Sensitive cell-based assay for determination of human immunodeficiency virus type 1 coreceptor tropism.

CCR5 antagonists are a powerful new class of antiretroviral drugs that require a companion assay to evaluate the presence of CXCR4-tropic (non-R5) viruses prior to use in human immunodeficiency virus (HIV)-infected individuals. In this study, we have developed, characterized, verified, and prevalidated a novel phenotypic test to determine HIV-1 coreceptor tropism (VERITROP) based on a sensitive cell-to-cell fusion assay. A proprietary vector was constructed containing a near-full-length HIV-1 genome with the yeast uracil biosynthesis (URA3) gene replacing the HIV-1 env coding sequence. Patient-derived HIV-1 PCR products were introduced by homologous recombination using an innovative yeast-based cloning strategy. The env-expressing vectors were then used in a cell-to-cell fusion assay to determine the presence of R5 and/or non-R5 HIV-1 variants within the viral population. Results were compared with (i) the original version of Trofile (Monogram Biosciences, San Francisco, CA), (ii) population sequencing, and (iii) 454 pyrosequencing, with the genotypic data analyzed using several bioinformatics tools, i.e., the 11/24/25 rule, Geno2Pheno (2% to 5.75%, 3.5%, or 10% false-positive rate [FPR]), and webPSSM. VERITROP consistently detected minority non-R5 variants from clinical specimens, with an analytical sensitivity of 0.3%, with viral loads of ≥1,000 copies/ml, and from B and non-B subtypes. In a pilot study, a 73.7% (56/76) concordance was observed with the original Trofile assay, with 19 of the 20 discordant results corresponding to non-R5 variants detected using VERITROP and not by the original Trofile assay. The degree of concordance of VERITROP and Trofile with population and deep sequencing results depended on the algorithm used to determine HIV-1 coreceptor tropism. Overall, VERITROP showed better concordance with deep sequencing/Geno2Pheno at a 0.3% detection threshold (67%), whereas Trofile matched better with population sequencing (79%). However, 454 sequencing using Geno2Pheno at a 10% FPR and 0.3% threshold and VERITROP more accurately predicted the success of a maraviroc-based regimen. In conclusion, VERITROP may promote the development of new HIV coreceptor antagonists and aid in the treatment and management of HIV-infected individuals prior to and/or during treatment with this class of drugs.

Novel method for simultaneous quantification of phenotypic resistance to maturation, protease, reverse transcriptase, and integrase HIV inhibitors based on 3'Gag(p2/p7/p1/p6)/PR/RT/INT-recombinant viruses: a useful tool in the multitarget era of antiretroviral therapy.

Twenty-six antiretroviral drugs (ARVs), targeting five different steps in the life cycle of the human immunodeficiency virus type 1 (HIV-1), have been approved for the treatment of HIV-1 infection. Accordingly, HIV-1 phenotypic assays based on common cloning technology currently employ three, or possibly four, different recombinant viruses. Here, we describe a system to assess HIV-1 resistance to all drugs targeting the three viral enzymes as well as viral assembly using a single patient-derived, chimeric virus. Patient-derived p2-INT (gag-p2/NCp7/p1/p6/pol-PR/RT/IN) products were PCR amplified as a single fragment (3,428 bp) or two overlapping fragments (1,657 bp and 2,002 bp) and then recombined into a vector containing a near-full-length HIV-1 genome with the Saccharomyces cerevisiae uracil biosynthesis gene (URA3) replacing the 3,428 bp p2-INT segment (Dudley et al., Biotechniques 46:458-467, 2009). P2-INT-recombinant viruses were employed in drug susceptibility assays to test the activity of protease (PI), nucleoside/nucleotide reverse transcriptase (NRTI), nonnucleoside reverse transcriptase (NNRTI), and integrase strand-transfer (INSTI) inhibitors. Using a single standardized test (ViralARTS HIV), this new technology permits the rapid and automated quantification of phenotypic resistance for all known and candidate antiretroviral drugs targeting all viral enzymes (PR, RT, including polymerase and RNase H activities, and IN), some of the current and potential assembly inhibitors, and any drug targeting Pol or Gag precursor cleavage sites (relevant for PI and maturation inhibitors) This novel assay may be instrumental (i) in the development and clinical assessment of novel ARV drugs and (ii) to monitor patients failing prior complex treatment regimens.

Assessment of acute kidney injury in neurologically and traumatically injured intensive care patients receiving large vancomycin doses.

BACKGROUND: Previous reports note that in a mixed patient population, vancomycin doses >4 g/day are associated with increased rates of acute kidney injury (AKI). OBJECTIVE: The objective of the study is to determine if vancomycin regimens >4 g/day are associated with a higher incidence of AKI in neurocritical care unit (NCCU) and trauma/burn Intensive Care Unit (TBICU) patients. MATERIALS AND METHODS: This single-centered, retrospective study enrolled adult patients initiated on vancomycin in the NCCU and TBICU at an academic medical center during 2016. Based on maximum steady-state dose exposure, patients were separated into two groups: ≤4 g/day and >4 g/day. The primary outcome of incidence of AKI was defined by the AKI Network criteria. RESULTS: A total of 284 patients were screened for eligibility; 165 patients met inclusion criteria, 98 patients received ≤4 g/day and 67 patients received >4 g/day. The >4 g/day group had a lower mean age (32.6±11.1 vs. 47.8±16.2, P < 0.001), included more male patients (81% vs. 60%, P = 0.008), were more often treated for a central nervous system infection (31% vs. 11%, P = 0.001), had, on average, more concomitant use of nephrotoxic drugs (2.2±1.2 vs. 1.8±0.9, P = 0.02) and had a higher exposure to contrast (94% vs. 79%, P < 0.001). The primary outcome of AKI occurred in 14 patients receiving ≤4 g/day and five patients receiving >4 g/day which was not statistically significant (14% vs. 7%, P = 0.22). CONCLUSIONS: Our results indicate that administering >4 g/day of vancomycin to achieve therapeutic vancomycin troughs does not appear to lead to an increased incidence of AKI in a mixed NCCU and TBICU population.

Time to get off your knees.

I have just read that the government is considering plans to allow Trusts to become completely run by private companies (Hospitals should be run by the private sector, News, April 21). What a disgrace.