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The aim of this study was to evaluate the difference in drug exposure of rifampicin in native versus non-native Paraguayan populations using dried blood spots (DBS) samples collected utilizing a limited sampling strategy. This was a prospective pharmacokinetic study that enrolled hospitalized tuberculosis (TB) patients from both native and non-native populations receiving oral rifampicin 10 mg/kg once-daily dosing. Steady-state DBS samples were collected at 2, 4, and 6 h after intake of rifampicin. The area under the time concentration curve 0-24 h (AUC0-24) was calculated using a Bayesian population PK model. Rifampicin AUC0-24 < 38.7 mg*h/L was considered as low. The probability of target attainment (PTA) was calculated using AUC0-24/MIC > 271 as a target and estimated MIC values of 0.125 and 0.25 mg/L. In total, 50 patients were included. Native patients (n = 30) showed comparable drug exposure to the non-natives (n = 20), median AUC0-24 24.7 (17.1-29.5 IQR) and 21.6 (15.0-35.4 IQR) mg*h/L (p = 0.66), respectively. Among total patients, only 16% (n = 8) had a rifampicin AUC0-24 > 38.7 mg*h/L. Furthermore, PTA analysis showed that only 12 (24%) of the patients met a target AUC0-24 /MIC ≥ 271, assuming an MIC of 0.125 mg/L, which plummeted to 0% at a wild-type MIC of 0.25 mg/L. We successfully used DBS and limited sampling for the AUC0-24 estimation of rifampicin. Currently, our group, the EUSAT-RCS consortium, is preparing a prospective multinational, multicenter phase IIb clinical trial evaluating the safety and efficacy of high-dose rifampicin (35 mg/kg) in adult subjects using the DBS technique for AUC0-24 estimation.

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BACKGROUND: Surveys for intestinal parasitic infections (IPIs) often involve samples from three sampling dates analysed by various microscopy techniques. However, analysis of three samples per individual is a huge burden on time and resources. We compared the value from analysing three or fewer samples. METHODS: In this cross-sectional study, three faecal samples were collected every other day from 332 children from two locations in Guantanamo province, Cuba. Samples were analysed by wet mount with Lugol stain, Willis flotation method and Kato-Katz thick smear. RESULTS: Most parasites were detected by wet mount, although helminth eggs not found by wet smear were detected by the Willis flotation method (in particular) and Kato-Katz smear. Blastocystis spp. was the most commonly detected parasite (about 65%), then Giardia duodenalis and then Entamoeba spp. Although analysis of two stool samples significantly increased occurrence data for Blastocystis, this was not so for the other parasites. For none of the protozoan parasites were results from analysing three samples significantly higher than results from analysing just two samples. CONCLUSIONS: Analysing two faecal samples by wet mount and the Willis flotation method provides useful data for estimating the prevalence of IPIs in relatively high prevalence settings. Analysing further samples provides limited additional information and adds an extra burden in terms of time and resources.

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INTRODUCTION: Due to its high specificity and sensitivity, liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) is the gold standard method for immunosuppressant quantification in therapeutic drug monitoring. In this context, dried blood spots (DBS) have become a promising strategy as a sample collection procedure. Although the advantages of DBS over venipuncture are well known, this approach has limitations that strongly influence the acceptance of analytical results. Among them, the most important is hematocrit (Ht). The easiest way of overcoming this problem is by analyzing complete spots. In this strategy, called dried matrix on paper discs (DMPD), blood is volumetrically applied on pre-punched discs. OBJECTIVES: To validate an LC-MS/MS method for the quantification of tacrolimus, sirolimus, everolimus and cyclosporin A using DMPD. METHODS: The procedure was validated according to international guidelines using a commercial kit. The following performance parameters were evaluated: selectivity, carryover, linearity, accuracy, precision, lower limit of quantitation, relative recovery, commutability and stability. In addition, a method comparison study was performed to evaluate the clinical influence of Ht on the results. RESULTS: All performance parameters were within acceptance criteria and, hence, it was determined that the validated method is fit for the intended purpose. Likewise, calculated bias values on medical decision levels showed that there was no clinical influence of Ht on the results. CONCLUSION: Unlike other similar methodologies that have been published, here, a simple method has been fully validated. This is the first LC-MS/MS methodology adapting a commercial kit to use DMPD as a sampling strategy.

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TDM of tacrolimus is usually performed with trough levels (C0h ). However, in pediatric patients, C0h may not be an adequate marker. The AUC is considered a more suitable indicator of drug exposure. As several blood samples are needed for the estimation of AUC, and LSS for predicting tacrolimus AUC and optimizing the dose adjustment have been proposed. Moreover, in emerging countries such as Mexico, non-innovator formulations, which bioequivalence has not been demonstrated, are frequently used. Hence, the aim of this study was to develop and validate a LSS to predict the tacrolimus AUC0-12h in Mexican pediatric kidney transplant recipients who received either Prograf® or non-innovator tacrolimus formulations. A total of 56 pharmacokinetic profiles were randomized into two groups: model development (n = 28) and model validation (n = 28). The limited sampling equations were obtained after a stepwise multiple regression using AUC as the dependent variable and tacrolimus blood concentrations, quantified by CMIA, at different time points as the independent variables. The final equation included observed concentrations at 1 hour (C1h ) and 4 hours (C4h ) after dose administration. The predictive performance of the model was adequate in terms of both, bias and precision. Results strongly suggest that the clinical use of this LSS could provide an ethical, cost-, and time-effective method in the TDM of tacrolimus in pediatric patients with kidney transplant. The model proved to be adequate with either Prograf® or non-innovator tacrolimus formulations of dubious bioequivalence.

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Cost reduction in plant breeding and conservation programs depends largely on correctly defining the minimal sample size required for the trustworthy assessment of intra- and inter-cultivar genetic variation. White clover, an important pasture legume, was chosen for studying this aspect. In clonal plants, such as the aforementioned, an appropriate sampling scheme eliminates the redundant analysis of identical genotypes. The aim was to define an optimal sampling strategy, i.e., the minimum sample size and appropriate sampling scheme for white clover cultivars, by using AFLP data (283 loci) from three popular types. A grid-based sampling scheme, with an interplant distance of at least 40 cm, was sufficient to avoid any excess in replicates. Simulations revealed that the number of samples substantially influenced genetic diversity parameters. When using less than 15 per cultivar, the expected heterozygosity (He) and Shannon diversity index (I) were greatly underestimated, whereas with 20, more than 95% of total intra-cultivar genetic variation was covered. Based on AMOVA, a 20-cultivar sample was apparently sufficient to accurately quantify individual genetic structuring. The recommended sampling strategy facilitates the efficient characterization of diversity in white clover, for both conservation and exploitation.

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Cost reduction in plant breeding and conservation programs depends largely on correctly defining the minimal sample size required for the trustworthy assessment of intra- and inter-cultivar genetic variation. White clover, an important pasture legume, was chosen for studying this aspect. In clonal plants, such as the aforementioned, an appropriate sampling scheme eliminates the redundant analysis of identical genotypes. The aim was to define an optimal sampling strategy, i.e., the minimum sample size and appropriate sampling scheme for white clover cultivars, by using AFLP data (283 loci) from three popular types. A grid-based sampling scheme, with an interplant distance of at least 40 cm, was sufficient to avoid any excess in replicates. Simulations revealed that the number of samples substantially influenced genetic diversity parameters. When using less than 15 per cultivar, the expected heterozygosity (He) and Shannon diversity index (I) were greatly underestimated, whereas with 20, more than 95 percent of total intra-cultivar genetic variation was covered. Based on AMOVA, a 20-cultivar sample was apparently sufficient to accurately quantify individual genetic structuring. The recommended sampling strategy facilitates the efficient characterization of diversity in white clover, for both conservation and exploitation.

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Sampling strategies for the difference are constructed when missing observations are present. Two different situations are analyzed. One of them is related with a non random device settled by the statistician for reducing costs. The other is a non response problem. An unbiased minimum variance estimator is obtained in the first case and an approximation to it is deduced. The unbiased estimation in the second is associated with subsampling tactics.