Measuring Enzymatic HIV-1 Susceptibility to Two Reverse Transcriptase Inhibitors as a Rapid and Simple Approach to HIV-1 Drug-Resistance Testing

Simple and cost-effective approaches for HIV drug-resistance testing are highly desirable for managing increasingly expanding HIV-1 infected populations who initiate antiretroviral therapy (ART), particularly in resource-limited settings. Non-nucleoside reverse trancriptase inhibitor (NNRTI)-based regimens with an NRTI backbone containing lamivudine (3TC) or emtricitabine (FTC) are preferred first ART regimens. Failure with these drug combinations typically involves the selection of NNRTI- and/or 3TC/FTC- resistant viruses. Therefore, the availability of simple assays to measure both types of drug resistance is critical. We have developed a high throughput screening test for assessing enzymatic resistance of the HIV-1 RT in plasma to 3TC/FTC and NNRTIs. The test uses the sensitive “Amp-RT” assay with a newly-developed real-time PCR format to screen biochemically for drug resistance in single reactions containing either 3TC-triphosphate (3TC-TP) or nevirapine (NVP). Assay cut-offs were defined based on testing a large panel of subtype B and non-subtype B clinical samples with known genotypic profiles. Enzymatic 3TC resistance correlated well with the presence of M184I/V, and reduced NVP susceptibility was strongly associated with the presence of K103N, Y181C/I, Y188L, and G190A/Q. The sensitivity and specificity for detecting resistance were 97.0% and 96.0% in samples with M184V, and 97.4% and 96.2% for samples with NNRTI mutations, respectively. We further demonstrate the utility of an HIV capture method in plasma by using magnetic beads coated with CD44 antibody that eliminates the need for ultracentifugation. Thus our results support the use of this simple approach for distinguishing WT from NNRTI- or 3TC/FTC-resistant viruses in clinical samples. This enzymatic testing is subtype-independent and can assist in the clinical management of diverse populations particularly in resource-limited settings

Postsurgical pain outcome of vertical and transverse abdominal incision: Design of a randomized controlled equivalence trial [ISRCTN60734227]

BACKGROUND: There are two ways to open the abdominal cavity in elective general surgery: vertically or transversely. Various clinical studies and a meta-analysis have postulated that the transverse approach is superior to other approaches as regards complications. However, in a recent survey it was shown that 90 % of all abdominal incisions in visceral surgery are still vertical incisions. This discrepancy between existing recommendations of clinical trials and clinical practice could be explained by the lack of acceptance of these results due to a number of deficits in the study design and analysis, subsequent low internal validity, and therefore limited external generalisability. The objective of this study is to address the issue from the patient's perspective. METHODS: This is an intraoperatively randomized controlled observer and patient-blinded two-group parallel equivalence trial. The study setting is the Department of General-, Visceral-, Trauma Surgery and Outpatient Clinic of the University of Heidelberg, Medical School. A total of 172 patients of both genders, aged over 18 years who are scheduled for an elective abdominal operation and are eligible for either a transverse or vertical incision. To show equivalence of the two approaches or the superiority of one of them from the perspective of the patient, a primary endpoint is defined: the pain experienced by the patient (VAS 0–100) on day two after surgery and the amount of analgesic required (piritramide [mg/h]). A confidence interval approach will be used for analysis. A global α-Level of 0.05 and a power of 0.8 is guaranteed, resulting in a size of 86 patients for each group. Secondary endpoints are: time interval to open and close the abdomen, early-onset complications (frequency of burst abdomen, postoperative pulmonary complications, and wound infection) and late complications (frequency of incisional hernias). Different outcome variables will be ranked by patients and surgeons to assess the relevance of possible endpoints from the patients' and surgeons' perspective. CONCLUSION: This is a randomized controlled observer and patient-blinded two-group parallel trial to answer the question if the transverse abdominal incision is equivalent to the vertical one due to the described endpoints

Different genes interact with particulate matter and tobacco smoke exposure in affecting lung function decline in the general population

BACKGROUND: Oxidative stress related genes modify the effects of ambient air pollution or tobacco smoking on lung function decline. The impact of interactions might be substantial, but previous studies mostly focused on main effects of single genes. OBJECTIVES: We studied the interaction of both exposures with a broad set of oxidative-stress related candidate genes and pathways on lung function decline and contrasted interactions between exposures. METHODS: For 12679 single nucleotide polymorphisms (SNPs), change in forced expiratory volume in one second (FEV(1)), FEV(1) over forced vital capacity (FEV(1)/FVC), and mean forced expiratory flow between 25 and 75% of the FVC (FEF(25-75)) was regressed on interval exposure to particulate matter >10 microm in diameter (PM10) or packyears smoked (a), additive SNP effects (b), and interaction terms between (a) and (b) in 669 adults with GWAS data. Interaction p-values for 152 genes and 14 pathways were calculated by the adaptive rank truncation product (ARTP) method, and compared between exposures. Interaction effect sizes were contrasted for the strongest SNPs of nominally significant genes (p(interaction)>0.05). Replication was attempted for SNPs with MAF<10% in 3320 SAPALDIA participants without GWAS. RESULTS: On the SNP-level, rs2035268 in gene SNCA accelerated FEV(1)/FVC decline by 3.8% (p(interaction) = 2.5x10(-6)), and rs12190800 in PARK2 attenuated FEV1 decline by 95.1 ml p(interaction) = 9.7x10(-8)) over 11 years, while interacting with PM10. Genes and pathways nominally interacting with PM10 and packyears exposure differed substantially. Gene CRISP2 presented a significant interaction with PM10 (p(interaction) = 3.0x10(-4)) on FEV(1)/FVC decline. Pathway interactions were weak. Replications for the strongest SNPs in PARK2 and CRISP2 were not successful. CONCLUSIONS: Consistent with a stratified response to increasing oxidative stress, different genes and pathways potentially mediate PM10 and tobac smoke effects on lung function decline. Ignoring environmental exposures would miss these patterns, but achieving sufficient sample size and comparability across study samples is challengin

A Template-Dependent Dislocation Mechanism Potentiates K65R Reverse Transcriptase Mutation Development in Subtype C Variants of HIV-1

Numerous studies have suggested that the K65R reverse transcriptase (RT) mutation develops more readily in subtype C than subtype B HIV-1. We recently showed that this discrepancy lies partly in the subtype C template coding sequence that predisposes RT to pause at the site of K65R mutagenesis. However, the mechanism underlying this observation and the elevated rates of K65R development remained unknown. Here, we report that DNA synthesis performed with subtype C templates consistently produced more K65R-containing transcripts than subtype B templates, regardless of the subtype-origin of the RT enzymes employed. These findings confirm that the mechanism involved is template-specific and RT-independent. In addition, a pattern of DNA synthesis characteristic of site-specific primer/template slippage and dislocation was only observed with the subtype C sequence. Analysis of RNA secondary structure suggested that the latter was unlikely to impact on K65R development between subtypes and that Streisinger strand slippage during DNA synthesis at the homopolymeric nucleotide stretch of the subtype C K65 region might occur, resulting in misalignment of the primer and template. Consequently, slippage would lead to a deletion of the middle adenine of codon K65 and the production of a -1 frameshift mutation, which upon dislocation and realignment of the primer and template, would lead to development of the K65R mutation. These findings provide additional mechanistic evidence for the facilitated development of the K65R mutation in subtype C HIV-1

Drug Resistance Mutations for Surveillance of Transmitted HIV-1 Drug-Resistance: 2009 Update

Programs that monitor local, national, and regional levels of transmitted HIV-1 drug resistance inform treatment guidelines and provide feedback on the success of HIV-1 treatment and prevention programs. To accurately compare transmitted drug resistance rates across geographic regions and times, the World Health Organization has recommended the adoption of a consensus genotypic definition of transmitted HIV-1 drug resistance. In January 2007, we outlined criteria for developing a list of mutations for drug-resistance surveillance and compiled a list of 80 RT and protease mutations meeting these criteria (surveillance drug resistance mutations; SDRMs). Since January 2007, several new drugs have been approved and several new drug-resistance mutations have been identified. In this paper, we follow the same procedures described previously to develop an updated list of SDRMs that are likely to be useful for ongoing and future studies of transmitted drug resistance. The updated SDRM list has 93 mutations including 34 NRTI-resistance mutations at 15 RT positions, 19 NNRTI-resistance mutations at 10 RT positions, and 40 PI-resistance mutations at 18 protease positions

Standing genetic variation and the evolution of drug resistance in HIV

Drug resistance remains a major problem for the treatment of HIV. Resistance can occur due to mutations that were present before treatment starts or due to mutations that occur during treatment. The relative importance of these two sources is unknown. We study three different situations in which HIV drug resistance may evolve: starting triple-drug therapy, treatment with a single dose of nevirapine and interruption of treatment. For each of these three cases good data are available from literature, which allows us to estimate the probability that resistance evolves from standing genetic variation. Depending on the treatment we find probabilities of the evolution of drug resistance due to standing genetic variation between 0 and 39%. For patients who start triple-drug combination therapy, we find that drug resistance evolves from standing genetic variation in approximately 6% of the patients. We use a population-dynamic and population-genetic model to understand the observations and to estimate important evolutionary parameters. We find that both, the effective population size of the virus before treatment, and the fitness of the resistant mutant during treatment, are key-parameters that determine the probability that resistance evolves from standing genetic variation. Importantly, clinical data indicate that both of these parameters can be manipulated by the kind of treatment that is used.Comment: 33 pages 6 figure

Transplantation of Specific Human Astrocytes Promotes Functional Recovery after Spinal Cord Injury

Repairing trauma to the central nervous system by replacement of glial support cells is an increasingly attractive therapeutic strategy. We have focused on the less-studied replacement of astrocytes, the major support cell in the central nervous system, by generating astrocytes from embryonic human glial precursor cells using two different astrocyte differentiation inducing factors. The resulting astrocytes differed in expression of multiple proteins thought to either promote or inhibit central nervous system homeostasis and regeneration. When transplanted into acute transection injuries of the adult rat spinal cord, astrocytes generated by exposing human glial precursor cells to bone morphogenetic protein promoted significant recovery of volitional foot placement, axonal growth and notably robust increases in neuronal survival in multiple spinal cord laminae. In marked contrast, human glial precursor cells and astrocytes generated from these cells by exposure to ciliary neurotrophic factor both failed to promote significant behavioral recovery or similarly robust neuronal survival and support of axon growth at sites of injury. Our studies thus demonstrate functional differences between human astrocyte populations and suggest that pre-differentiation of precursor cells into a specific astrocyte subtype is required to optimize astrocyte replacement therapies. To our knowledge, this study is the first to show functional differences in ability to promote repair of the injured adult central nervous system between two distinct subtypes of human astrocytes derived from a common fetal glial precursor population. These findings are consistent with our previous studies of transplanting specific subtypes of rodent glial precursor derived astrocytes into sites of spinal cord injury, and indicate a remarkable conservation from rat to human of functional differences between astrocyte subtypes. In addition, our studies provide a specific population of human astrocytes that appears to be particularly suitable for further development towards clinical application in treating the traumatically injured or diseased human central nervous system

Echocardiographic prediction of outcome after cardiac resynchronization therapy: conventional methods and recent developments

Echocardiography plays an important role in patient assessment before cardiac resynchronization therapy (CRT) and can monitor many of its mechanical effects in heart failure patients. Encouraged by the highly variable individual response observed in the major CRT trials, echocardiography-based measurements of mechanical dyssynchrony have been extensively investigated with the aim of improving response prediction and CRT delivery. Despite recent setbacks, these techniques have continued to develop in order to overcome some of their initial flaws and limitations. This review discusses the concepts and rationale of the available echocardiographic techniques, highlighting newer quantification methods and discussing some of the unsolved issues that need to be addressed

Potential Impact of Antiretroviral Chemoprophylaxis on HIV-1 Transmission in Resource-Limited Settings

Background. The potential impact of pre-exposure chemoprophylaxis (PrEP) an heterosexual transmission of HIV-1 infection in resource-limited settings is uncertain. Methodology/Principle Findings. A deterministic mathematical model was used to simulate the effects of antiretroval PreP on an HIV-1 epidemic in sub-Saharan Africa under different scenarios (optimistic neutral and pessimistic) both with and without sexual disinhibition. Sensitivity analyses were used to evaluate the effect of uncertainty in input parameters on model output and included calculation of partial rank correlations and standardized rank regressions. In the scenario without sexual disinhibition after PrEP initiation, key parameters influencing infections prevented were effectiveness of PrEP (partial rank correlation coefficient (PRCC) = 0.94), PrEP discontinuation rate (PRCC=-0.94), level of coverage (PRCC=0.92), and time to achieve target coverage (PRCC=-082). In the scenario with sexual disinhibition, PrEP effectiveness and the extent of sexual disinhibition had the greatest impact on prevention. An optimistic scenario of PrEP with 90% effectiveness and 75% coverage of the general population predicted a 74% decline in cumulative HIV-1 infections after 10 years, and a 28.8% decline with PrEP targeted to the highest risk groups (16% of the population). Even With a 100% increase in at-risk behavior from sexual disinhibition, a beneficial effect (23.4%-62.7% decrease in infections) was seen with 90% effective PrEP across a broad range of coverage (25%-75%). Similar disinhibition led to a rise in infections with lower effectiveness of PrEP (≤50%). Conclusions/Significance. Mathematical modeling supports the potential public health benefit of PrEP. Approximately 2.7 to 3.2 million new HIV-1 infections could be averaged in southern sub-Saharan Africa over 10 years by targeting PrEP (having 90% effectiveness) to those at highest behavioral risk and by preventing sexual disinhibition. This benefit could be lost, however, by sexual disinhibition and by high PrEP discontinuation, especially with lower PrEP effectiveness (≤:50%). © 2007 Abbas et al

Identifying a Window of Vulnerability during Fetal Development in a Maternal Iron Restriction Model

It is well acknowledged from observations in humans that iron deficiency during pregnancy can be associated with a number of developmental problems in the newborn and developing child. Due to the obvious limitations of human studies, the stage during gestation at which maternal iron deficiency causes an apparent impairment in the offspring remains elusive. In order to begin to understand the time window(s) during pregnancy that is/are especially susceptible to suboptimal iron levels, which may result in negative effects on the development of the fetus, we developed a rat model in which we were able to manipulate and monitor the dietary iron intake during specific stages of pregnancy and analyzed the developing fetuses. We established four different dietary-feeding protocols that were designed to render the fetuses iron deficient at different gestational stages. Based on a functional analysis that employed Auditory Brainstem Response measurements, we found that maternal iron restriction initiated prior to conception and during the first trimester were associated with profound changes in the developing fetus compared to iron restriction initiated later in pregnancy. We also showed that the presence of iron deficiency anemia, low body weight, and changes in core body temperature were not defining factors in the establishment of neural impairment in the rodent offspring