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Background: The purpose of this study is to compare the effect of different systems for eliminating duplicates in order to optimize the calculation of the prevalence of methicillin-resistant Staphylococcus aureus (MRSA) infection. Methods: We compare the Clinical and Laboratory Standards Institute (CLSI) criterion, time criteria and the criterion recommended by the European Antimicrobial Surveillance System (EARSS). Results: Multiple isolates of MRSA are frequently recovered from successive cultures from the same patient (the average isolation rate of MRSA is 2.72), which demonstrates the importance of eliminating duplicates. When CLSI criterion data are compared to those obtained using other criteria, a significant increase in the number of S. aureus isolates was found applying time criteria (up to 36%) or the EARSS criterion (13%). There is also an increase in the methicillin resistance rate (between 3.31 and 3.96%; p < 0.01). Conclusions: We believe that the EARSS method, with the proper quality controls and latest software tools available, is the best for determining the true situation of MRSA.

Resumen La deteccion y la especificidad de los autoanticuerpos contra antigenos extraibles del nucleo (ENA) tienen un importante papel en la ayuda al diagnostico de ciertas enfermedades autoinmunes. Existen diversos metodos para detectar estos autoanticuerpos entre los que se encuentran el enzimoinmunoanalisis (ELISA) y el “Inmunodot” como los mas utilizados. En este estudio evaluamos cuatro metodos para la deteccion de ENAs, comparando un ensayo Inmunodot frente a tres ensayos ELISA. Se analizaron muestras de 26 pacientes en las que fueron determinados los autoanticuerpos antinucleares (ANAs) por Inmunofluorescencia (IFI) y los anticuerpos frente a ENAs (Ro, La, Sm-RNP, Sm, Scl-70 y Jo-1) por Inmunodot y por los tres ensayos de ELISA. Se obtuvo que en un 27% de los casos todas las tecnicas eran coincidentes en el resultado y se correlacionaron con los sintomas clinicos del paciente. La concordancia entre las tecnicas de ELISA evaluadas fue de un 88% para ELISA 1 vs 2, 73% para ELISA 1 vs 3 y 73% para ELISA 2 vs 3, y la de los metodos de ELISA frente el metodo de Inmunodot fueron de un 38%, 42% y 27% (vs ELISA1, 2 y 3 respectivamente). Las tecnicas de ELISA tienen un porcentaje de concordancia entre si mas alto que con Inmunodot, y ademas se correlacionan mejor con los sintomas clinicos y el patron ANA del paciente. Los resultados muestran que la presencia de autoanticuerpos ENA no siempre tiene relacion con la presentacion clinica del paciente, por ello el laboratorio y el clinico deben ser conscientes de la sensibilidad y la especificidad de cada metodo empleado en el laboratorio clinico.

Background: The ε4 allele of Apolipoprotein E is involved in lipid metabolism. Oxidative stress produces an increase in lipid peroxidation that has been implicated in the pathogenic cascade in Alzheimer’s disease. This study estimated the effect of the ε4 allele, malondialdehyde and lipid levels on the risk for Alzheimer’s disease. Methods: A total of 41 control subjects and 73 patients with Alzheimer’s disease were recruited. The Apolipoprotein E genotype was determined by amplification of exon 4 of the Apolipoprotein E by polymerase chain reaction (PCR); malondialdehyde concentration was determined by high-pressure liquid chromatography, and serum lipids were measured by routine photometric techniques. Results: Malondialdehyde levels were significantly higher in Alzheimer’s disease patients independent of the Apolipoprotein E genotype and ε4 allele. The ε4 allele increases the risk of Alzheimer’s disease by 5.114 times and elevated malondialdehyde levels increase the risk by 9.342. Conclusion: The presence of ε4 allele and elevated malondialdehyde levels are independent risk factors for Alzheimer’s disease. These findings support the hypothesis that lipid peroxidation and ε4 allele contribute to the pathogenic cascade in Alzheimer’s disease by different pathways.

Sir, The increase in resistance of Gram-negative bacilli is a serious public health problem and our aim was to determine the effect of different duplicate elimination criteria on the evolution of fluoroquinolone resistance in Escherichia coli and Klebsiella pneumoniae in our setting over a period of 18 years. The criteria used are described in Figure 1. Antibiotic susceptibility was studied using microdilution with the Wider semiautomatic system (Soria Melguizo, Spain) and Etest (AB Biodisk, Solna, Sweden), following CLSI (formerly NCCLS) recommendations. In E. coli, the mean percentage of fluoroquinolone-resistant strains according to the EARS-Net criterion was greater than that detected using the CLSI criterion, both when the total number of strains and when the number of extended-spectrum b-lactamase (ESBL)-producing strains was considered (28.4% versus 22.4% and 65.2% versus 60.5%, respectively). With regard to K. pneumoniae, we obtained 7.2% versus 6.3% and 52.2% versus 40.0%. Analysis of the criterion of time showed that when the period considered for elimination of duplicates increased, the ciprofloxacin resistance rate also increased compared with the CLSI criterion. When the EARS-Net criterion was applied, the prevalence of these microorganisms increased in both hospitalized and non-hospitalized patients, although this phenomenon was more important in the former than in the latter. All data are shown in Table 1. The increase in fluoroquinolone resistance is a serious public health problem, especially in ESBL-producing strains, for which the therapeutic options are far more limited, and the local epidemiology of resistance has been considered a key tool in the control of this process. It is essential to apply a duplicate elimination criterion, since analysis of the data based on the total criterion does not reflect the real situation of resistance. What has not yet been definitively established is which criterion is the best to use in each case. The first isolate from each patient is an objective criterion, but has serious limitations since the same patient may frequently be infected by different strains of the same species during the course of their disease and hospital stay, or a strain may become resistant during treatment of the patient due to the selection of resistant mutants during antibiotic therapy. The EARS-Net criterion reflects more faithfully the real situation of resistance, but suggests that it is necessary to apply strict quality control in phenotypic microbiological studies to detect resistance. Our study suggests that the new EARS-Net method of studying duplicates should be applied in order to detect changes in resistance patterns that may occur during the course of disease due to mutations of the microorganism causing the infection or superinfections by more resistant microorganisms.

The purpose of this study is to compare the effect of different systems for eliminating duplicates in order to optimize the calculation of the prevalence of methicillin-resistant Staphylococcus aureus (MRSA) infection.We compare the Clinical and Laboratory Standards Institute (CLSI) criterion, time criteria and the criterion recommended by the European Antimicrobial Surveillance System (EARSS).Multiple isolates of MRSA are frequently recovered from successive cultures from the same patient (the average isolation rate of MRSA is 2.72), which demonstrates the importance of eliminating duplicates. When CLSI criterion data are compared to those obtained using other criteria, a significant increase in the number of S. aureus isolates was found applying time criteria (up to 36%) or the EARSS criterion (13%). There is also an increase in the methicillin resistance rate (between 3.31 and 3.96%; p0.01).We believe that the EARSS method, with the proper quality controls and latest software tools available, is the best for determining the true situation of MRSA.

Circulating tumor cell (CTC) enumeration is important clinically for identifying prognostic and predictive factors in patients with solid cancers. The CellSearch device (Veridex) is an immunomagnetic CTC selection and enumeration system used in clinical practice. The ImageStream (Amnis) combines the strengths of flow cytometry and fluorescent microscopy in a single platform and has potential application for CTC counting. The performance in CTC enumeration was compared between the ImageStream and CellSearch systems. Various numbers of PANC-1 tumor cells were spiked into 7.5 mL of peripheral blood from a healthy donor. Before cell analysis by the ImageStream, tumor cell enrichment was performed by immunomagnetic selection with anti-EpPCAM. Anti-CD45 and anti-CK markers were used to discriminate between tumor cells and leukocytes. The ratios of tumor cells recovered from each dilution were calculated for both methods. The Wilcoxon rank test was applied to compare the results of the two methods and the reference value. The results of the two tested methods differed significantly from the reference value, but did not differ between them. Nevertheless, lower level of trueness and precision was observed in ImageStream when fewer numbers of CTCs were analyzed. Our results suggest that ImageStream platform for CTC enumeration has a potential value for the early diagnosis of disseminated disease, but needs an improvement of precision for the enumeration of low number of CTC.

The ICP-MS for simultaneous trace element determination in human blood has prevailed as the most suitable methodology for clinical aims because of its rapidity, detection limits and minimal sample quantity needed for analysis. As the proteic matrix is high, it is necessary to fine-tune the ICP-MS Agilent 7500i with autosampler CETAC ASX-500 and ISIS System connected, and further we have to the sample pre-treatment in order to obtain good results. The study of the results shows that the best pre-treatment for blood serum samples consists of a basic treatment by 1/10 dilution with a solution of EDTA and NH4OH, with a detection limit of the order of μg/L and a reduction of the necessary patient sample volume to 2 mL.

[Background]: Colorectal carcinoma is a common cause of cancer. Adjuvant treatments include: 5-fluorouracil administered together with folinic acid, or more recently, oral fluoropyrimidines such as capecitabine, in combination with oxaliplatin or irinotecan. Metastatic colorectal cancer patients can benefit from other additional treatments such as cetuximab or bevacizumab., [Methods]: Using cell culture techniques, we isolated clonal populations from primary cultures of ascitic effusion derived from a colon cancer patient and after several passages an established cell line, HGUE-C-1, was obtained. Genetic analysis of HGUE-C-1 cells was performed by PCR of selected exons and sequencing. Cell proliferation studies were performed by MTT assays and cell cycle analyses were performed by flow cytometry. Retinoblastoma activity was measured by luciferase assays and proteins levels and activity were analysed by Western blot or immunohistochemistry., [Results]: We have established a new cell line from ascitic efussion of a colon cancer patient who did not respond to 5-fluorouracil or irinotecan. HGUE-C-1 cells did not show microsatellite instability and did not harbour mutations in KRAS, BRAF, PI3KCA or TP53. However, these cells showed loss of heterozygosity affecting Adenomatous Polyposis Coli and nuclear staining of β-catenin protein. The HGUE-C-1 cell line was sensitive to erlotinib, gefitinib, NVP-BEZ235, rapamycin and trichostatin, among other drugs, but partially resistant to heat shock protein inhibitors and highly resistant to AZD-6244 and oxaliplatin, even though the patient from which this cell line was derived had not been exposed to these drugs. Molecular characterization of this cell line revealed low expression levels and activity of Retinoblastoma protein and elevated basal levels of Erk1/2 activity and p70S6K expression and activity, which may be related to chemoresistance mechanisms., [Conclusions]: HGUE-C-1 represents a novel and peculiar colon carcinoma model to study chemoresistance to chemotherapeutic agents and to novel anti-neoplasic drugs that interrupt signalling pathways such as the APC/βcatenin, Ras/Raf/Mek/Erk, PI3K/mTOR/p70S6K pathways as well as histone regulation mechanisms., This article has been funded by grants from the Instituto de Salud Carlos III FIS PI080901and FIS PI01202025 to Miguel Saceda.