Imaging of Postoperative Infection at the Knee Joint.

Postoperative infections of the knee are uncommon but may occur with joint arthroplasties, fracture fixation, or after arthroscopic procedures. The ultimate diagnosis is made by joint aspiration or tissue sampling. Joint aspiration and tissue sampling can be performed under imaging guidance or intraoperatively. Imaging is an important adjunct to clinical and laboratory findings and should start with radiographs. Cross-sectional imaging including magnetic resonance (MR) imaging, computed tomography (CT), nuclear studies, and ultrasound (US) are frequently used if the diagnosis is in doubt and to evaluate the extent of disease. We discuss the current algorithm in the diagnosis of various postoperative infections of the knee joint. The article addresses the utility of radiography, MR imaging, CT, US, and the most commonly used nuclear studies in the diagnosis of various postoperative knee infections and the imaging appearances of these infections on each of these diagnostic modalities.

Tenofovir alafenamide demonstrates broad cross-genotype activity against wild-type HBV clinical isolates and maintains susceptibility to drug-resistant HBV isolates in vitro.

Tenofovir alafenamide (TAF) is a novel prodrug of tenofovir (TFV). This study evaluated the antiviral activity of TAF against wild-type genotype A-H HBV clinical isolates as well as adefovir-resistant, lamivudine-resistant, and entecavir-resistant HBV isolates. Full length HBV genomes or the polymerase/reverse transcriptase (pol/RT) region from treatment-naïve patients infected with HBV genotypes A-H were amplified and cloned into an expression vector under the control of a CMV promoter. In addition, 11 drug resistant HBV constructs were created by site-directed mutagenesis of a full length genotype D construct. Activity of TAF was measured by transfection of each construct into HepG2 cells and assessment of HBV DNA levels following treatment across a range of TAF concentrations. TAF activity in vitro was similar against wild-type genotype A-H HBV clinical isolates. All lamivudine- and entecavir-resistant isolates and 4/5 adefovir-resistant isolates were found to be sensitive to inhibition by TAF in vitro as compared to the wild-type isolate. The adefovir-resistant isolate rtA181V + rtN236T exhibited low-level reduced susceptibility to TAF. TAF is similarly active in vitro against wild-type genotype A-H HBV clinical isolates. The TAF sensitivity results for all drug-resistant isolates are consistent with what has been observed with the parent drug TFV. The in vitro cell-based HBV phenotyping assay results support the use of TAF in treatment of HBV infected subjects with diverse HBV genotypes, in both treatment-naive and treatment-experienced HBV infected patients.

Chemoprophylaxis With Oral Emtricitabine and Tenofovir Alafenamide Combination Protects Macaques From Rectal Simian/Human Immunodeficiency Virus Infection.

Tenofovir alafenamide (TAF) is a novel prodrug of tenofovir that efficiently delivers tenofovir diphosphate to lymphoid cells following oral administration. We investigated whether the combination of TAF and emtricitabine (FTC) could prevent simian/human immunodeficiency virus (SHIV) infection in macaques to determine the potential use of TAF for pre-exposure prophylaxis (PrEP) to prevent human immunodeficiency virus infection. Macaques were exposed rectally to SHIV once per week for up to 19 weeks and received saline or FTC/TAF 24 hours before and 2 hours after each virus inoculation. All 6 controls were infected, while the 6 PrEP-treated animals were protected from infection. Our results support the clinical investigation of FTC/TAF for PrEP.

A Novel Assay to Measure the Magnitude of the Inducible Viral Reservoir in HIV-infected Individuals.

BACKGROUND: Quantifying latently infected cells is critical to evaluate the efficacy of therapeutic strategies aimed at reducing the size of the long-lived viral reservoir, but the low frequency of these cells makes this very challenging. METHODS: We developed TILDA (Tat/rev Induced Limiting Dilution Assay) to measure the frequency of cells with inducible multiply-spliced HIV RNA, as these transcripts are usually absent in latently infected cells but induced upon viral reactivation. TILDA requires less than a million cells, does not require RNA extraction and can be completed in two days. FINDINGS: In suppressed individuals on ART, we found the median frequency of latently infected CD4 + T cells as estimated by TILDA to be 24 cells/million, which was 48 times more than the frequency measured by the quantitative viral outgrowth assay, and 6-27 times less than the frequencies of cells harbouring viral DNA measured by PCR-based assays. TILDA measurements strongly correlated with most HIV DNA assays. The size of the latent reservoir measured by TILDA was lower in subjects who initiated ART during the early compared to late stage of infection (p = 0.011). In untreated HIV disease, the frequency of CD4 + cells carrying latent but inducible HIV largely exceeded the frequency of actively producing cells, demonstrating that the majority of infected cells are transcriptionally silent even in the absence of ART. INTERPRETATIONS: Our results suggest that TILDA is a reproducible and sensitive approach to measure the frequency of productively and latently infected cells in clinical settings. We demonstrate that the latent reservoir represents a substantial fraction of all infected cells prior to ART initiation. RESEARCH IN CONTEXT: In this manuscript, we describe the development of a novel assay that measures the magnitude of the latent HIV reservoir, the main barrier to HIV eradication. This novel assay, termed TILDA for Tat/rev Induced Limiting Dilution Assay, requires only 10 ml of blood, does not necessitate extraction of viral nucleic acids, is highly reproducible, covers a wide dynamic range of reservoir sizes and can be completed in two days. As such, TILDA may represent an alternative to existing assays used to evaluate the efficacy of therapeutic strategies aimed at reducing the size of the latent HIV reservoir.

Week 144 resistance analysis of elvitegravir/cobicistat/emtricitabine/tenofovir DF versus atazanavir+ritonavir+emtricitabine/tenofovir DF in antiretroviral-naïve patients.

OBJECTIVE: To describe baseline and emergent HIV-1 resistance to elvitegravir/ cobicistat/emtricitabine/tenofovir DF (EVG/COBI/FTC/TDF) and ritonavir-boosted atazanavir/emtricitabine/tenofovir DF (ATV+RTV+FTC/TDF) in HIV-1-infected, treatment-naïve subjects through 144 weeks. METHOD: This was a randomized, double-blind, phase 3 study. HIV-1 protease (PR) and reverse transcriptase (RT) were sequenced at screening. Genotypic and phenotypic analyses were performed at virologic failure confirmation and retrospectively at baseline for PR, RT, and integrase (IN) for patients with virologic failure through week 144. RESULTS: In the EVG/ COBI/FTC/TDF group through week 144, HIV-1 from 8 patients (2.3%; 8/353 treated patients) developed primary IN strand transfer inhibitor (INSTI) (n = 6) and/or nucleoside RT inhibitor (NRTI) resistance substitutions (n = 7). The emergence of resistance decreased after the first year, with 5 patients developing HIV-1 resistance through week 48, 1 from weeks 48-96, and 2 from weeks 96-144. Emergent substitutions were E92Q, N155H, or Q148R (n = 2 each) and T66I or T97A (n = 1 each) in IN and M184V/I (n = 7) and K65R (n = 1) in RT. All 8 isolates had reduced susceptibility to EVG, FTC, or TDF. Virus with EVG phenotypic resistance showed cross-resistance to raltegravir. In the ATV+RTV+FTC/TDF group, HIV-1 from 2 patients (0.6%; 2/355 treated patients; both at week 144) developed the resistance substitution M184V/I in RT. CONCLUSIONS: Resistance development to EVG/COBI/FTC/TDF was infrequent (2.3%) through 144 weeks of therapy and decreased over time, consistent with durable efficacy.

Isolated Lactobacillus chronic prosthetic knee infection.

Lactobacillus is a gram-positive rod bacteria found primarily in the gastrointestinal and female genital tracts. Prosthetic infections in implants are being increasingly reported. The authors present a case of a 58-year-old patient with Lactobacillus septic prosthetic knee joint infection. To the authors' knowledge, this is the first reported case of chronic prosthetic knee infection with isolated Lactobacillus species. Lactobacillus has been most commonly implicated with bacteremia and endocarditis and rarely with pneumonia, meningitis, and endovascular infection, and a vast majority of the cases are reported in immunocompromised patients. In the current case, diabetes mellitus, hepatitis, malnutrition, anemia, and liver failure were comorbid conditions, placing the patient at increased risk of infection. The findings suggest that further case series are necessary to establish the significance of Lactobacillus as an etiologic agent in chronic low-virulence, and potentially vancomycin-resistant, prosthetic joint infection. The need also exists for further research aimed at the risk of prosthetic joint infection with oral intake of certain probiotic foods and supplements. The goal of this case report is to bring to light the potential of this organism to be a cause of subtle chronic prosthetic joint infection.

In vitro characterization of MK-1439, a novel HIV-1 nonnucleoside reverse transcriptase inhibitor.

Nonnucleoside reverse transcriptase inhibitors (NNRTIs) are a mainstay of therapy for treating human immunodeficiency type 1 virus (HIV-1)-infected patients. MK-1439 is a novel NNRTI with a 50% inhibitory concentration (IC50) of 12, 9.7, and 9.7 nM against the wild type (WT) and K103N and Y181C reverse transcriptase (RT) mutants, respectively, in a biochemical assay. Selectivity and cytotoxicity studies confirmed that MK-1439 is a highly specific NNRTI with minimum off-target activities. In the presence of 50% normal human serum (NHS), MK-1439 showed excellent potency in suppressing the replication of WT virus, with a 95% effective concentration (EC95) of 20 nM, as well as K103N, Y181C, and K103N/Y181C mutant viruses with EC95 of 43, 27, and 55 nM, respectively. MK-1439 exhibited similar antiviral activities against 10 different HIV-1 subtype viruses (a total of 93 viruses). In addition, the susceptibility of a broader array of clinical NNRTI-associated mutant viruses (a total of 96 viruses) to MK-1439 and other benchmark NNRTIs was investigated. The results showed that the mutant profile of MK-1439 was superior overall to that of efavirenz (EFV) and comparable to that of etravirine (ETR) and rilpivirine (RPV). Furthermore, E138K, Y181C, and K101E mutant viruses that are associated with ETR and RPV were susceptible to MK-1439 with a fold change (FC) of <3. A two-drug in vitro combination study indicated that MK-1439 acts nonantagonistically in the antiviral activity with each of 18 FDA-licensed drugs for HIV infection. Taken together, these in vitro data suggest that MK-1439 possesses the desired properties for further development as a new antiviral agent.

Lower prevalence of drug resistance mutations at first-line virological failure to first-line therapy with atripla vs. tenofovir + emtricitabine/lamivudine + efavirenz administered on a multiple tablet therapy.

BACKGROUND: Fixed-dose combination antiretroviral therapy administered as a single-tablet regimen (STR) may improve virologic suppression rates. The effect of STRs on development of resistance when virologic failure occurs on STRs is not known. OBJECTIVES: To compare the rate of emergent drug resistance mutations (DRMs) on first-line therapy with coformulated tenofovir disoproxil fumarate (TDF)/emtricitabine (FTC)/efavirenz (EFV) as an STR versus TDF, lamivudine (3TC) or FTC, and EFV given as non-STR. METHODS: Patients from eight cohorts and four randomized clinical trials who received first-line antiretroviral therapy with Atripla (STR group) or with TDF + FTC/3TC + EFV (non-STR group) were eligible if a genotypic resistance test was available immediately after the first episode of viral failure. The DRM list from the 2013 version of IAS-USA was used. RESULTS: One hundred and eighty-six patients were included in the final analysis, 122 (65.6%) from eight cohorts and 64 (34.1%) from four randomized clinical trials. The overall proportion of patients with at least one DRM at viral failure was 67.7%, including 53.4% (31 of 58) in the STR group vs. 74.2% (95 of 128) in the non-STR group (P = 0.005). Among patients exclusively from cohorts, at least one DRM was detected in 53.4% (31 of 58) in the STR group vs. 78.1% (50 of 64) in the non-STR group (P = 0.004). DRMs for individual drugs were: TDF, 15.5 vs. 16.4% (P = 0.87); 3TC/FTC, 31 vs. 35.2% (P = 0.58); and NNRTI, 51.7 vs. 65.6% (P = 0.07). The proportion of patients with an M184V/I among the 128 patients who received FTC was 32.8 vs. 36.2% among the 58 treated with 3TC (P = 0.65). CONCLUSIONS: Compared to patients receiving the STR-Atripla, those receiving the same components individually in a non-STR regimen have a statistically significantly increased risk of selecting for DRMs associated with their drugs on failure.

Characterization of resistance to the protease inhibitor GS-9451 in hepatitis C virus-infected patients.

GS-9451, a novel hepatitis C virus (HCV) nonstructural 3/4a (NS3/4a) protease inhibitor, is highly active in patients infected with HCV genotype 1 (GT 1). The aim of this study is to characterize the clinical resistance profile of GS-9451 in GT 1 HCV-infected patients in a phase 1, 3-day monotherapy study. The full-length NS3/4A gene was population sequenced at baseline, on the final treatment day, and at follow-up time points. NS3 protease domains from patient isolates with emerging mutations were cloned into an NS3 shuttle vector, and their susceptibilities to GS-9451 and other HCV inhibitors were determined using a transient replication assay. No resistance mutations at NS3 position 155, 156, or 168 were detected in any of the baseline samples or in viruses from patients treated with 60 mg of GS-9451 once daily. Among patients who received 200 mg and 400 mg of GS-9451, viruses with mutations at position D168 (D168E/G/V) and R155 (R155K), which confer high-level resistance to GS-9451, were detected in those with GT 1b and GT 1a virus, respectively. Viruses with D168 mutations were no longer detected in any GT 1b patient at day 14 and subsequent time points. In GT 1a patients, R155K mutants were replaced by the wild type in 57% of patients at week 24. These NS3 clinical mutants were sensitive to NS5B and NS5A inhibitors, as well as alpha interferon (IFN-α) and ribavirin. The lack of cross-resistance between GS-9451 and other classes of HCV inhibitors supports the utility of combination therapy.

Mechanism of resistance to GS-9148 conferred by the Q151L mutation in HIV-1 reverse transcriptase.

GS-9148 is an investigational phosphonate nucleotide analogue inhibitor of reverse transcriptase (RT) (NtRTI) of human immunodeficiency virus type 1 (HIV-1). This compound is an adenosine derivative with a 2',3'-dihydrofuran ring structure that contains a 2'-fluoro group. The resistance profile of GS-9148 is unique in that the inhibitor can select for the very rare Q151L mutation in HIV-1 RT as a pathway to resistance. Q151L is not stably selected by any of the approved nucleoside or nucleotide analogues; however, it may be a transient intermediate that leads to the related Q151M mutation, which confers resistance to multiple compounds that belong to this class of RT inhibitors. Here, we employed pre-steady-state kinetics to study the impact of Q151L on substrate and inhibitor binding and the catalytic rate of incorporation. Most importantly, we found that the Q151L mutant is unable to incorporate GS-9148 under single-turnover conditions. Interference experiments showed that the presence of GS-9148-diphosphate, i.e., the active form of the inhibitor, does not reduce the efficiency of incorporation for the natural counterpart. We therefore conclude that Q151L severely compromises binding of GS-9148-diphosphate to RT. This effect is highly specific, since we also demonstrate that another NtRTI, tenofovir, is incorporated with selectivity similar to that seen with wild-type RT. Incorporation assays with other related compounds and models based on the RT/DNA/GS-9148-diphosphate crystal structure suggest that the 2'-fluoro group of GS-9148 may cause steric hindrance with the side chain of the Q151L mutant.

No resistance to tenofovir disoproxil fumarate detected after up to 144 weeks of therapy in patients monoinfected with chronic hepatitis B virus.

UNLABELLED: Tenofovir disoproxil fumarate (TDF) is a nucleotide analogue with potent activity against human immunodeficiency virus type 1 and hepatitis B virus (HBV). To date, no reports of HBV clinical resistance to TDF have been confirmed. In two phase 3 studies (GS-US-174-0102 and GS-US-174-0103), 375 hepatitis B e antigen-negative (HBeAg(-) ) patients and 266 HBeAg(+) patients with chronic hepatitis B (some nucleoside-naive and some lamivudine-experienced) were randomized 2:1 to receive TDF (n = 426) or adefovir dipivoxil (ADV; n = 215) for 48 weeks. After week 48, eligible patients received open-label TDF with no interruption. The studies are being continued through week 384/year 8; week 144 data are presented here. Per protocol, viremic patients (HBV DNA level ≥ 400 copies/mL or 69 IU/mL) had the option of adding emtricitabine (FTC) at or after week 72. Resistance analyses of HBV polymerase/reverse transcriptase (pol/RT) were based on population dideoxy sequencing. Phenotypic analyses were conducted in HepG2 cells with recombinant HBV derived from patient serum. Most patients maintained TDF monotherapy treatment across both studies (607/641, 95%). A resistance analysis of HBV pol/RT was performed at the baseline for all patients, for viremic patients at week 144 or at the last time when they were on TDF monotherapy (34 on TDF and 19 on ADV-TDF), and for patients who remained viremic after the addition of FTC (7/20 on TDF and 5/14 on ADV-TDF). No patient developed amino acid substitutions associated with resistance to TDF. Virological breakthrough on TDF monotherapy was infrequent over 144 weeks (13/426, 3%) and was attributed to documented nonadherence in most cases (11/13, 85%). Persistent viremia (≥400 copies/mL) through week 144 was rare (5/641, 0.8%) and was not associated with virological resistance to TDF by population or clonal analyses. CONCLUSION: No nucleoside-naive or nucleoside-experienced patient developed HBV pol/RT mutations associated with TDF resistance after up to 144 weeks of exposure to TDF monotherapy.

Susceptibility of treatment-naive hepatitis C virus (HCV) clinical isolates to HCV protease inhibitors.

In order to assess the natural variation in susceptibility to hepatitis C virus (HCV) NS3 protease inhibitors (PIs) among untreated HCV patient samples, the susceptibilities of 39 baseline clinical isolates were determined using a transient-replication assay on a panel of HCV PIs, including two α-ketoamides (VX-950 and SCH-503034) and three macrocyclic inhibitors (MK-7009, ITMN-191, and TMC-435350). Some natural variation in susceptibility to all HCV PIs tested was observed among the baseline clinical isolates. The susceptibility to VX-950 correlated strongly with the susceptibility to SCH-503034. A moderate correlation was observed between the susceptibilities to ITMN-191 and MK-7009. In contrast, the phenotypic correlations between the α-ketoamides and macrocyclic inhibitors were significantly lower. This difference is partly attributable to reduced susceptibility of the HCV variants containing the NS3 polymorphism Q80K (existing in 47% of genotype 1a isolates) to the macrocyclic compounds but no change in the sensitivity of the same variants to the α-ketoamides tested. Our results suggest that the natural variation in baseline susceptibility may contribute to different degrees of antiviral response among patients in vivo, particularly at lower doses.

Vaccination with peptide mimetics of the gp41 prehairpin fusion intermediate yields neutralizing antisera against HIV-1 isolates.

Eliciting a broadly neutralizing polyclonal antibody response against HIV-1 remains a major challenge. One approach to vaccine development is prevention of HIV-1 entry into cells by blocking the fusion of viral and cell membranes. More specifically, our goal is to elicit neutralizing antibodies that target a transient viral entry intermediate (the prehairpin intermediate) formed by the HIV-1 gp41 protein. Because this intermediate is transient, a stable mimetic is required to elicit an immune response. Previously, a series of engineered peptides was used to select a mAb (denoted D5) that binds to the surface of the gp41 prehairpin intermediate, as demonstrated by x-ray crystallographic studies. D5 inhibits the replication of HIV-1 clinical isolates, providing proof-of-principle for this vaccine approach. Here, we describe a series of peptide mimetics of the gp41 prehairpin intermediate designed to permit a systematic analysis of the immune response generated in animals. To improve the chances of detecting weak neutralizing polyclonal responses, two strategies were employed in the initial screening: use of a neutralization-hypersensitive virus and concentration of the IgG fraction from immunized animal sera. This allowed incremental improvements through iterative cycles of design, which led to vaccine candidates capable of generating a polyclonal antibody response, detectable in unfractionated sera, that neutralize tier 1 HIV-1 and simian HIV primary isolates in vitro. Our findings serve as a starting point for the design of more potent immunogens to elicit a broadly neutralizing response against the gp41 prehairpin intermediate.

Phenotypic susceptibility to bevirimat in isolates from HIV-1-infected patients without prior exposure to bevirimat.

Bevirimat (BVM) is the first of a new class of anti-HIV drugs with a novel mode of action known as maturation inhibitors. BVM inhibits the last cleavage of the Gag polyprotein by HIV-1 protease, leading to the accumulation of the p25 capsid-small peptide 1 (SP1) intermediate and resulting in noninfectious HIV-1 virions. Early clinical studies of BVM showed that over 50% of the patients treated with BVM did not respond to treatment. We investigated the impact of prior antiretroviral (ARV) treatment and/or natural genetic diversity on BVM susceptibility by conducting in vitro phenotypic analyses of viruses made from patient samples. We generated 31 recombinant viruses containing the entire gag and protease genes from 31 plasma samples from HIV-1-infected patients with (n = 21) or without (n = 10) prior ARV experience. We found that 58% of the patient isolates tested had a >10-fold reduced susceptibility to BVM, regardless of the patient's ARV experience or the level of isolate resistance to protease inhibitors. Analysis of mutants with site-directed mutations confirmed the role of the V370A SP1 polymorphism (SP1-V7A) in resistance to BVM. Furthermore, we demonstrated for the first time that a capsid polymorphism, V362I (CA protein-V230I), is also a major mutation conferring resistance to BVM. In contrast, none of the previously defined resistance-conferring mutations in Gag selected in vitro (H358Y, L363M, L363F, A364V, A366V, or A366T) were found to occur among the viruses that we analyzed. Our results should be helpful in the design of diagnostics for prediction of the potential benefit of BVM treatment in HIV-1-infected patients.

CD8+ lymphocytes control viral replication in SIVmac239-infected rhesus macaques without decreasing the lifespan of productively infected cells.

While CD8+ T cells are clearly important in controlling virus replication during HIV and SIV infections, the mechanisms underlying this antiviral effect remain poorly understood. In this study, we assessed the in vivo effect of CD8+ lymphocyte depletion on the lifespan of productively infected cells during chronic SIVmac239 infection of rhesus macaques. We treated two groups of animals that were either CD8+ lymphocyte-depleted or controls with antiretroviral therapy, and used mathematical modeling to assess the lifespan of infected cells either in the presence or absence of CD8+ lymphocytes. We found that, in both early (day 57 post-SIV) and late (day 177 post-SIV) chronic SIV infection, depletion of CD8+ lymphocytes did not result in a measurable increase in the lifespan of either short- or long-lived productively infected cells in vivo. This result indicates that the presence of CD8+ lymphocytes does not result in a noticeably shorter lifespan of productively SIV-infected cells, and thus that direct cell killing is unlikely to be the main mechanism underlying the antiviral effect of CD8+ T cells in SIV-infected macaques with high virus replication.

PD-1 blockade in rhesus macaques: impact on chronic infection and prophylactic vaccination.

Programmed Cell Death 1 (PD-1) plays a crucial role in immunomodulation. Binding of PD-1 to its ligand receptors down-regulates immune responses, and published reports suggest that this immune modulation is exploited in cases of tumor progression or chronic viral infection to evade immune surveillance. Thus, blockade of this signal could restore or enhance host immune functions. To test this hypothesis, we generated a panel of mAbs specific to human PD-1 that block PD ligand 1 and tested them for in vitro binding, blocking, and functional T cell responses, and evaluated a lead candidate in two in vivo rhesus macaque (Macaca mulatta) models. In the first therapeutic model, chronically SIV-infected macaques were treated with a single infusion of anti-PD-1 mAb; viral loads increased transiently before returning to, or falling below, pretreatment baselines. In the second prophylactic model, naive macaques were immunized with an SIV-gag adenovirus vector vaccine. Induced PD-1 blockade caused a statistically significant (p<0.05) increase in the peak percentage of T cells specific for the CM9 Gag epitope. These new results on PD-1 blockade in nonhuman primates point to a broader role for PD-1 immunomodulation and to potential applications in humans.

Comparison of opioid requirements and analgesic response in opioid-tolerant versus opioid-naïve patients after total knee arthroplasty.

STUDY OBJECTIVES: To compare opioid requirements in opioid-tolerant and opioid-naïve patients after total knee arthroplasty, and to compare pain scores, sedation scores, and adverse effects between the groups. DESIGN: Prospective, observational study. SETTING: Academic medical center. PATIENTS: Twenty-nine patients aged 18 years or older who underwent elective total knee arthroplasty between October 1, 2005, and June 31, 2007. MEASUREMENTS AND MAIN RESULTS: Patients were classified on the basis of their daily opioid consumption during the week before surgery: those who required 10 mg or less of oral morphine equivalent were considered opioid naïve (20 patients), and those who required at least 30 mg of oral morphine equivalent were opioid tolerant (nine patients). Postoperative opioid consumption, pain scores, sedation scores, and adverse effects were compared between the two groups up to 48 hours after discharge from the postanesthesia care unit (PACU). Postoperative opioid consumption (in intravenous morphine equivalents) was significantly greater in the opioid-tolerant group than in the opioid-naïve group in the PACU (median 56 vs 8.2 mg, p=0.0013), during the first 24 hours after discharge from the PACU (108 vs 20.5 mg, p=0.0004), and 24-48 hours after discharge from the PACU (152.3 vs 25 mg, p=0.0001). Pain scores, assessed by using a verbal numeric scale from 0-10, were significantly greater in the opioid-tolerant group than in the opioid-naïve group during the first 24 hours after discharge from the PACU (5.9 vs 4.1, p=0.03). We observed no significant difference in pain scores during the other time periods studied. Sedation scores and adverse effects were similar between groups. CONCLUSION: After total knee arthroplasty, patients tolerant to opioids required significantly more opioids in the PACU and up to 48 hours after discharge from the PACU than did opioid-naïve patients. Opioid-tolerant patients also experienced greater pain during the first 24 hours after discharge from the PACU; however, sedation scores and adverse effects did not appear to be significantly different at any of the time periods studied. Clinicians need to be aggressive with pain management immediately after surgery and ensure that patients restart any opioid treatment at home as soon as possible.

Effect of nucleoside and nucleotide reverse transcriptase inhibitors of HIV on endogenous nucleotide pools.

BACKGROUND: Alterations in endogenous nucleotide pools as a result of HIV therapy with nucleoside and nucleotide reverse transcriptase inhibitors (N[t]RTIs) is a proposed mechanism for therapy-related adverse events and drug interactions resulting in treatment failure. In vitro studies were performed in order to understand the effect of N(t)RTIs on endogenous nucleotide pools. METHODS: The T-cell line CEM-CCRF was treated with control antimetabolites or the N(t)RTIs abacavir, didanosine, lamivudine, tenofovir (TFV) and zidovudine (AZT), either alone or in combination. The levels of natural 2'-deoxynucleoside triphosphates (dNTP) and ribonucleoside triphophosphates were determined by liquid chromatography coupled with triple quadrupole mass spectrometry. RESULTS: Antimetabolites altered nucleotide pools in a manner consistent with their known mechanisms of action. AZT was the only N(t)RTI that significantly altered dNTP pools. incubation of 10 microM AZT, either alone or in combination with other N(t)RTIs, increased 2'-deoxyadenosine triphosphate, 2'-deoxyguanosine triphosphate and thymidine triphosphate levels by up to 1.44-fold the concentrations observed in untreated cells. At higher than pharmacological concentrations of AZT, evidence for inhibition of 2'-deoxycytidylate deaminase and enzymes involved in the salvage of thymidine was also observed. Phosphorylated metabolites of TFV are known to inhibit purine nucleoside phosphorylase (PNP). However, in contrast to a potent PNP inhibitor, TFV was unable to alter intracellular dNTP pools upon addition of exogenous 2'-deoxyguanosine. CONCLUSIONS: N(t)RTIs have the potential to alter nucleotide pools; however, at the pharmacologically relevant concentrations, tested N(t)RTI or their combinations did not have an effect on nucleotide pools with the notable exception of AZT.

The A62V and S68G mutations in HIV-1 reverse transcriptase partially restore the replication defect associated with the K65R mutation.

BACKGROUND: The K65R mutation in human immunodeficiency virus type 1 reverse transcriptase can be selected by abacavir, didanosine, tenofovir, and stavudine in vivo resulting in reduced susceptibility to these drugs and decreased viral replication capacity. In clinical isolates, K65R is frequently accompanied by the A62V and S68G reverse transcriptase mutations. METHODS: The role of A62V and S68G in combination with K65R was investigated using phenotypic, viral growth competition, pre-steady-state kinetic, and excision analyses. RESULTS: Addition of A62V and S68G to K65R caused no significant change in human immunodeficiency virus type 1 resistance to abacavir, didanosine, tenofovir, or stavudine but partially restored the replication defect of virus containing K65R. The triple mutant K65R+A62V+S68G still showed some replication defect compared with wild-type virus. Pre-steady-state kinetic analysis demonstrated that K65R resulted in a decreased rate of incorporation (kpol) for all natural dNTPs, which were partially restored to wild-type levels by addition of A62V and S68G. When added to K65R and S68G, the A62V mutation seemed to restore adenosine triphosphate-mediated excision of tenofovir to wild-type levels. CONCLUSIONS: A62V and S68G serve as partial compensatory mutations for the K65R mutation in reverse transcriptase by improving the viral replication capacity, which is likely due to increased incorporation efficiency of the natural substrates.