Evaluation of the new advanced 15-loci MIRU-VNTR genotyping tool in Mycobacterium tuberculosis molecular epidemiology studies

Background. During the last few years, PCR-based methods have been developed to simplify and reduce the time required for genotyping Mycobacterium tuberculosis (MTB) by standard approaches based on IS6110-Restriction Fragment Length Polymorphism (RFLP). Of these, MIRU-12-VNTR (Mycobacterial interspersed repetitive units- variable number of tandem repeats) (MIRU-12) has been considered a good alternative. Nevertheless, some limitations and discrepancies with RFLP, which are minimized if the technique is complemented with spoligotyping, have been found. Recently, a new version of MIRU-VNTR targeting 15 loci (MIRU-15) has been proposed to improve the MIRU-12 format. Results. We evaluated the new MIRU-15 tool in two different samples. First, we analyzed the same convenience sample that had been used to evaluate MIRU-12 in a previous study, and the new 15-loci version offered higher discriminatory power (Hunter-Gaston discriminatory index [HGDI]: 0.995 vs 0.978; 34.4% of clustered cases vs 57.5%) and better correlation (full or high correlation with RFLP for 82% of the clusters vs 47%). Second, we evaluated MIRU-15 on a population-based sample and, once again, good correlation with the RFLP clustering data was observed (for 83% of the RFLP clusters). To understand the meaning of the discrepancies still found between MIRU-15 and RFLP, we analyzed the epidemiological data for the clustered patients. In most cases, splitting of RFLP-clustered patients by MIRU-15 occurred for those without epidemiological links, and RFLP-clustered patients with epidemiological links were also clustered by MIRU-15, suggesting a good epidemiological background for clustering defined by MIRU-15. Conclusion. The data obtained by MIRU-15 suggest that the new design is very efficient at assigning clusters confirmed by epidemiological data. If we add this to the speed with which it provides results, MIRU-15 could be considered a suitable tool for real-time genotyping.

Quantitative electroencephalography reveals different physiological profiles between benign and remitting-relapsing multiple sclerosis patients

BACKGROUND A possible method of finding physiological markers of multiple sclerosis (MS) is the application of EEG quantification (QEEG) of brain activity when the subject is stressed by the demands of a cognitive task. In particular, modulations of the spectral content that take place in the EEG of patients with multiple sclerosis remitting-relapsing (RRMS) and benign multiple sclerosis (BMS) during a visuo-spatial task need to be observed. METHODS The sample consisted of 19 patients with RRMS, 10 with BMS, and 21 control subjects. All patients were free of medication and had not relapsed within the last month. The power spectral density (PSD) of different EEG bands was calculated by Fast-Fourier-Transformation (FFT), those analysed being delta, theta, alpha, beta and gamma. Z-transformation was performed to observe individual profiles in each experimental group for spectral modulations. Lastly, correlation analyses was performed between QEEG values and other variables from participants in the study (age, EDSS, years of evolution and cognitive performance). RESULTS Nearly half (42%) the RRMS patients showed a statistically significant increase of two or more standard deviations (SD) compared to the control mean value for the beta-2 and gamma bands (F = 2.074, p = 0.004). These alterations were localized to the anterior regions of the right hemisphere, and bilaterally to the posterior areas of the scalp. None of the BMS patients or control subjects had values outside the range of +/- 2 SD. There were no significant correlations between these values and the other variables analysed (age, EDSS, years of evolution or behavioural performance). CONCLUSION During the attentional processing, changes in the high EEG spectrum (beta-2 and gamma) in MS patients exhibit physiological alterations that are not normally detected by spontaneous EEG analysis. The different spectral pattern between pathological and controls groups could represent specific changes for the RRMS patients, indicative of compensatory mechanisms or cortical excitatory states representative of some phases during the RRMS course that are not present in the BMS group.

Validation of the spanish version of the multiple sclerosis international quality of life (musiqol) questionnaire

BACKGROUND: The Multiple Sclerosis International Quality Of Life (MusiQoL) questionnaire, a 31-item, multidimensional, self-administrated questionnaire that is available in 14 languages including Spanish, has been validated using a large international sample. We investigated the validity and reliability of the Spanish version of MusiQoL in Spain. METHODS: Consecutive patients with different types and severities of multiple sclerosis (MS) were recruited from 22 centres across Spain. All patients completed the MusiQoL questionnaire, the 36-Item Short Form (SF-36) health survey, and a symptoms checklist at baseline and 21 days later. External validity, internal consistency, reliability and reproducibility were tested. RESULTS: A total of 224 Spanish patients were evaluated. Dimensions of MusiQoL generally demonstrated a high internal consistency (Cronbach's alpha: 0.70-0.92 for all but two MusiQoL domain scores). External validity testing revealed that the MusiQoL index score correlated significantly with all SF-36 dimension scores (Pearson's correlation: 0.46-0.76), reproducibility was satisfactory (intraclass correlation coefficient: 0.60-0.91), acceptability was high, and the time taken to complete the 31-item questionnaire was reasonable (mean [standard deviation]: 9.8 [11.8] minutes). CONCLUSIONS: The Spanish version of the MusiQoL questionnaire appears to be a valid and reliable instrument for measuring quality of life in patients with MS in Spain and constitutes a useful instrument to measure health-related quality of life in the clinical setting.

Validity and reliability of the Spanish version of the DN4 (Douleur Neuropathique 4 questions) questionnaire for differential diagnosis of pain syndromes associated to a neuropathic or somatic component

BACKGROUND This study assesses the validity and reliability of the Spanish version of DN4 questionnaire as a tool for differential diagnosis of pain syndromes associated to a neuropathic (NP) or somatic component (non-neuropathic pain, NNP). METHODS A study was conducted consisting of two phases: cultural adaptation into the Spanish language by means of conceptual equivalence, including forward and backward translations in duplicate and cognitive debriefing, and testing of psychometric properties in patients with NP (peripheral, central and mixed) and NNP. The analysis of psychometric properties included reliability (internal consistency, inter-rater agreement and test-retest reliability) and validity (ROC curve analysis, agreement with the reference diagnosis and determination of sensitivity, specificity, and positive and negative predictive values in different subsamples according to type of NP). RESULTS A sample of 164 subjects (99 women, 60.4%; age: 60.4 +/- 16.0 years), 94 (57.3%) with NP (36 with peripheral, 32 with central, and 26 with mixed pain) and 70 with NNP was enrolled. The questionnaire was reliable [Cronbach's alpha coefficient: 0.71, inter-rater agreement coefficient: 0.80 (0.71-0.89), and test-retest intra-class correlation coefficient: 0.95 (0.92-0.97)] and valid for a cut-off value > or = 4 points, which was the best value to discriminate between NP and NNP subjects. DISCUSSION This study, representing the first validation of the DN4 questionnaire into another language different than the original, not only supported its high discriminatory value for identification of neuropathic pain, but also provided supplemental psychometric validation (i.e. test-retest reliability, influence of educational level and pain intensity) and showed its validity in mixed pain syndromes.

Early and late skin reactions to radiotherapy for breast cancer and their correlation with radiation-induced DNA damage in lymphocytes

INTRODUCTION Radiotherapy outcomes might be further improved by a greater understanding of the individual variations in normal tissue reactions that determine tolerance. Most published studies on radiation toxicity have been performed retrospectively. Our prospective study was launched in 1996 to measure the in vitro radiosensitivity of peripheral blood lymphocytes before treatment with radical radiotherapy in patients with breast cancer, and to assess the early and the late radiation skin side effects in the same group of patients. We prospectively recruited consecutive breast cancer patients receiving radiation therapy after breast surgery. To evaluate whether early and late side effects of radiotherapy can be predicted by the assay, a study was conducted of the association between the results of in vitro radiosensitivity tests and acute and late adverse radiation effects. METHODS Intrinsic molecular radiosensitivity was measured by using an initial radiation-induced DNA damage assay on lymphocytes obtained from breast cancer patients before radiotherapy. Acute reactions were assessed in 108 of these patients on the last treatment day. Late morbidity was assessed after 7 years of follow-up in some of these patients. The Radiation Therapy Oncology Group (RTOG) morbidity score system was used for both assessments. RESULTS Radiosensitivity values obtained using the in vitro test showed no relation with the acute or late adverse skin reactions observed. There was no evidence of a relation between acute and late normal tissue reactions assessed in the same patients. A positive relation was found between the treatment volume and both early and late side effects. CONCLUSION After radiation treatment, a number of cells containing major changes can have a long survival and disappear very slowly, becoming a chronic focus of immunological system stimulation. This stimulation can produce, in a stochastic manner, late radiation-related adverse effects of varying severity. Further research is warranted to identify the major determinants of normal tissue radiation response to make it possible to individualize treatments and improve the outcome of radiotherapy in cancer patients.

Neural Correlates of the Severity of Cocaine, Heroin, Alcohol, MDMA and Cannabis Use in Polysubstance Abusers: A Resting-PET Brain Metabolism Study

INTRODUCTION Functional imaging studies of addiction following protracted abstinence have not been systematically conducted to look at the associations between severity of use of different drugs and brain dysfunction. Findings from such studies may be relevant to implement specific interventions for treatment. The aim of this study was to examine the association between resting-state regional brain metabolism (measured with 18F-fluorodeoxyglucose Positron Emission Tomography (FDG-PET) and the severity of use of cocaine, heroin, alcohol, MDMA and cannabis in a sample of polysubstance users with prolonged abstinence from all drugs used. METHODS Our sample consisted of 49 polysubstance users enrolled in residential treatment. We conducted correlation analyses between estimates of use of cocaine, heroin, alcohol, MDMA and cannabis and brain metabolism (BM) (using Statistical Parametric Mapping voxel-based (VB) whole-brain analyses). In all correlation analyses conducted for each of the drugs we controlled for the co-abuse of the other drugs used. RESULTS The analysis showed significant negative correlations between severity of heroin, alcohol, MDMA and cannabis use and BM in the dorsolateral prefrontal cortex (DLPFC) and temporal cortex. Alcohol use was further associated with lower metabolism in frontal premotor cortex and putamen, and stimulants use with parietal cortex. CONCLUSIONS Duration of use of different drugs negatively correlated with overlapping regions in the DLPFC, whereas severity of cocaine, heroin and alcohol use selectively impact parietal, temporal, and frontal-premotor/basal ganglia regions respectively. The knowledge of these associations could be useful in the clinical practice since different brain alterations have been associated with different patterns of execution that may affect the rehabilitation of these patients.

Characterization of Mycobacterium tuberculosis Beijing isolates from the Mediterranean area

BACKGROUND. The Beijing lineage of Mycobacterium tuberculosis is causing concern due to its global distribution and its involvement in severe outbreaks. Studies focused on this lineage are mainly restricted to geographical settings where its prevalence is high, whereas those in other areas are scarce. In this study, we analyze Beijing isolates in the Mediterranean area, where this lineage is not prevalent and is mainly associated with immigrant cases. RESULTS. Only 1% (N = 26) of the isolates from two population-based studies in Spain corresponded to Beijing strains, most of which were pan-susceptible and from Peruvian and Ecuadorian patients. Restriction fragment length polymorphism typing with the insertion sequence IS6110 identified three small clusters (2-3 cases). Mycobacterial interspersed repetitive unit-variable number tandem repeat typing (MIRU-15) offered low discriminatory power, requiring the introduction of five additional loci. A selection of the Beijing isolates identified in the Spanish sample, together with a sample of Beijing strains from Italy, to broaden the analysis context in the Mediterranean area, were assayed in an infection model with THP-1 cells. A wide range of intracellular growth rates was observed with only two isolates showing an increased intracellular replication, in both cases associated with contained production of TNF-alpha. No correlation was observed between virulence and the Beijing phylogenetic group, clustered/orphan status, or resistance. The Beijing strain responsible for extensive spread on Gran Canaria Island was also identified in Madrid, but did not lead to secondary cases and did not show high infectivity in the infection model. CONCLUSIONS. The Beijing lineage in our area is a non-homogeneous family, with only certain highly virulent representatives. The specific characterization of Beijing isolates in different settings could help us to accurately identify the virulent representatives before making general assumptions about this lineage.

Validation of a new tool to assess health-related quality of life in psoriasis: the PSO-LIFE questionnaire.

BACKGROUND Several questionnaires have been used to measure health related quality of life (HRQoL) in patients with psoriasis, few have been adapted for use in Spain; none of them was developed specifically for the Spanish population. The purpose of the study was to validate and assess the sensitivity to change of a new questionnaire to measure HRQOL in patients with psoriasis (PSO-LIFE). METHODS Observational, prospective, multicenter study performed in centers around Spain. Patients with active or inactive psoriasis completed the PSO-LIFE together with other Dermatology Quality of Life Index (DLQI) and Psoriasis Disability Index (PDI). A control group of patients with urticaria or atopic dermatitis was also included. Internal consistency and test-retest reliability of the PSO-LIFE were assessed by calculating Cronbach's alpha and Intraclass Correlation Coefficient (ICC). Validity was assessed by examining factorial structure, the capacity to discriminate between groups, and correlations with other measures. Sensitivity to change was measured using effect sizes. RESULTS The final sample included for analysis consisted of 304 patients and 56 controls. Mean (SD) age of psoriasis patients was 45.3 (14.5) years compared to 38.8 (14) years for controls (p < 0.01). Cronbach's alpha for the PSO-LIFE was 0.95 and test-retest reliability using the ICC was 0.98. Factor analysis showed the questionnaire to be unidimensional. Mean (SD) PSO-LIFE scores differed between patients with psoriasis and controls (64.9 [22.5] vs 69.4 [17.3]; p < 0.05), between those with active and inactive disease (57.4 [20.4] vs 76.4 [20.6]; p < 0.01), and between those with visible and non-visible lesions (63.0 [21.9] vs. 74.8 [23.9]; p < 0.01). The correlation between PSO-LIFE and PASI scores was moderate (r = -0.43) while correlations with DLQI and PDI dimensions ranged from moderate to high (between 0.4 and 0.8). Effect size on the PSO-LIFE in patients reporting 'much improved' health status at study completion was 1.01 (large effect size). CONCLUSIONS The present results provide substantial support for the reliability, validity, and responsiveness of the PSO-LIFE questionnaire in the population for which it was designed.

Human Papillomavirus (HPV) Type Distribution in Females with Abnormal Cervical Cytology. A Correlation with Histological Study

The aim of this study was to determine human papillomavirus (HPV) types distribution in cervical preneoplasic lesions in a Southern Spanish population and their relationship between HPV type and grade of histopathological abnormality. Finally, 232 cervical samples from 135 women with previous cytological abnormalities were included in this study. Colposcopy studies and biopsies were performed. Haematoxylin-eosin stained slides were observed and detection of HPV DNA in cervical swabs was carried out with use of a polymerase chain reaction and microarrays technology. The relationship between the presence of HPV infection and diagnostic variables was evaluated. HPV 16 was the most common type followed by HPV 58, 51, 33 and 31. However, the two HPV types targeted in the prophylactic vaccines such as HPV type 16 and 18 were detected in only 37 (21.2%) and 2 (1.1%) cases respectively. Thirty-three (18.9%) of samples were infected with multiple types, the majority of them with two types. In addition, during the follow-up of patients many changes in type distribution were observed. Several studies will be necessary in order to evaluate the HPV type distribution for therapeutically and prophylactic purposes such as vaccine treatment. Also, because of the differences obtained depending of use of various DNA technologies, the performance of some comparative studies of the different methods from detection of HPV would be advisable in a high population of patients and with the most homogeneous conditions possible.

Association between magnesium-deficient status and anthropometric and clinical-nutritional parameters in posmenopausal women.

Background: During menopause occurs weight gain and bone loss occurs due to the hormone decline during this period and other factors such as nutrition. Magnesium deficiency suggests a risk factor for obesity and osteoporosis. OBJECTIVE: To evaluate the clinical and nutritional magnesium status in a population of postmenopausal women, assessing intake and serum levels of magnesium in the study population and correlation with anthropometric parameters such as body mass index(BMI) and body fat, and biochemical parameters associated. SUBJECTS AND METHOD: The study involved 78 healthy women aged 44-76, with postmenopausal status, from the province of Grenade, Spain. The sample was divided into two age groups: group1, aged < 58, and group 2 aged >/= 58. Anthropometric parameters were recorded and nutritional intake was assessed by 72-hour recall, getting the RDAs through Nutriber(R) program. To assess the biochemical parameters was performed a blood sample was taken. Magnesium was analyzed by flame atomic absorption spectrophotometry (FAAS) in erythrocyte and plasma wet-mineralized samples. RESULTS: Our results show that 37.85% of the total subjects have an overweight status. Magnesium intake found in our population is insufficient in 36% of women,while plasma magnesium deficiency corresponds to 23% of the population and 72% of women have deficient levels of magnesium in erythrocyte. Positive correlations were found between magnesium intake and dietary intake of calcium, of phosphorus,and with prealbumin plasma levels, as well as with a lower waist / hip ratio Magnesium levels in erythrocyte were correlated with lower triglycerides and urea values. CONCLUSION: It is important to control and monitor the nutritional status of magnesium in postmenopausal women to prevent nutritional alterations and possible clinical and chronic degenerative diseases associated with magnesium deficiency and with menopause.

ESR1 gene promoter region methylation in free circulating DNA and its correlation with estrogen receptor protein expression in tumor tissue in breast cancer patients.

BACKGROUND Tumor expression of estrogen receptor (ER) is an important marker of prognosis, and is predictive of response to endocrine therapy in breast cancer. Several studies have observed that epigenetic events, such methylation of cytosines and deacetylation of histones, are involved in the complex mechanisms that regulate promoter transcription. However, the exact interplay of these factors in transcription activity is not well understood. In this study, we explored the relationship between ER expression status in tumor tissue samples and the methylation of the 5' CpG promoter region of the estrogen receptor gene (ESR1) isolated from free circulating DNA (fcDNA) in plasma samples from breast cancer patients. METHODS Patients (n = 110) with non-metastatic breast cancer had analyses performed of ER expression (luminal phenotype in tumor tissue, by immunohistochemistry method), and the ESR1-DNA methylation status (fcDNA in plasma, by quantitative methylation specific PCR technique). RESULTS Our results showed a significant association between presence of methylated ESR1 in patients with breast cancer and ER negative status in the tumor tissue (p = 0.0179). There was a trend towards a higher probability of ESR1-methylation in those phenotypes with poor prognosis i.e. 80% of triple negative patients, 60% of HER2 patients, compared to 28% and 5.9% of patients with better prognosis such as luminal A and luminal B, respectively. CONCLUSION Silencing, by methylation, of the promoter region of the ESR1 affects the expression of the estrogen receptor protein in tumors of breast cancer patients; high methylation of ESR1-DNA is associated with estrogen receptor negative status which, in turn, may be implicated in the patient's resistance to hormonal treatment in breast cancer. As such, epigenetic markers in plasma may be of interest as new targets for anticancer therapy, especially with respect to endocrine treatment.

Association of 25-hydroxyvitamin D with metabolic syndrome in patients with psoriasis: a case-control study.

Vitamin D deficiency is associated with higher cardiovascular risk and metabolic syndrome (MeS) criteria. The main objective of this study was to analyse the association of 25-hydroxyvitamin D (25-OHD) serum levels with MeS (National Cholesterol Education Program-Adult Treatment Panel-III criteria) in 46 Spanish patients with psoriasis, but without arthritis and systemic treatment, and 46 control subjects, matched by sex and age. The patients with psoriasis showed significantly lower level of 25-OHD than controls (30.5 vs. 38.3 ng/ml; p = 0.0001). Patients with MeS had significantly lower serum levels of 25-OHD than those without MeS (24.1 ± 7.5 vs. 32.8 ± 8.9, p = 0.007), and a negative correlation was found between 25-OHD and waist circumference, diastolic blood pressure, fasting glucose, and triglyceridaemia. In the control group no significant correlation between 25-OHD and MeS was found. Al-though the sample was small, our results suggest a potential protective role for 25-OHD in the metabolic profile of patients with psoriasis without arthritis.

Comparison of oxidative stress markers in HIV-infected patients on efavirenz or atazanavir/ritonavir-based therapy.

INTRODUCTION Chronic low-grade inflammation and immune activation may persist in HIV patients despite effective antiretroviral therapy (ART). These abnormalities are associated with increased oxidative stress (OS). Bilirubin (BR) may have a beneficial role in counteracting OS. Atazanavir (ATV) inhibits UGT1A1, thus increasing unconjugated BR levels, a distinctive feature of this drug. We compared changes in OS markers in HIV patients on ATV/r versus efavirenz (EFV)-based first-line therapies. MATERIALS AND METHODS Cohort of the Spanish Research Network (CoRIS) is a multicentre, open, prospective cohort of HIV-infected patients naïve to ART at entry and linked to a biobank. We identified hepatitis C virus/hepatitis B virus (HCV/HBV) negative patients who started first-line ART with either ATV/r or EFV, had a baseline biobank sample and a follow-up sample after at least nine months of ART while maintaining initial regimen and being virologically suppressed. Lipoprotein-associated Phospholipase A2 (Lp-PLA2), Myeloperoxidase (MPO) and Oxidized LDL (OxLDL) were measured in paired samples. Marker values at one year were interpolated from available data. Multiple imputations using chained equations were used to deal with missing values. Change in the OS markers was modelled using multiple linear regressions adjusting for baseline marker values and baseline confounders. Correlations between continuous variables were explored using Pearson's correlation tests. RESULTS 145 patients (97 EFV; 48 ATV/r) were studied. Mean (SD) baseline values for OS markers in EFV and ATV/r groups were: Lp-PLA2 [142.2 (72.8) and 150.1 (92.8) ng/mL], MPO [74.3 (48.2) and 93.9 (64.3) µg/L] and OxLDL [76.3 (52.3) and 82.2 (54.4) µg/L]. After adjustment for baseline variables patients on ATV/r had a significant decrease in Lp-PLA2 (estimated difference -16.3 [CI 95%: -31.4, -1.25; p=0.03]) and a significantly lower increase in OxLDL (estimated difference -21.8 [-38.0, -5.6; p<0.01] relative to those on EFV, whereas no differences in MPO were found. Adjusted changes in BR were significantly higher for the ATV/r group (estimated difference 1.33 [1.03, 1.52; p<0.01]). Changes in BR and changes in OS markers were significantly correlated. CONCLUSIONS In virologically suppressed patients on stable ART, OS was lower in ATV/r-based regimens compared to EFV. We hypothesize these changes could be in part attributable to increased BR plasma levels.

Lack of correlation between the optimal glycaemic control and coronary micro vascular dysfunction in patients with diabetes mellitus: a cross sectional study.

Background Coronary microvascular dysfunction (CMD) is associated with cardiovascular events in type 2 diabetes mellitus (T2DM). Optimal glycaemic control does not always preclude future events. We sought to assess the effect of the current target of HBA1c level on the coronary microcirculatory function and identify predictive factors for CMD in T2DM patients. Methods We studied 100 patients with T2DM and 214 patients without T2DM. All of them with a history of chest pain, non-obstructive angiograms and a direct assessment of coronary blood flow increase in response to adenosine and acetylcholine coronary infusion, for evaluation of endothelial independent and dependent CMD. Patients with T2DM were categorized as having optimal (HbA1c < 7 %) vs. suboptimal (HbA1c ≥ 7 %) glycaemic control at the time of catheterization. Results Baseline characteristics and coronary endothelial function parameters differed significantly between T2DM patients and control group. The prevalence of endothelial independent CMD (29.8 vs. 39.6 %, p = 0.40) and dependent CMD (61.7 vs. 62.2 %, p = 1.00) were similar in patients with optimal vs. suboptimal glycaemic control. Age (OR 1.10; CI 95 % 1.04–1.18; p < 0.001) and female gender (OR 3.87; CI 95 % 1.45–11.4; p < 0.01) were significantly associated with endothelial independent CMD whereas glomerular filtrate (OR 0.97; CI 95 % 0.95–0.99; p < 0.05) was significantly associated with endothelial dependent CMD. The optimal glycaemic control was not associated with endothelial independent (OR 0.60, CI 95 % 0.23–1.46; p 0.26) or dependent CMD (OR 0.99, CI 95 % 0.43–2.24; p = 0.98). Conclusions The current target of HBA1c level does not predict a better coronary microcirculatory function in T2DM patients. The appropriate strategy for prevention of CMD in T2DM patients remains to be addressed. Keywords: Endothelial dysfunction; Diabetes mellitus; Coronary microcirculation