Introduction of an automated system for the diagnosis and quantification of hepatitis B and hepatitis C viruses.

Hepatitis B virus (HBV) and Hepatitis C virus (HCV) infections pose major public health problems because of their prevalence worldwide. Consequently, screening for these infections is an important part of routine laboratory activity. Serological and molecular markers are key elements in diagnosis, prognosis and treatment monitoring for HBV and HCV infections. Today, automated chemiluminescence immunoassay (CLIA) analyzers are widely used for virological diagnosis, particularly in high-volume clinical laboratories. Molecular biology techniques are routinely used to detect and quantify viral genomes as well as to analyze their sequence; in order to determine their genotype and detect resistance to antiviral drugs. Real-time PCR, which provides high sensitivity and a broad dynamic range, has gradually replaced other signal and target amplification technologies for the quantification and detection of nucleic acid. The next-generation DNA sequencing techniques are still restricted to research laboratories.The serological and molecular marker methods available for HBV and HCV are discussed in this article, along with their utility and limitations for use in Chronic Hepatitis B (CHB) diagnosis and monitoring.

Differential outcome of concurrent radiotherapy plus epidermal growth factor receptor inhibitors versus radiotherapy plus cisplatin in patients with human papillomavirus-related head and neck cancer.

BACKGROUND Human papillomavirus (HPV)-related head and neck cancer has been associated with an improved prognosis in patients treated with radiotherapy (RT) +/- chemotherapy (CT); however, RT combined with epidermal growth factor receptor (EGFR) inhibitors has not been fully studied in this group of patients. METHODS Immunohistochemical expression of p16 and PCR of HPV16 DNA were retrospectively analyzed in tumor blocks from 108 stage III/IV head and neck cancer patients treated with RT+CT (56) or RT+EGFR inhibitors (52). Disease-free survival (DFS) and overall survival (OS) were analyzed by the Kaplan-Meier method. RESULTS DNA of HPV16 was found in 12 of 108 tumors (11%) and p16 positivity in 18 tumors (17%), with similar rates in both arms of treatment. After a median follow-up time of 35 months (range 6-135), p16-positive patients treated with RT+EGFR inhibitors showed improved survival compared with those treated with RT+CT (2-year OS 88% vs. 60%, HR 0.18; 95% CI 0.04 to 0.88; p = 0.01; and 2-year DFS 75% vs. 47%, HR 0.17; 95% CI 0.03 to 0.8; p = 0.01). However, no differences were observed in p16-negative patients (2-year OS 56% vs. 53%, HR 0.97; 95% CI 0.55 to 1.7; p = 0.9; and 2-year DFS 43% vs. 45%, HR 0.99; 95% CI 0.57 to 1.7; p = 0.9). CONCLUSIONS This is the first study to show that p16-positive patients may benefit more from RT+EGFR inhibitors than conventional RT+CT. These results are hypothesis-generating and should be confirmed in prospective trials.

Malnutrition prevalence in hospitalized elderly diabetic patients.

Background & aims: Malnutrition prevalence is unknown among elderly patients with diabetes mellitus. Our objectives were to determine malnutrition prevalence in elderly in patients with diabetes, and to describe their impact on prognosis. Methods: An observational multicenter study was conducted in 35 Spanish hospitals. Malnutrition was assessed with the Mini Nutritional Assessment (MNA) tool. Patients were followed until discharge. Results: 1,090 subjects were included (78 ± 7.1 years; 50% males). 39.1% had risk of malnutrition, and 21.2% malnutrition. A 15.5% of the malnourished subjects and 31.9 % of those at risk had a BMI =?30 kg/m2. In multivariate analysis, female gender (OR = 1.38; 95% CI: 1.19- 1.11), age (OR = 1.04; 95% CI: 1.02-1.06) and presence of diabetic complications (OR = 1.97; 95% CI: 1.52-2.56) were associated with malnutrition. Length of stay (LOS) was longer in at-risk and malnourished patients than in well-nourished (12.7 ± 9.9 and 15.7 ± 12.8 days vs 10.7 ± 9.9 days; p < 0.0001). After adjustment by age and gender, MNA score (OR = 0.895; 95% CI 0.814-0.985) and albumin (OR = 0.441; 95% CI 0.212-0.915) were associated with mortality. MNA score was associated with the probability of home discharge (OR = 1.150; 95% CI 1.084-1.219). Conclusion: A high prevalence of malnutrition among elderly in patients with diabetes was observed, regardless of BMI. Malnutrition, albumin, and MNA score were related to LOS, mortality and home discharge.

R-ESHAP as salvage therapy for patients with relapsed or refractory diffuse large B-cell lymphoma: the influence of prior exposure to rituximab on outcome. A GEL/TAMO study.

BACKGROUND The role of re-treatment with rituximab in aggressive B-cell lymphomas still needs to be defined. This study evaluated the influence of prior exposure to rituximab on response rates and survival in patients with diffuse large B-cell lymphoma treated with rituximab plus etoposide, cytarabine, cisplatinum and methylprednisolone (R-ESHAP). DESIGN AND METHODS We retrospectively analyzed 163 patients with relapsed or refractory diffuse large B-cell lymphoma who received R-ESHAP as salvage therapy with a curative purpose. Patients were divided into two groups according to whether rituximab had been administered (n=94, "R+" group) or not (n=69, "R-" group) prior to R-ESHAP. RESULTS Response rates were significantly higher in the R- group in the univariate but not in the multivariate analysis. In the analysis restricted to the R+ group, we observed very low complete remission and overall response rates in patients with primary refractory disease (8% and 33%, respectively), as compared to those in patients who were in first partial remission (41% and 86%) or who had relapsed disease (50% and 75%) (p<0.01 in both cases). Overall, 60% and 65% of patients in the R+ and R- groups, respectively, underwent stem-cell transplantation after the salvage therapy. With a median follow-up of 29 months (range, 6-84), patients in the R+ group had significantly worse progression-free survival (17% vs. 57% at 3 years, p<0.0001) and overall survival (38% v 67% at 3 years, p=0.0005) than patients in the R- group. Prior exposure to rituximab was also an independent adverse prognostic factor for both progression-free survival (RR: 2.0; 95% CI: 1.2-3.3, p=0.008) and overall survival (RR: 2.2; 95% CI: 1.3-3.9, p=0.004). CONCLUSIONS R-ESHAP was associated with a high response rate in patients who were not refractory to upfront rituximab-based chemotherapy. However, the survival outcome was poor for patients previously exposed to rituximab, as compared to in those who had not previously been treated with rituximab.

Epigenetic regulation of human cancer/testis antigen gene, HAGE, in chronic myeloid leukemia.

BACKGROUND AND OBJECTIVES Cancer testis antigens (CTA) provide attractive targets for cancer-specific immunotherapy. Although CTA genes are expressed in some normal tissues, such as the testis, this immunologically protected site lacks MHC I expression and as such, does not present self antigens to T cells. To date, CTA genes have been shown to be expressed in a range of solid tumors via demethylation of their promoter CpG islands, but rarely in chronic myeloid leukemia (CML) or other hematologic malignancies. DESIGN AND METHODS In this study, the methylation status of the HAGE CTA gene promoter was analyzed by quantitative methylation-specific polymerase chain reaction (MSP) and sequencing in four Philadelphia-positive cell lines (TCC-S, K562, KU812 and KYO-1) and in CML samples taken from patients in chronic phase (CP n=215) or blast crisis (BC n=47). HAGE expression was assessed by quantitative reverse transcriptase-polymerase chain reaction. RESULTS The TCC-S cell line showed demethylation of HAGE that was associated with over-expression of this gene. HAGE hypomethylation was significantly more frequent in BC (46%) than in CP (22%) (p=0.01) and was correlated with high expression levels of HAGE transcripts (p<0.0001). Of note, in CP-CML, extensive HAGE hypomethylation was associated with poorer prognosis in terms of cytogenetic response to interferon (p=0.01) or imatinib (p=0.01), molecular response to imatinib (p=0.003) and progression-free survival (p=0.05). INTERPRETATIONS AND CONCLUSION: The methylation status of the HAGE promoter directly correlates with its expression in both CML cell lines and patients and is associated with advanced disease and poor outcome.

Prospective phase II trial of extended treatment with rituximab in patients with B-cell post-transplant lymphoproliferative disease.

BACKGROUND AND OBJECTIVES The elective treatment of patients with post-transplant lymphoproliferative disorders is controversial. The purpose of this trial was to evaluate the efficacy of treatment with extended doses of rituximab adapted to the response in patients with post-transplant lymphoproliferative disorders after solid organ transplantation. DESIGN AND METHODS This was a prospective, multicenter, phase II trial. Patients were treated with reduction of immunosuppression and four weekly infusions of rituximab. Those patients who did not achieve complete remission (CR) received a second course of four rituximab infusions. The primary end-point of the study was the CR rate. RESULTS Thirty-eight patients were assesable. One episode of grade 4 neutropenia was the only severe adverse event observed. After the first course of rituximab, 13 (34.2%) patients achieved CR, 8 patients did not respond, and 17 patients achieved partial remission. Among those 17 patients, 12 could be treated with a second course of rituximab, and 10 (83.3%) achieved CR, yielding an intention-to-treat CR rate of 60.5%. Eight patients excluded from the trial because of absence of CR were treated with rituximab combined with chemotherapy, and six (75%) achieved CR. Event-free survival was 42% and overall survival was 47% at 27.5 months. Fourteen patients died, ten of progression of their post-transplant lymphoproliferative disorder. INTERPRETATION AND CONCLUSIONS These results confirm that extended treatment with rituximab can obtain a high rate of CR in patients with post-transplant lymphoproliferative disorders after solid organ transplantation without increasing toxicity, and should be recommended as initial therapy for these patients.

Survival, classifications, and desmosomal plaque genes in non-small cell lung cancer.

Novel biomarkers are required to improve prognostic predictions obtained with lung cancer staging systems. This study of 62 surgically-treated Non-Small Cell Lung Cancer (NSCLC) patients had two objectives: i) to compare the predictive value of T-stage classifications between the 6(th) and 7(th) editions of the Tumor, Node, and Metastasis staging system (TNM); and ii) to examine the association of Pkp1 and/or Krt15 gene expression with survival and outcomes. Multivariate and Kaplan-Meier survival analyses were performed, examining the relationship of survival with T-stage, recurrence, and TNM-stage (by each TNM edition) and with the single/combined expression of Pkp1 and/or Krt15 genes. Five-year survival rates only significantly differed as a function of T-stage in patients without recurrence when estimated using the 6(th) edition of the TNM classification and only in patients in pathologic TNM-stage IA using the 7(th). Overall survival for patients with elevated expression of both genes was 13.5 months in those with adenocarcinoma and 34.6 months in those with squamous cell carcinoma. Overall survival was 30.4 months in patients with Pkp1 gene upregulation and 30.9 months in those with Krt15 gene upregulation. In conclusion, survival estimations as a function of T-staging differed between the 6(th) and 7(th) editions of TNM. Overall survival differed according to the expression of Pkp1 and/or Krt15 genes, although this relationship did not reach statistical significance.

Effectiveness of Pregabalin as Monotherapy or Combination Therapy for Neuropathic Pain in Patients Unresponsive to Previous Treatments in a Spanish Primary Care Setting

BACKGROUND AND OBJECTIVE Patients from a previous study of neuropathic pain (NP) in the Spanish primary care setting still had symptoms despite treatment. Subsequently, patients were treated as prescribed by their physician and followed up for 3 months. Since pregabalin has been shown to be effective in NP, including refractory cases, the objective of this study was to assess the effectiveness of pregabalin therapy in patients with NP refractory to previous treatments. METHODS This was a post hoc analysis of pregabalin-naïve NP patients treated with pregabalin in a 3-month follow-up observational multicenter study to assess symptoms and satisfaction with treatment. Patients were evaluated with the Douleur Neuropathique en 4 questions (DN4), the Brief Pain Inventory (BPI) and the Treatment Satisfaction for Medication Questionnaire (SATMED-Q) overall satisfaction domain. RESULTS 1,670 patients (mean age 58 years, 59 % women), previously untreated or treated with ≥1 drug other than pregabalin, were treated with pregabalin (37 % on monotherapy). At 3 months, pain intensity and its interference with activities decreased by half (p < 0.0001), while the number of days with no or mild pain increased by a mean of 4.5 days (p < 0.0001). Treatment satisfaction increased twofold (p < 0.0001). Patients with a shorter history of pain and those with neuralgia and peripheral nerve compression syndrome (PCS) as etiologies had the highest proportion on monotherapy and showed the greatest improvements in pain-related parameters in their respective group categories. CONCLUSION Treatment with pregabalin (as monotherapy or combination therapy) provides benefits in pain and treatment satisfaction in patients with NP, including refractory cases. Shorter disease progression and neuralgia and PCS etiologies are favorable factors for pregabalin treatment response.

Pulmonary embolism and 3-month outcomes in 4036 patients with venous thromboembolism and chronic obstructive pulmonary disease: data from the RIETE registry.

BACKGROUND Patients with chronic obstructive pulmonary disease (COPD) have a modified clinical presentation of venous thromboembolism (VTE) but also a worse prognosis than non-COPD patients with VTE. As it may induce therapeutic modifications, we evaluated the influence of the initial VTE presentation on the 3-month outcomes in COPD patients. METHODS COPD patients included in the on-going world-wide RIETE Registry were studied. The rate of pulmonary embolism (PE), major bleeding and death during the first 3 months in COPD patients were compared according to their initial clinical presentation (acute PE or deep vein thrombosis (DVT)). RESULTS Of the 4036 COPD patients included, 2452 (61%; 95% CI: 59.2-62.3) initially presented with PE. PE as the first VTE recurrence occurred in 116 patients, major bleeding in 101 patients and mortality in 443 patients (Fatal PE: first cause of death). Multivariate analysis confirmed that presenting with PE was associated with higher risk of VTE recurrence as PE (OR, 2.04; 95% CI: 1.11-3.72) and higher risk of fatal PE (OR, 7.77; 95% CI: 2.92-15.7). CONCLUSIONS COPD patients presenting with PE have an increased risk for PE recurrences and fatal PE compared with those presenting with DVT alone. More efficient therapy is needed in this subtype of patients.

Efficacy and safety of a multifactor intervention to improve therapeutic adherence in patients with chronic obstructive pulmonary disease (COPD): protocol for the ICEPOC study.

Low therapeutic adherence to medication is very common. Clinical effectiveness is related to dose rate and route of administration and so poor therapeutic adherence can reduce the clinical benefit of treatment. The therapeutic adherence of patients with chronic obstructive pulmonary disease (COPD) is extremely poor according to most studies. The research about COPD adherence has mainly focussed on quantifying its effect, and few studies have researched factors that affect non-adherence. Our study will evaluate the effectiveness of a multifactor intervention to improve the therapeutic adherence of COPD patients.

Skin infection caused by Scedosporium apiospermum in immunocompromised patients. Report of two cases.

Scedosporium apiospermum is a filamentous fungus that can cause cutaneous or extracutaneous disease. A large number of cases have been published over the last decades, mainly in patients immunocompromised as a result of their disease or treatment. These kinds of infections can progress rapidly and become disseminated, leading to very serious or even fatal complications. We report two new cases of skin infection by Scedosporium apiospermum from our hospital.

Angiographic demonstration of neoangiogenesis after intra-arterial infusion of autologous bone marrow mononuclear cells in diabetic patients with critical limb ischemia.

Critical limb ischemia in diabetic patients is associated with high rates of morbidity and mortality. Suboptimal responses to the available medical and surgical treatments are common in these patients, who also demonstrate limited vascular homeostasis. Neovasculogenesis induced by stem cell therapy could be a useful approach for these patients. Neovasculogenesis and clinical improvement were compared at baseline and at 3 and 12 months after autologous bone marrow-derived mononuclear cell (BMMNC) transplantation in diabetic patients with peripheral artery disease. We conducted a prospective study to evaluate the safety and efficacy of intra-arterial administration of autologous BMMNCs (100-400 × 10(6) cells) in 20 diabetic patients with severe below-the-knee arterial ischemia. Although the time course of clinical effects differed among patients, after 12 months of follow-up all patients presented a notable improvement in the Rutherford-Becker classification, the University of Texas diabetic wound scales, and the Ankle-Brachial Index in the target limb. The clinical outcome was consistent with neovasculogenesis, which was assessed at 3 months by digital subtraction angiography and quantified by MetaMorph software. Unfortunately, local cell therapy in the target limb had no beneficial effect on the high mortality rate in these patients. In diabetic patients with critical limb ischemia, intra-arterial perfusion of BMMNCs is a safe procedure that generates a significant increase in the vascular network in ischemic areas and promotes remarkable clinical improvement.

Effectiveness of holistic neuropsychological rehabilitation for Spanish population with acquired brain injury measured using Rasch analysis.

INTRODUCTION The Rasch model is increasingly used in the field of rehabilitation because it improves the accuracy of measurements of patient status and their changes after therapy. OBJECTIVE To determine the long-term effectiveness of a holistic neuropsychological rehabilitation program for Spanish outpatients with acquired brain injury (ABI) using Rasch analysis. METHODS Eighteen patients (ten with long evolution - patients who started the program > 6 months after ABI- and eight with short evolution) and their relatives attended the program for 6 months. Patients' and relatives' answers to the European Brain Injury Questionnaire and the Frontal Systems Behavior Scale at 3 time points (pre-intervention. post-intervention and 12 month follow-up) were transformed into linear measures called logits. RESULTS The linear measures revealed significant improvements with large effects at the follow-up assessment on cognitive and executive functioning, social and emotional self-regulation, apathy and mood. At follow-up, the short evolution group achieved greater improvements in mood and cognitive functioning than the long evolution patients. CONCLUSIONS The program showed long-term effectiveness for most of the variables, and it was more effective for mood and cognitive functioning when patients were treated early. Relatives played a key role in the effectiveness of the rehabilitation program.

Estudio comparativo del estatus clínico-nutricional en mujeres obesas posmenopáusicas incluídas en un programa de pérdida de peso basado en platos preparados.

INTRODUCTION Few studies have evaluated the efficacy and reliability of weight loss-focussed prepared food dishes in obese post-menopausal women. OBJECTIVE To compare the efficacy of a weight loss programme based on a balanced hypocaloric diet using prepared dishes* with that of a similar programme based on standard commercially available foods and with a non-intervened control group. A further aim was to evaluate the subjectivity of participants in the preparation of the diet-adjusted dishes based on usually consumed products. SUBJECTS Obese post-menopausal women aged between 55 and 65 years. DESIGN Controlled longitudinal interventional study. METHOD The sample of 75 female volunteers were divided into three groups of 25 women: a control group, who continued to consume their usual non-dietary adjusted meals (CG), an intervened group, treated with a diet adjusted to their individual requirements and based on standard commercially available food (SG), and another intervened group, treated with a similarly adjusted diet but based on prepared dishes (PG). Data were gathered on anthropometric variables, consumption habits and physical activity levels, and clinical-nutritional controls were conducted at the start and every two weeks to the end of the 8-week study in order to evaluate biochemical changes. RESULTS The weight loss was slightly higher in the prepared-dishes group (PG) than in the standard food diet group (SG), but the difference was not statistically significant, whereas it was considerably higher in both groups than in the non-dietary adjusted control group (CG) and this difference was highly significant (losses of 7.60 kg in PG and 7.01 kg in SG versus 2.10 kg in CG (p < 0.01). However, the PG showed a significantly higher (p < 0.01) loss of fatty mass and abdominal circumference versus the SG women. CONCLUSION More weight was lost by the two groups treated with a diet based on prepared dishes or usual food items in comparison to untreated controls, but the diet based on prepared dishes obtained more reliable and higher quality outcomes, achieving a positive change at fatty compartment level and in the abdominal circumference.

Amplicon DNA melting analysis for the simultaneous detection of Brucella spp and Mycobacterium tuberculosis complex. Potential use in rapid differential diagnosis between extrapulmonary tuberculosis and focal complications of brucellosis.

Some sites of extrapulmonary tuberculosis and focal complications of brucellosis are very difficult to differentiate clinically, radiologically, and even histopathologically. Conventional microbiological methods for the diagnosis of extrapulmonary tuberculosis and complicated brucellosis not only lack adequate sensitivity, they are also time consuming, which could lead to an unfavourable prognosis. The aim of this work was to develop a multiplex real-time PCR assay based on SYBR Green I to simultaneously detect Brucella spp and Mycobacterium tuberculosis complex and evaluate the efficacy of the technique with different candidate genes. The IS711, bcsp31 and omp2a genes were used for the identification of Brucella spp and the IS6110, senX3-regX3 and cfp31 genes were targeted for the detection of the M. tuberculosis complex. As a result of the different combinations of primers, nine different reactions were evaluated. A test was defined as positive only when the gene combinations were capable of co-amplifying both pathogens in a single reaction tube and showed distinguishable melting temperatures for each microorganism. According to the melting analysis, only three combinations of amplicons (senX3-regX3+bcsp31, senX3-regX3+IS711 and IS6110+IS711) were visible. Detection limits of senX3-regX3+bcsp31 and senX3-regX3+IS711 were of 2 and 3 genome equivalents for M. tuberculosis complex and Brucella while for IS6110+IS711 they were of 200 and 300 genome equivalents, respectively. The three assays correctly identified all the samples, showing negative results for the control patients. The presence of multicopy elements and GC content were the components most influencing the efficiency of the test; this should be taken into account when designing a multiplex-based SYBR Green I assay. In conclusion, multiplex real time PCR assays based on the targets senX3-regX3+bcsp31 and senX3-regX3+IS711 using SYBR Green I are highly sensitive and reproducible. This may therefore be a practical approach for the rapid differential diagnosis between extrapulmonary tuberculosis and complicated brucellosis.