single, and Hotdeck, Heckman modelling, multivariate imputation by chained equations (MICE), and a Bayesian approach as multiple imputation methods. Predictive mean matching was used to account for non-normality, where instead of imputing regression estimates, "real" observed values from similar cases are imputed. Methods were also compared by means of a simulated dataset. The simulation showed that the Bayesian approach yielded the most unbiased estimates for imputation. The finding of no increase in consumption levels despite a higher availability remained unaltered. Copyright (C) 2011 John Wiley & Sons, Ltd.

Risky single occasion drinking frequency and alcohol-related consequences: can abstinence during early adulthood lead to alcohol problems?

QUESTIONS UNDER STUDY: the main purpose of this longitudinal study was to determine the impact of risky single occasion drinking (RSOD) frequency on alcohol dependence and drinking consequences reported 15 months later. METHODS: As a baseline sample, 5,990 young men were assessed on their drinking habits including the frequency of RSOD. Of them, 5,196 were reassessed at follow-up 15 months later on RSOD frequency, alcohol dependence and alcohol related consequences in thze interceding year. Drop out biases were investigated. RESULTS: Around 45% of the baseline participants reported regular RSOD (every month or more frequently). Despite the fact that RSOD distribution was generally stable during the initial sample, 47.4% reported a variation of their RSOD frequency 15 months later. Around 25% of the sample reported reduced RSOD frequency. Nonetheless, occasional RS drinkers were more likely to become regular (monthly) RSO drinkers at follow up. Daily and weekly RSOD were associated with high proportions of alcohol dependence and detrimental consequences of drinking. Surprisingly, abstainers at baseline were more likely to be at risk of alcohol dependence and consequences at follow up than non-RSO drinkers. CONCLUSIONS: Despite the fact that alcohol abstinence is logically the best way to avoid the detrimental consequences of alcohol drinking, abstainers at baseline reported as many problems due to alcohol use at follow up as occasional or monthly RSO drinkers. The few participants who had become RSO drinkers during the follow up period were indeed likely to engage in detrimental behaviour. Non-RSO drinkers had the fewest problems due to alcohol use. This substantiates the early occurrence of drinking consequences among inexperienced RSO drinkers.

Application of colon capsule endoscopy (CCE) to evaluate the whole gastrointestinal tract: a comparative study of single-camera and dual-camera analysis.

BACKGROUND AND STUDY AIMS Colon capsule endoscopy (CCE) was developed for the evaluation of colorectal pathology. In this study, our aim was to assess if a dual-camera analysis using CCE allows better evaluation of the whole gastrointestinal (GI) tract compared to a single-camera analysis. PATIENTS AND METHODS We included 21 patients (12 males, mean age 56.20 years) submitted for a CCE examination. After standard colon preparation, the colon capsule endoscope (PillCam Colon™) was swallowed after reinitiation from its "sleep" mode. Four physicians performed the analysis: two reviewed both video streams at the same time (dual-camera analysis); one analyzed images from one side of the device ("camera 1"); and the other reviewed the opposite side ("camera 2"). We compared numbers of findings from different parts of the entire GI tract and level of agreement among reviewers. RESULTS A complete evaluation of the GI tract was possible in all patients. Dual-camera analysis provided 16% and 5% more findings compared to camera 1 and camera 2 analysis, respectively. Overall agreement was 62.7% (kappa = 0.44, 95% CI: 0.373-0.510). Esophageal (kappa = 0.611) and colorectal (kappa = 0.595) findings had a good level of agreement, while small bowel (kappa = 0.405) showed moderate agreement. CONCLUSION The use of dual-camera analysis with CCE for the evaluation of the GI tract is feasible and detects more abnormalities when compared with single-camera analysis.

First-line gefitinib in Caucasian EGFR mutation-positive NSCLC patients: a phase-IV, open-label, single-arm study.

BACKGROUND Phase-IV, open-label, single-arm study (NCT01203917) to assess efficacy and safety/tolerability of first-line gefitinib in Caucasian patients with stage IIIA/B/IV, epidermal growth factor receptor (EGFR) mutation-positive non-small-cell lung cancer (NSCLC). METHODS Treatment: gefitinib 250 mg day(-1) until progression. Primary endpoint: objective response rate (ORR). Secondary endpoints: disease control rate (DCR), progression-free survival (PFS), overall survival (OS) and safety/tolerability. Pre-planned exploratory objective: EGFR mutation analysis in matched tumour and plasma samples. RESULTS Of 1060 screened patients with NSCLC (859 known mutation status; 118 positive, mutation frequency 14%), 106 with EGFR sensitising mutations were enrolled (female 70.8%; adenocarcinoma 97.2%; never-smoker 64.2%). At data cutoff: ORR 69.8% (95% confidence interval (CI) 60.5-77.7), DCR 90.6% (95% CI 83.5-94.8), median PFS 9.7 months (95% CI 8.5-11.0), median OS 19.2 months (95% CI 17.0-NC; 27% maturity). Most common adverse events (AEs; any grade): rash (44.9%), diarrhoea (30.8%); CTC (Common Toxicity Criteria) grade 3/4 AEs: 15%; SAEs: 19%. Baseline plasma 1 samples were available in 803 patients (784 known mutation status; 82 positive; mutation frequency 10%). Plasma 1 EGFR mutation test sensitivity: 65.7% (95% CI 55.8-74.7). CONCLUSION First-line gefitinib was effective and well tolerated in Caucasian patients with EGFR mutation-positive NSCLC. Plasma samples could be considered for mutation analysis if tumour tissue is unavailable.

Use of Two-Part Regression Calibration Model to Correct for Measurement Error in Episodically Consumed Foods in a Single-Replicate Study Design: EPIC Case Study.

In epidemiologic studies, measurement error in dietary variables often attenuates association between dietary intake and disease occurrence. To adjust for the attenuation caused by error in dietary intake, regression calibration is commonly used. To apply regression calibration, unbiased reference measurements are required. Short-term reference measurements for foods that are not consumed daily contain excess zeroes that pose challenges in the calibration model. We adapted two-part regression calibration model, initially developed for multiple replicates of reference measurements per individual to a single-replicate setting. We showed how to handle excess zero reference measurements by two-step modeling approach, how to explore heteroscedasticity in the consumed amount with variance-mean graph, how to explore nonlinearity with the generalized additive modeling (GAM) and the empirical logit approaches, and how to select covariates in the calibration model. The performance of two-part calibration model was compared with the one-part counterpart. We used vegetable intake and mortality data from European Prospective Investigation on Cancer and Nutrition (EPIC) study. In the EPIC, reference measurements were taken with 24-hour recalls. For each of the three vegetable subgroups assessed separately, correcting for error with an appropriately specified two-part calibration model resulted in about three fold increase in the strength of association with all-cause mortality, as measured by the log hazard ratio. Further found is that the standard way of including covariates in the calibration model can lead to over fitting the two-part calibration model. Moreover, the extent of adjusting for error is influenced by the number and forms of covariates in the calibration model. For episodically consumed foods, we advise researchers to pay special attention to response distribution, nonlinearity, and covariate inclusion in specifying the calibration model.

First-line gefitinib in Caucasian EGFR mutation-positive NSCLC patients: a phase-IV, open-label, single-arm study.

BACKGROUND: Phase-IV, open-label, single-arm study (NCT01203917) to assess efficacy and safety/tolerability of first-line gefitinib in Caucasian patients with stage IIIA/B/IV, epidermal growth factor receptor (EGFR) mutation-positive non-small-cell lung cancer (NSCLC). METHODS: TREATMENT: gefitinib 250 mg day(-1) until progression. Primary endpoint: objective response rate (ORR). Secondary endpoints: disease control rate (DCR), progression-free survival (PFS), overall survival (OS) and safety/tolerability. Pre-planned exploratory objective: EGFR mutation analysis in matched tumour and plasma samples. RESULTS: Of 1060 screened patients with NSCLC (859 known mutation status; 118 positive, mutation frequency 14%), 106 with EGFR sensitising mutations were enrolled (female 70.8%; adenocarcinoma 97.2%; never-smoker 64.2%). At data cutoff: ORR 69.8% (95% confidence interval (CI) 60.5-77.7), DCR 90.6% (95% CI 83.5-94.8), median PFS 9.7 months (95% CI 8.5-11.0), median OS 19.2 months (95% CI 17.0-NC; 27% maturity). Most common adverse events (AEs; any grade): rash (44.9%), diarrhoea (30.8%); CTC (Common Toxicity Criteria) grade 3/4 AEs: 15%; SAEs: 19%. Baseline plasma 1 samples were available in 803 patients (784 known mutation status; 82 positive; mutation frequency 10%). Plasma 1 EGFR mutation test sensitivity: 65.7% (95% CI 55.8-74.7). CONCLUSION: First-line gefitinib was effective and well tolerated in Caucasian patients with EGFR mutation-positive NSCLC. Plasma samples could be considered for mutation analysis if tumour tissue is unavailable.

Application of colon capsule endoscopy (CCE) to evaluate the whole gastrointestinal tract: a comparative study of single-camera and dual-camera analysis.

BACKGROUND AND STUDY AIMS Colon capsule endoscopy (CCE) was developed for the evaluation of colorectal pathology. In this study, our aim was to assess if a dual-camera analysis using CCE allows better evaluation of the whole gastrointestinal (GI) tract compared to a single-camera analysis. PATIENTS AND METHODS We included 21 patients (12 males, mean age 56.20 years) submitted for a CCE examination. After standard colon preparation, the colon capsule endoscope (PillCam Colon™) was swallowed after reinitiation from its "sleep" mode. Four physicians performed the analysis: two reviewed both video streams at the same time (dual-camera analysis); one analyzed images from one side of the device ("camera 1"); and the other reviewed the opposite side ("camera 2"). We compared numbers of findings from different parts of the entire GI tract and level of agreement among reviewers. RESULTS A complete evaluation of the GI tract was possible in all patients. Dual-camera analysis provided 16% and 5% more findings compared to camera 1 and camera 2 analysis, respectively. Overall agreement was 62.7% (kappa = 0.44, 95% CI: 0.373-0.510). Esophageal (kappa = 0.611) and colorectal (kappa = 0.595) findings had a good level of agreement, while small bowel (kappa = 0.405) showed moderate agreement. CONCLUSION The use of dual-camera analysis with CCE for the evaluation of the GI tract is feasible and detects more abnormalities when compared with single-camera analysis.

Clinical and epidemiological features and prognosis of complicated pyelonephritis: a prospective observational single hospital-based study.

BACKGROUND Complicated pyelonephritis (cPN), a common cause of hospital admission, is still a poorly-understood entity given the difficulty involved in its correct definition. The aim of this study was to analyze the main epidemiological, clinical, and microbiological characteristics of cPN and its prognosis in a large cohort of patients with cPN. METHODS We conducted a prospective, observational study including 1325 consecutive patients older than 14 years diagnosed with cPN and admitted to a tertiary university hospital between 1997-2013. After analyzing the main demographic, clinical and microbiological data, covariates found to be associated with attributable mortality in univariate analysis were included in a multivariate logistic regression model. RESULTS Of the 1325 patients, 689 (52%) were men and 636 (48%) women; median age 63 years, interquartile range [IQR] (46.5-73). Nine hundred and forty patients (70.9%) had functional or structural abnormalities in the urinary tract, 215 (16.2%) were immunocompromised, 152 (11.5%) had undergone a previous urinary tract instrumentation, and 196 (14.8%) had a long-term bladder catheter, nephrostomy tube or ureteral catheter. Urine culture was positive in 813 (67.7%) of the 1251 patients in whom it was done, and in the 1032 patients who had a blood culture, 366 (34%) had bacteraemia. Escherichia coli was the causative agent in 615 episodes (67%), Klebsiella spp in 73 (7.9%) and Proteus ssp in 61 (6.6%). Fourteen point one percent of GNB isolates were ESBL producers. In total, 343 patients (25.9%) developed severe sepsis and 165 (12.5%) septic shock. Crude mortality was 6.5% and attributable mortality was 4.1%. Multivariate analysis showed that an age >75 years (OR 2.77; 95% CI, 1.35-5.68), immunosuppression (OR 3.14; 95% CI, 1.47-6.70), and septic shock (OR 58.49; 95% CI, 26.6-128.5) were independently associated with attributable mortality. CONCLUSIONS cPN generates a high morbidity and mortality and likely a great consumption of healthcare resources. This study highlights the factors directly associated with mortality, though further studies are needed in the near future aimed at identifying subgroups of low-risk patients susceptible to outpatient management.

Robust synchronization of coupled circadian and cell cycle oscillators in single mammalian cells.

Circadian cycles and cell cycles are two fundamental periodic processes with a period in the range of 1 day. Consequently, coupling between such cycles can lead to synchronization. Here, we estimated the mutual interactions between the two oscillators by time-lapse imaging of single mammalian NIH3T3 fibroblasts during several days. The analysis of thousands of circadian cycles in dividing cells clearly indicated that both oscillators tick in a 1:1 mode-locked state, with cell divisions occurring tightly 5 h before the peak in circadian Rev-Erbα-YFP reporter expression. In principle, such synchrony may be caused by either unidirectional or bidirectional coupling. While gating of cell division by the circadian cycle has been most studied, our data combined with stochastic modeling unambiguously show that the reverse coupling is predominant in NIH3T3 cells. Moreover, temperature, genetic, and pharmacological perturbations showed that the two interacting cellular oscillators adopt a synchronized state that is highly robust over a wide range of parameters. These findings have implications for circadian function in proliferative tissues, including epidermis, immune cells, and cancer.

Continuous versus single bolus enteral nutrition: comparison of energy metabolism in humans.

Continuous respiratory exchange measurements were performed on five women and five men for 1 h before and 6 h after the administration of a milkshake (53% carbohydrates, 30% lipid, and 17% protein energy) given either as a single bolus dose or continuously during 3 h using a nasogastric tube. The energy administered corresponded to 2.3 times the postabsorptive resting energy expenditure. Resting energy expenditure, respiratory quotient, plasma glucose, and insulin concentrations increased sooner and steeper, and plasma free fatty acids levels decreased earlier with the meal ingested as a single dose than with continuous administration. The magnitude of nutrient-induced thermogenesis was greater (P less than 0.01) with the single dose (means +/- SE, 10.0 +/- 0.6%) than with the continuous administration (8.1 +/- 0.5%). The overall (6 h) substrate balances were not significantly different between the two modes of administration. It is concluded that the mode of enteral nutrient administration influences the immediate thermogenic response as well as changes in respiratory quotient, glycemia, and insulinemia; however, the overall nutrient balance was not affected by the mode of enteral nutrient administration.

Longitudinal and cross-sectional changes in active commuting to school among Brazilian schoolchildren.

OBJECTIVE: The objective of the study is to evaluate cross-sectional and longitudinal changes in children's commuting to school in a representative sample of a Brazilian city. METHODS: Two school-based studies were carried out in 2002 (n=2936; 7-10years old) and 2007 (n=1232; 7-15years old) in Florianopolis, Brazil. Cross-sectional data were collected from children aged 7 to 10years in 2002 and 2007. Longitudinal analyses were performed with data from 733 children participating in both surveys. Children self-reported their mode of transportation to school using a validated illustrated questionnaire. Changes were tested with chi square statistics and McNemar's test. RESULTS: Cross-sectional data showed a 17% decline in active commuting; a decrease from 49% in 2002 to 41% in 2007. On the other hand, active commuting among the 733 children increased as they entered adolescence 5years later, rising from 40% to 49%. CONCLUSION: Active commuting to school decreased in Brazilian children aged 7-10years over a five year period; whereas, it increased among children entering adolescence. Policies should focus on safety and environmental determinants to increase active commuting.

Limitations of stimulated echo acquisition mode (STEAM) techniques in cardiac applications.

Stimulated echoes are widely used for imaging functional tissue parameters such as diffusion coefficient, perfusion, and flow rates. They are potentially interesting for the assessment of various cardiac functions. However, severe limitations of the stimulated echo acquisition mode occur, which are related to the special dynamic properties of the beating heart and flowing blood. To the well-known signal decay due to longitudinal relaxation and through-plane motion between the preparation and the read-out period of the stimulated echoes, additional signal loss is often observed. As the prepared magnetization is fixed with respect to the tissue, this signal loss is caused by the tissue deformation during the cardiac cycle, which leads to a modification of the modulation frequency of the magnetization. These effects are theoretically derived and corroborated by phantom and in vivo experiments.