New Directions in Infection-Associated Ischemic Stroke.

The relationship between infections and stroke has not been fully characterized, probably delaying the development of specific treatments. This narrative review addresses mechanisms of stroke linked to infections, including hypercoagulability, endothelial dysfunction, vasculitis, and impaired thrombolysis. SARS-CoV-2, the virus that causes COVID-19, may promote the development of stroke, which may represent its most severe neurological complication. The development of specific therapies for infection-associated stroke remains a profound challenge. Perhaps the most important remaining issue is the distinction between infections that trigger a stroke versus infections that are truly incidental. This distinction likely requires the establishment of appropriate biomarkers, candidates of which are elevated levels of fibrin D-dimer and anticardiolipin/antiphospholipid antibodies. These candidate biomarkers might have potential use in identifying pathogenic infections preceding stroke, which is a precursor to establishing specific therapies for this syndrome.

The development and application of a high-sensitivity immunoassay for cardiac myosin-binding protein C.

Cardiac troponins (cTns) are released and cleared slowly after myocardial injury. Cardiac myosin-binding protein C (cMyC) is a similar cardiac-restricted protein that has more rapid release and clearance kinetics. Direct comparisons are hampered by the lack of an assay for cMyC that matches the sensitivity of the contemporary assays for cTnI and cTnT. Using a novel pair of monoclonal antibodies, we generated a sensitive assay for MyC on the Erenna platform (Singulex) and compared serum concentrations with those of cTnI (Abbott) and cTnT (Roche) in stable ambulatory cardiac patients without evidence of acute cardiac injury or significant coronary artery stenoses. The assay for cMyC had a lower limit of detection of 0.4 ng/L, a lower limit of quantification (LLoQ) of 1.2 ng/L (LLoQ at 20% coefficient of variation [CV]) and reasonable recovery (107.1 ± 3.7%; mean ± standard deviation), dilutional linearity (101.0 ± 7.7%), and intraseries precision (CV, 11 ± 3%) and interseries precision (CV, 13 ± 3%). In 360 stable patients, cMyC was quantifiable in 359 patients and compared with cTnT and cTnI measured using contemporary high-sensitivity assays. cMyC concentration (median, 12.2 ng/L; interquartile range [IQR], 7.9-21.2 ng/L) was linearly correlated with those for cTnT (median, <3.0 ng/L; IQR, <3.0-4.9 ng/L; R = 0.56, P < 0.01) and cTnI (median, 2.10 ng/L; IQR, 1.3-4.2 ng/L; R = 0.77, P < 0.01) and showed similar dependencies on age, renal function, and left ventricular function. We have developed a high-sensitivity assay for cMyC. Concentrations of cMyC in clinically stable patients are highly correlated with those of cTnT and cTnI. This high correlation may enable ratiometric comparisons between biomarkers to distinguish clinical instability.

High Altitude and Acute Mountain Sickness and Changes in Circulating Endothelin-1, Interleukin-6, and Interleukin-17a.

INTRODUCTION: Hypoxia induces an inflammatory response, which is enhanced by exercise. High altitude (HA) leads to endothelial activation and may be proinflammatory. The relationship between endothelial activation, inflammation, and acute mountain sickness (AMS) and its severity has never been examined. METHODS: Forty-eight trekkers were studied during a progressive trek at 3833, 4450, and 5129 m at rest postascent (exercise), and then again at rest 24 hours later. Twenty of the subjects were also tested at rest pre- and postexercise at sea level (SL) at 6 weeks preascent. We examined plasma levels of the interleukin 6 (IL-6), 17a (IL-17a), and endothelin-1 (ET-1) along with oxygen saturation (SpO2) and Lake Louise scores (LLS). RESULTS: ET-1 (5.7 ± 2.1 vs. 4.3 ± 1.9 pg/mL; p < 0.001), IL-6 (3.3 ± 3.3 vs. 2.4 ± 2.3 pg/mL; p = 0.007), and IL-17a (1.3 ± 3.0 vs. 0.46 ± 0.4 pg/mL; p < 0.001) were all overall significantly higher at HA versus SL. There was a paired increase in ET-1 and IL-6 with exercise versus rest at SL, 3833, 4450, and 5129 m (p < 0.05). There was a negative correlation between LLS and SpO2 (r = -0.32; 95% confidence interval [CI] -0.21 to -0.42; p < 0.001) and a positive correlation between LLS and IL-6 (r = 0.16; 0.0-0.27; p = 0.007) and ET-1 levels (r = 0.29; 0.18-0.39; p < 0.001. Altitude, ET-1, IL-6, and SpO2 were all univariate predictors of AMS. On multivariate analysis, ET-1 (p = 0.002) and reducing SpO2 (p = 0.02) remained as the only independent predictors (overall r(2) = 0.16; p < 0.001) of AMS. ET-1 (p = 03) and SpO2 were (p = 0.01) also independent predictors of severe AMS (overall r(2) = 0.19; p < 0.001). CONCLUSIONS: HA leads to endothelial activation and an inflammatory response. The rise in ET-1 and IL-6 is heavily influenced by the degree of exercise and hypoxia. ET-1 is an independent predictor of both AMS and its severity.

HIV-1 RNA concentration and cognitive performance in a cohort of HIV-positive people.

OBJECTIVE: To determine whether higher viral concentrations in the cerebrospinal fluid (CSF) and/or peripheral blood were associated with greater severity of cognitive impairment in HIV-1-seropositive subjects with cognitive-motor impairment. METHODS: Cognitive performance measurements and viral load were obtained from HIV-1-seropositive individuals with cognitive-motor impairment entering a clinical trial before the introduction of highly active antiretroviral therapy (HAART). CSF viral load (UltraSensitive Roche HIV-1 Monitor test with detection limit of 50 copies/ml) was available from 179 patients, and peripheral (plasma or serum) viral load from 111 patients. Of these patients, 62% met the 1993 Centers for Disease Control (CDC) criteria for AIDS, and 19% had clinically significant cognitive impairment (i.e., global deficit score > or = 0.5). Possible associations between viral load and cognitive scores were examined with general linear regression models with and without adjustment for age, education, study site, antiretroviral use, CD4 cell count, and CDC stage. RESULTS: The mean CSF viral load was 2.83 log(10)/ml +/- 0.94 (SD) (undetectable in 19.5%). Mean peripheral viral load was 4.11 log(10)/ml +/- 0.90 (SD). No statistically significant associations emerged between either CSF or peripheral viral load and the global deficit score, or any of the seven cognitive domain deficit scores. CONCLUSIONS: Among these HIV-1-sero-positive individuals with mainly minor HIV-1-associated cognitive deficits and not receiving HAART, no association between CSF or blood concentration of HIV-1 RNA and cognitive performance could be found. These results suggest that the severity of HIV-1-associated cognitive impairment is not directly related to concurrent viral concentration in the CSF or the peripheral blood.

Cerebrospinal and peripheral human immunodeficiency virus type 1 load in a multisite, randomized, double-blind, placebo-controlled trial of D-Ala1-peptide T-amide for HIV-1-associated cognitive-motor impairment.

D-Ala1-peptide T-amide (DAPTA) has shown neuroprotection in vitro against gp120-induced loss of dendritic arborization and is promulgated as a CCR5 antagonist. A multisite, randomized, double-blind clinical trial of DAPTA versus placebo prior to combination antiretroviral therapy conducted with human immunodeficiency virus (HIV)-1 seropositive participants having cognitive impairment showed no overall cognitive effect, though subgroups with greater impairment and CD4 cell counts of 201 to 500 cells/mm3 at baseline showed significant improvement. The objective of this study was to examine whether intranasal administration of DAPTA at a dose of 2 mg three times per day (tid) was associated with a reduction of cerebrospinal fluid (CSF) and peripheral (plasma and serum) viral load among a subgroup of participants completing 6 months of treatment. Baseline and 6-month CSF (n = 92) and peripheral (plasma n = 33; serum n = 24) viral load were measured by the Roche Ultrasensitive assay, version 1.5, with reflexive use of the AMPLICOR assay and preservation of the blind. A DAPTA treatment indicator variable was tested using generalized linear models on change in viral load. Peripheral load (combined plasma and serum) was significantly reduced in the DAPTA-treated group. No group differences in CSF viral load were found. This retrospective study on a limited subgroup of the original trial sample indicated that DAPTA treatment may reduce peripheral viral load without concomitant CSF effects. Future studies should be undertaken to confirm the existence of this result and the CSF-periphery dissociation observed with respect to HIV-1-associated cognitive-motor impairment.

New two-amino acid insertion near codon 70 of the HIV type 1 protease gene.

A two-amino acid insertion near codon 70 of the HIV-1 protease gene was found in an individual failing protease inhibitor therapy. Susceptibility of this strain to protease inhibitors was similar to that of non-insert-containing strains with comparable resistance mutations and one or more other major PI mutations.

Structural analysis of an HIV-1 protease I47A mutant resistant to the protease inhibitor lopinavir.

We have identified a rare HIV-1 protease (PR) mutation, I47A, associated with a high level of resistance to the protease inhibitor lopinavir (LPV) and with hypersusceptibility to the protease inhibitor saquinavir (SQV). The I47A mutation was found in 99 of 112,198 clinical specimens genotyped after LPV became available in late 2000, but in none of 24,426 clinical samples genotyped from 1998 to October 2000. Phenotypic data obtained for five I47A mutants showed unexpected resistance to LPV (86- to >110-fold) and hypersusceptibility to SQV (0.1- to 0.7-fold). Molecular modeling and energy calculations for these mutants using our structural phenotyping methodology showed an increase in the binding energy of LPV by 1.9-3.1 kcal/mol with respect to the wild type complex, corresponding to a 20- to >100-fold decrease in binding affinity, consistent with the observed high levels of LPV resistance. In the WT PR-LPV complex, the Ile 47 side chain is positioned close to the phenoxyacetyl moiety of LPV and its van der Waals interactions contribute significantly to the ligand binding. These interactions are lost for the smaller Ala 47 residue. Calculated binding energy changes for SQV ranged from -0.4 to -1.2 kcal/mol. In the mutant I47A PR-SQV complexes, the PR flaps are packed more tightly around SQV than in the WT complex, resulting in the formation of additional hydrogen bonds that increase binding affinity of SQV consistent with phenotypic hypersusceptibility. The emergence of mutations at PR residue 47 strongly correlates with increasing prescriptions of LPV (Spearman correlation r(s) = 0.96, P < .0001).

Rare one and two amino acid inserts adjacent to codon 103 of the HIV-1 reverse transcriptase (RT) affect susceptibility to non-nucleoside RT inhibitors.

HIV-1 strains that possess a one or two amino acid insert between codons 102 and 103 of the reverse transcriptase (RT) gene were identified in three HIV-1-infected individuals. Each strain also had one or more known mutations associated with nucleoside RT inhibitors (NRTIs) and non-nucleoside RT inhibitors (NNRTIs). Recombinant viruses from these strains had reduced susceptibility to efavirenz and nevirapine, and homology modelling predicted a loss of binding contacts with efavirenz. Mutagenesis studies indicated that replication of insert-containing strains was dependent on RT gene mutations and polymorphisms that co-evolved with the insert. These results suggest that inserts in the NNRTI-binding pocket contribute to NNRTI resistance, but are tolerated only under specific genetic conditions.

HIV type 1 genotypic resistance in a clinical database correlates with antiretroviral utilization.

We established a database of HIV-1 reverse transcriptase (RT) and protease (PR) sequences and mutations to monitor the prevalence of antiretroviral drug resistance and mutational patterns in clinical samples submitted for testing to a major U.S. reference laboratory. At the end of 1998, 80% of the clinical samples tested harbored HIV strains with genotypically predicted resistance to at least one antiretroviral (ARV) drug. By the third quarter of 2002, the frequency of genotypically predicted resistance declined to 65% of samples tested. The prevalence of both PR and nucleoside RT inhibitor resistance declined over this period, while an increase in resistance to nonnucleoside RT inhibitors was found. These genotypic results strongly correlated with a nationwide decrease in the prescription of PR and nucleoside RT inhibitors, and an increase in the prescription of nonnucleoside RT inhibitors over the time period. The increased number of strains that were genotypically sensitive to all classes of ARV probably indicates an increase in genotypic assay use in ARV-naive individuals, however, the trends and correlations in this data set were similar when evaluated after removal of genotypically sensitive strains. Continued monitoring of ARV resistance prevalence, patterns, and utilization trends in clinical databases provides insight into the evolving relationship between clinical practice and ARV resistance.

Structure-based phenotyping predicts HIV-1 protease inhibitor resistance.

Mutations in HIV-1 drug targets lead to resistance and consequent therapeutic failure of antiretroviral drugs. Phenotypic resistance assays are time-consuming and costly, and genotypic rules-based interpretations may fail to predict the effects of multiple mutations. We have developed a computational procedure that rapidly evaluates changes in the binding energy of inhibitors to mutant HIV-1 PR variants. Models of WT complexes were produced from crystal structures. Mutant complexes were built by amino acid substitutions in the WT complexes with subsequent energy minimization of the ligand and PR binding site residues. Accuracy of the models was confirmed by comparison with available crystal structures and by prediction of known resistance-related mutations. PR variants from clinical isolates were modeled in complex with six FDA-approved PIs, and changes in the binding energy (DeltaE(bind)) of mutant versus WT complexes were correlated with the ratios of phenotypic 50% inhibitory concentration (IC(50)) values. The calculated DeltaE(bind) of five PIs showed significant correlations (R(2) = 0.7-0.8) with IC(50) ratios from the Virco Antivirogram assay, and the DeltaE(bind) of six PIs showed good correlation (R(2) = 0.76-0.85) with IC(50) ratios from the Virologic PhenoSense assay. DeltaE(bind) cutoffs corresponding to a four-fold increase in IC(50) were used to define the structure-based phenotype as susceptible, resistant, or equivocal. Blind predictions for 78 PR variants gave overall agreement of 92% (kappa = 0.756) and 86% (kappa = 0.666) with PhenoSense and Antivirogram phenotypes, respectively. The structural phenotyping predicted drug resistance of clinical HIV-1 PR variants with an accuracy approaching that of frequently used cell-based phenotypic assays.

The use of data mining to investigate a possible quality problem with ultrasensitive HIV viral load data at a large reference laboratory.

Suppression of HIV viral load to <50 copies/mL, the lower limit of detection for the ultrasensitive assays, has been shown to correlate with favorable clinical outcome. Patients periodically exhibited transient or sustained low-level viremia based on this test. In order to investigate a possible quality concern, we used our corporate data warehouse to examine the patterns in our data over time as well as across geographic regions.