Long-Lasting Protection of Activity of Nucleoside Reverse Transcriptase Inhibitors and Protease Inhibitors (PIs) by Boosted PI Containing Regimens.

BACKGROUND: The accumulation of mutations after long-lasting exposure to a failing combination antiretroviral therapy (cART) is problematic and severely reduces the options for further successful treatments. METHODS: We studied patients from the Swiss HIV Cohort Study who failed cART with nucleoside reverse transcriptase inhibitors (NRTIs) and either a ritonavir-boosted PI (PI/r) or a non-nucleoside reverse transcriptase inhibitor (NNRTI). The loss of genotypic activity <3, 3-6, >6 months after virological failure was analyzed with Stanford algorithm. Risk factors associated with early emergence of drug resistance mutations (<6 months after failure) were identified with multivariable logistic regression. RESULTS: Ninety-nine genotypic resistance tests from PI/r-treated and 129 from NNRTI-treated patients were analyzed. The risk of losing the activity of ≥1 NRTIs was lower among PI/r- compared to NNRTI-treated individuals <3, 3-6, and >6 months after failure: 8.8% vs. 38.2% (p = 0.009), 7.1% vs. 46.9% (p<0.001) and 18.9% vs. 60.9% (p<0.001). The percentages of patients who have lost PI/r activity were 2.9%, 3.6% and 5.4% <3, 3-6, >6 months after failure compared to 41.2%, 49.0% and 63.0% of those who have lost NNRTI activity (all p<0.001). The risk to accumulate an early NRTI mutation was strongly associated with NNRTI-containing cART (adjusted odds ratio: 13.3 (95% CI: 4.1-42.8), p<0.001). CONCLUSIONS: The loss of activity of PIs and NRTIs was low among patients treated with PI/r, even after long-lasting exposure to a failing cART. Thus, more options remain for second-line therapy. This finding is potentially of high relevance, in particular for settings with poor or lacking virological monitoring.

Anatomical correlates for category-specific naming of objects and actions: A brain stimulation mapping study.

The production of object and action words can be dissociated in aphasics, yet their anatomical correlates have been difficult to distinguish in functional imaging studies. To investigate the extent to which the cortical neural networks underlying object- and action-naming processing overlap, we performed electrostimulation mapping (ESM), which is a neurosurgical mapping technique routinely used to examine language function during brain-tumor resections. Forty-one right-handed patients who had surgery for a brain tumor were asked to perform overt naming of object and action pictures under stimulation. Overall, 73 out of the 633 stimulated cortical sites (11.5%) were associated with stimulation-induced language interferences. These interference sites were very much localized (<1 cm(2) ), and showed substantial variability across individuals in their exact localization. Stimulation interfered with both object and action naming over 44 sites, whereas it specifically interfered with object naming over 19 sites and with action naming over 10 sites. Specific object-naming sites were mainly identified in Broca's area (Brodmann area 44/45) and the temporal cortex, whereas action-naming specific sites were mainly identified in the posterior midfrontal gyrus (Brodmann area 6/9) and Broca's area (P = 0.003 by the Fisher's exact test). The anatomical loci we emphasized are in line with a cortical distinction between objects and actions based on conceptual/semantic features, so the prefrontal/premotor cortex would preferentially support sensorimotor contingencies associated with actions, whereas the temporal cortex would preferentially underpin (functional) properties of objects. Hum Brain Mapp 35:429-443, 2014. © 2012 Wiley Periodicals, Inc.

Performance of indirect immunofluorescence assay, immunochromatography assay and reverse transcription-polymerase chain reaction for detecting human respiratory syncytial virus in nasopharyngeal aspirate samples

Comparison of the use of indirect immunofluorescence assay (IFA), immunochromatography assay (ICA-BD) and reverse transcription-polymerase chain reaction (RT-PCR) for detecting human respiratory syncytial virus (HRSV) in 306 nasopharyngeal aspirates samples (NPA) was performed in order to assess their analytical performance. By comparing the results obtained using ICA-BD with those using IFA, we found relative indices of 85.0% for sensitivity and 91.2% for specificity, and the positive (PPV) and negative (NPV) predictive values were 85.0% and 91.2%, respectively. The relative indices for sensitivity and specificity as well as the PPV and NPV for RT-PCR were 98.0%, 89.0%, 84.0% and 99.0%, respectively, when compared to the results of IFA. In addition, comparison of the results of ICA-BD and those of RT-PCR yielded relative indices of 79.5% for sensitivity and 95.4% for specificity, as well as PPV and NPV of 92.9% and 86.0%, respectively. Although RT-PCR has shown the best performance, the substantial agreement between the ICA-BD and IFA results suggests that ICA-BD, also in addition to being a rapid and facile assay, could be suitable as an alternative diagnostic screening for HRSV infection in children.

Added Value of Deep Sequencing Relative to Population Sequencing in Heavily Pre-Treated HIV-1-Infected Subjects.

OBJECTIVE: To explore the potential of deep HIV-1 sequencing for adding clinically relevant information relative to viral population sequencing in heavily pre-treated HIV-1-infected subjects. METHODS: In a proof-of-concept study, deep sequencing was compared to population sequencing in HIV-1-infected individuals with previous triple-class virological failure who also developed virologic failure to deep salvage therapy including, at least, darunavir, tipranavir, etravirine or raltegravir. Viral susceptibility was inferred before salvage therapy initiation and at virological failure using deep and population sequencing genotypes interpreted with the HIVdb, Rega and ANRS algorithms. The threshold level for mutant detection with deep sequencing was 1%. RESULTS: 7 subjects with previous exposure to a median of 15 antiretrovirals during a median of 13 years were included. Deep salvage therapy included darunavir, tipranavir, etravirine or raltegravir in 4, 2, 2 and 5 subjects, respectively. Self-reported treatment adherence was adequate in 4 and partial in 2; one individual underwent treatment interruption during follow-up. Deep sequencing detected all mutations found by population sequencing and identified additional resistance mutations in all but one individual, predominantly after virological failure to deep salvage therapy. Additional genotypic information led to consistent decreases in predicted susceptibility to etravirine, efavirenz, nucleoside reverse transcriptase inhibitors and indinavir in 2, 1, 2 and 1 subject, respectively. Deep sequencing data did not consistently modify the susceptibility predictions achieved with population sequencing for darunavir, tipranavir or raltegravir. CONCLUSIONS: In this subset of heavily pre-treated individuals, deep sequencing improved the assessment of genotypic resistance to etravirine, but did not consistently provide additional information on darunavir, tipranavir or raltegravir susceptibility. These data may inform the design of future studies addressing the clinical value of minority drug-resistant variants in treatment-experienced subjects.

HIV-1 reverse transcriptase connection domain mutations: dynamics of emergence and implications for success of combination antiretroviral therapy.

BACKGROUND: Factors promoting the emergence of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) connection domain mutations and their effect on antiretroviral therapy (ART) are still largely undetermined. We investigated this matter by analyzing genotypic resistance tests covering 400 amino acid positions in the RT of HIV-1 subtype B viruses and corresponding treatment histories and laboratory measurements. METHODS: The emergence of connection domain mutations was studied in 334 patients receiving monotherapy or dual therapy with thymidine analogues at the time of the genotypic resistance test. Response to subsequent combination ART (cART) was analyzed using Cox regression for 291 patients receiving unboosted protease inhibitors. Response was defined by ever reaching an HIV RNA level <50 copies/mL during the first cART. RESULTS: The connection domain mutations N348I, R356K, R358K, A360V, and A371V were more frequently observed in ART-exposed than ART-naive patients, of which only N348I and A360V were nonpolymorphic (with a prevalence of <1.5% in untreated patients). N348I correlated with M184V and predominantly occurred in patients receiving lamivudine and zidovudine concomitantly. A360V was not associated with specific drug combinations and was found to emerge later than M184V or thymidine analogue mutations. Nonpolymorphic connection domain mutations were rarely detected in the absence of established drug resistance mutations in ART-exposed individuals (prevalence, <1%). None of the 5 connection domain mutations associated with treatment showed a statistically significant effect on response to cART. CONCLUSIONS: Despite their frequent emergence, connection domain mutations did not show large detrimental effects on response to cART. Currently, routine implementation of connection domain sequencing seems unnecessary for developed health care settings.

Revisiting steroid treatment for septic shock: molecular actions and clinical effects - a review

Corticosteroids are widely used to treat a diversity of pathological conditions including allergic, autoimmune and some infectious diseases. These drugs have complex mechanisms of action involving both genomic and non-genomic mechanisms and interfere with different signal transduction pathways in the cell. The use of corticosteroids to treat critically ill patients with acute respiratory distress syndrome and severe infections, such as sepsis and pneumonia, is still a matter of intense debate in the scientific and medical community with evidence both for and against its use in these patients. Here, we review the basic molecular mechanisms important for corticosteroid action as well as current evidence for their use, or not, in septic patients. We also present an analysis of the reasons why this is still such a controversial point in the literature.

In house colorimetric reverse hybridisation assay for detection of the mutation most frequently associated with resistance to isoniazid in Mycobacterium tuberculosis

Mutations in the katG gene have been identified and correlated with isoniazid (INH) resistance in Mycobacterium tuberculosis isolates. The mutation AGC→ACC (Ser→Thr) at katG315 has been reported to be the most frequent and is associated with transmission and multidrug resistance. Rapid detection of this mutation could therefore improve the choice of an adequate anti-tuberculosis regimen, the epidemiological monitoring of INH resistance and, possibly, the tracking of transmission of resistant strains. An in house reverse hybridisation assay was designed in our laboratory and evaluated with 180 isolates of M. tuberculosis. It could successfully characterise the katG315 mutation in 100% of the samples as compared to DNA sequencing. The test is efficient and is a promising alternative for the rapid identification of INH resistance in regions with a high prevalence of katG315 mutants.

Detection of rifampin-resistant genotypes in Mycobacterium tuberculosis by reverse hybridization assay

We used a colorimetric reverse dot blot hybridization (CRDH) assay to detect the presence of mutations in a specific region of the rpoB gene, associated with rifampin (RIF) resistance, in a panel of 156 DNAs extracted from 103 RIF-sensitive and 53 RIF-resistant cultures of Mycobacterium tuberculosis. When compared with the antimicrobial susceptibility test (AST), the sensitivity and specificity of the CRDH were 92.3% and 98.1%, respectively. When compared with sequencing, the sensitivity and specificity of the CRDH were 90.6% and 100%, respectively. To evaluate the performance of the assay directly in clinical specimens, 30 samples from tuberculosis patients were used. For these samples, the results of the CRDH were 100% consistent with the results of the AST and sequencing. These results indicate that the rate of concordance of the CRDH is high when compared to conventional methods and sequencing data. The CRDH can be successfully applied when a rapid test is required for the identification of RIF resistance in M. tuberculosis.

Colorimetric microwell plate reverse-hybridization assay for Mycobacterium tuberculosis detection

Direct smear examination using Ziehl-Neelsen staining for pulmonary tuberculosis (PTB) diagnosis is inexpensive and easy to use, but has the major limitation of low sensitivity. Rapid molecular methods are becoming more widely available in centralized laboratories, but they depend on timely reporting of results and strict quality assurance obtainable only from costly commercial kits available in high burden nations. This study describes a pre-commercial colorimetric method, Detect-TB, for detecting Mycobacterium tuberculosis DNA in which an oligonucleotide probe is fixed onto wells of microwell plates and hybridized with biotinylated polymerase chain reaction amplification products derived from clinical samples. The probe is capable of hybridising with the IS6110 insertion element and was used to specifically recognise the M. tuberculosis complex. When combined with an improved silica-based DNA extraction method, the sensitivity of the test was 50 colony-forming units of the M. tuberculosis reference strain H37Rv. The results that were in agreement with reference detection methods were observed in 95.2% (453/476) of samples included in the analysis. Sensitivity and specificity for 301 induced sputum samples and 175 spontaneous sputum samples were 85% and 98%, and 94% and 100%, respectively. This colorimetric method showed similar specificity to that described for commercially available kits and may provide an important contribution for PTB diagnosis.

Long-term assessment of didanosine, lamivudine, and efavirenz in antiretroviral-naive patients: 3-year follow-up.

The aim of this study was to evaluate the long-term efficacy and safety of didadosine (ddI), lamivudine (3TC), and efavirenz (EFV). This was a follow-up to the VESD study, a 12-month open-label, observational, multicenter study of adult patients with HIV infection who started antiretroviral treatment with the ddI-3TC-EFV once-daily regimen. Of the 167 patients originally included, 106 patients remained on the same triple therapy at the end of the study (1 year), and they were offered an extra 24 months of follow-up; 96 were enrolled in this study (VESD-2). Seventy patients out of the initial cohort were still on the same regimen at month 36, with 97% of them with plasma viral load <50 copies /ml. An intention-to-treat analysis showed that the percentage of patients with plasma viral load <50 copies/ml was 73% at 36 months. CD4 cell counts increased 344 cells/microl over the 36 months. Safety and tolerance were good with no unexpected long-term toxicity. After 3 years of treatment with ddI-3TC-EFV, more than 40% of the patients were still receiving the initial antiretroviral therapy with sustained, durable immunovirological benefit and good acceptance. Long-term toxicity and virological failure were low.

Rab18 dynamics in adipocytes in relation to lipogenesis, lipolysis and obesity.

Lipid droplets (LDs) are organelles that coordinate lipid storage and mobilization, both processes being especially important in cells specialized in managing fat, the adipocytes. Proteomic analyses of LDs have consistently identified the small GTPase Rab18 as a component of the LD coat. However, the specific contribution of Rab18 to adipocyte function remains to be elucidated. Herein, we have analyzed Rab18 expression, intracellular localization and function in relation to the metabolic status of adipocytes. We show that Rab18 production increases during adipogenic differentiation of 3T3-L1 cells. In addition, our data show that insulin induces, via phosphatidylinositol 3-kinase (PI3K), the recruitment of Rab18 to the surface of LDs. Furthermore, Rab18 overexpression increased basal lipogenesis and Rab18 silencing impaired the lipogenic response to insulin, thereby suggesting that this GTPase promotes fat accumulation in adipocytes. On the other hand, studies of the β-adrenergic receptor agonist isoproterenol confirmed and extended previous evidence for the participation of Rab18 in lipolysis. Together, our data support the view that Rab18 is a common mediator of lipolysis and lipogenesis and suggests that the endoplasmic reticulum (ER) is the link that enables Rab18 action on these two processes. Finally, we describe, for the first time, the presence of Rab18 in human adipose tissue, wherein the expression of this GTPase exhibits sex- and depot-specific differences and is correlated to obesity. Taken together, these findings indicate that Rab18 is involved in insulin-mediated lipogenesis, as well as in β-adrenergic-induced lipolysis, likely facilitating interaction of LDs with ER membranes and the exchange of lipids between these compartments. A role for Rab18 in the regulation of adipocyte biology under both normal and pathological conditions is proposed.

MicroRNA expression profiling in Imatinib-resistant Chronic Myeloid Leukemia patients without clinically significant ABL1-mutations.

The development of Imatinib Mesylate (IM), the first specific inhibitor of BCR-ABL1, has had a major impact in patients with Chronic Myeloid Leukemia (CML), establishing IM as the standard therapy for CML. Despite the clinical success obtained with the use of IM, primary resistance to IM and molecular evidence of persistent disease has been observed in 20-25% of IM treated patients. The existence of second generation TK inhibitors, which are effective in patients with IM resistance, makes identification of predictors of resistance to IM an important goal in CML. In this study, we have identified a group of 19 miRNAs that may predict clinical resistance to IM in patients with newly diagnosed CML.

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.

Construction and characterisation of a complete reverse genetics system of dengue virus type 3

Dengue virulence and fitness are important factors that determine disease outcome. However, dengue virus (DENV) molecular biology and pathogenesis are not completely elucidated. New insights on those mechanisms have been facilitated by the development of reverse genetic systems in the past decades. Unfortunately, instability of flavivirus genomes cloned in Escherichia coli has been a major problem in these systems. Here, we describe the development of a complete reverse genetics system, based on the construction of an infectious clone and replicon for a low passage DENV-3 genotype III of a clinical isolate. Both constructs were assembled into a newly designed yeast- E. coli shuttle vector by homologous recombination technique and propagated in yeast to prevent any possible genome instability in E. coli . RNA transcripts derived from the infectious clone are infectious upon transfection into BHK-21 cells even after repeated passages of the plasmid in yeast. Transcript-derived DENV-3 exhibited growth kinetics, focus formation size comparable to original DENV-3 in mosquito C6/36 cell culture. In vitro characterisation of DENV-3 replicon confirmed its identity and ability to replicate transiently in BHK-21 cells. The reverse genetics system reported here is a valuable tool that will facilitate further molecular studies in DENV replication, virus attenuation and pathogenesis.