David W. Haas

Cryptococcosis in Human Immunodeficiency Virus-Negative Patients in the Era of Effective Azole Therapy

We conducted a case study of human immunodeficiency virus (HIV)-negative patients with cryptococcosis at 15 United States medical centers from 1990 through 1996 to understand the demographics, therapeutic approach, and factors associated with poor prognosis in this population. Of 306 patients with cryptococcosis, there were 109 with pulmonary involvement, 157 with central nervous system (CNS) involvement, and 40 with involvement at other sites. Seventy-nine percent had a significant underlying condition. Patients with pulmonary disease were usually treated initially with fluconazole (63%); patients with CNS disease generally received amphotericin B (92%). Fluconazole was administered to approximately two-thirds of patients with CNS disease for consolidation therapy. Therapy was successful for 74% of patients. Significant predictors of mortality in multivariate analysis included age > or =60 years, hematologic malignancy, and organ failure. Overall mortality was 30%, and mortality attributable to cryptococcosis was 12%. Cryptococcosis continues to be an important infection in HIV-negative patients and is associated with substantial overall and cause-specific mortality.

HIV infection of naturally occurring and genetically reprogrammed human regulatory T-cells.

A T-cell subset, defined as CD4+CD25hi (regulatory T-cells [Treg cells]), was recently shown to suppress T-cell activation. We demonstrate that human Treg cells isolated from healthy donors express the HIV-coreceptor CCR5 and are highly susceptible to HIV infection and replication. Because Treg cells are present in very few numbers and are difficult to expand in vitro, we genetically modified conventional human T-cells to generate Treg cells in vitro by ectopic expression of FoxP3, a transcription factor associated with reprogramming T-cells into a Treg subset. Overexpression of FoxP3 in naïve human CD4+ T-cells recapitulated the hyporesponsiveness and suppressive function of naturally occurring Treg cells. However, FoxP3 was less efficient in reprogramming memory T-cell subset into regulatory cells. In addition, FoxP3-transduced T-cells also became more susceptible to HIV infection. Remarkably, a portion of HIV-positive individuals with a low percentage of CD4+ and higher levels of activated T-cells have greatly reduced levels of FoxP3+CD4+CD25hi T-cells, suggesting disruption of the Treg cells during HIV infection. Targeting and disruption of the T-cell regulatory system by HIV may contribute to hyperactivation of conventional T-cells, a characteristic of HIV disease progression. Moreover, the ability to reprogram human T-cells into Treg cells in vitro will greatly aid in decoding their mechanism of suppression, their enhanced susceptibility to HIV infection, and the unique markers expressed by this subset.

Pharmacogenetics of Long-Term Responses to Antiretroviral Regimens Containing Efavirenz and/or Nelfinavir: An Adult AIDS Clinical Trials Group Study

BACKGROUND Efavirenz and nelfinavir are metabolized by cytochrome P-450 (CYP) 2B6 and CYP2C19, respectively, with some involvement by CYP3A. Nelfinavir is a substrate for P-glycoprotein, which is encoded by MDR1. The present study examined associations between genetic variants and long-term responses to treatment. METHODS Adult AIDS Clinical Trials Group study 384 randomized antiretroviral-naive subjects to receive efavirenz and/or nelfinavir plus 2 nucleoside analogues, with follow-up lasting up to 3 years. Population pharmacokinetics were estimated from a nonlinear mixed-effects model. Polymorphisms in CYP2B6, CYP2C19, CYP3A4, CYP3A5, and MDR1 were characterized. RESULTS The 504 participants in the genetic study included 340 efavirenz recipients and 348 nelfinavir recipients (184 of the 504 participants received both efavirenz and nelfinavir). Of the participants, 49% were white, 31% were black, and 19% were Hispanic. Plasma exposure to efavirenz and nelfinavir in each population was significantly associated with the polymorphisms CYP2B6 516G-->T and CYP2C19 681G-->A, respectively. Among efavirenz recipients, the MDR1 position 3435 TT genotype was associated with decreased likelihood of virologic failure and decreased emergence of efavirenz-resistant virus but not with plasma efavirenz exposure. Among nelfinavir recipients, a trend toward decreased virologic failure was associated with the polymorphism CYP2C19 681G-->A. CONCLUSIONS Genetic variants predict plasma exposure to efavirenz and nelfinavir, and they may predict virologic failure and/or emergence of drug-resistant virus. These associations with treatment responses must be validated in other studies.

Metabolic Outcomes in a Randomized Trial of Nucleoside, Nonnucleoside and Protease Inhibitor-Sparing Regimens for Initial HIV Treatment

Background:The metabolic effects of initial therapy for HIV-1 infection are important determinants of regimen selection. Methods:Open-label study in 753 subjects randomized equally to efavirenz or lopinavir/ritonavir(r) plus two nucleoside reverse-transcriptase inhibitor (NRTI) vs. the NRTI-sparing regimen of lopinavir/r plus efavirenz. Zidovudine, stavudine, or tenofovir with lamivudine was selected prior to randomization. Metabolic outcomes through 96 weeks were lipoatrophy, defined as at least 20% loss in extremity fat, and fasting serum lipids. Results:Lipoatrophy by dual-energy X-ray absorptiometry at week 96 occurred in 32% [95% confidence interval (CI) 25–39%] of subjects in the efavirenz plus two NRTIs arm, 17% (95% CI 12–24) in the lopinavir/r plus two NRTIs arm, and 9% (95% CI 5–14) in the NRTI-sparing arm (P ≤ 0.023 for all comparisons). Varying the definition of lipoatrophy (≥10 to ≥40% fat loss) and correction for baseline risk factors did not affect the significant difference in lipoatrophy between the NRTI-containing regimens. Lipoatrophy was most frequent with stavudine-containing regimens and least frequent with tenofovir-containing regimens (P < 0.001), which were not significantly different from the NRTI-sparing regimen. Total cholesterol increases at week 96 were greatest in the NRTI-sparing arm (median +57 mg/dl) compared with the other two arms (+32–33 mg/dl; P < 0.001). Use of lipid-lowering agents was more common (25 vs. 11–13%) in the NRTI-sparing arm. Conclusion:Lipoatrophy was more frequent with efavirenz than lopinavir/r when combined with stavudine or zidovudine, and less frequent when either drug was combined with tenofovir. Lipoatrophy was least frequent with the NRTI-sparing regimen, but this benefit was offset by greater cholesterol elevations and the need for lipid-lowering agents.

Pharmacogenetics of Plasma Efavirenz Exposure after Treatment Discontinuation: An Adult AIDS Clinical Trials Group Study

BACKGROUND Efavirenz has a long plasma half-life and a low genetic barrier to resistance. Simultaneously stopping treatment with all agents in efavirenz-containing regimens may result in functional efavirenz monotherapy that selects for drug-resistant human immunodeficiency virus type 1. Lower plasma efavirenz clearance is associated with a cytochrome P450 2B6 gene (CYP2B6) polymorphism (516G-->T) that is more frequent among African American individuals than among European American individuals. METHODS We characterized relationships between this polymorphism and predicted plasma efavirenz concentration-time profiles after discontinuation of therapy with use of data obtained from subjects receiving therapy. Pharmacokinetic parameters were estimated using population-based methods. Concentrations after discontinuation of therapy were predicted from subject-specific estimates. RESULTS. Median estimated efavirenz half-lives were 23, 27, and 48 h for patients with CYP2B6 position 516 GG (78 patients), GT (60), and TT (14) genotypes, respectively (P<.001). After therapy was stopped, plasma efavirenz concentrations in patients with GG, GT, and TT genotypes were predicted to exceed 46.7 ng/mL (the estimated protein-adjusted 95% inhibitory concentration for wild-type virus) for a median of 5.8 days (interquartile range [IQR], 4.4-8.3 days), 7.0 days (IQR, 5.0-8.0 days), and 14 days (IQR, 11.1-21.2 days), respectively (P<.001). Plasma efavirenz levels were predicted to exceed 46.7 ng/mL for >21 days in 5% of subjects with GG genotype, 5% of subjects with GT genotype, and 29% of subjects with TT genotype. CONCLUSIONS The CYP2B6 position 516 TT genotype or a prolonged measured elimination half-life may predict increased risk of developing drug resistance among patients who discontinue efavirenz-containing regimens. This has implications for strategies to safely discontinue antiretroviral regimens while avoiding the emergence of drug resistance.

CD1d-restricted Human Natural Killer T Cells Are Highly Susceptible to Human Immunodeficiency Virus 1 Infection

Human natural killer (NK) T cells are unique T lymphocytes that express an invariant T cell receptor (TCR) Vα24-Vβ11 and have been implicated to play a role in various diseases. A subset of NKT cells express CD4 and hence are potential targets for human immunodeficiency virus (HIV)-1 infection. We demonstrate that both resting and activated human Vα24+ T cells express high levels of the HIV-1 coreceptors CCR5 and Bonzo (CXCR6), but low levels of CCR7, as compared with conventional T cells. Remarkably NKT cells activated with α-galactosylceramide (α-GalCer)-pulsed dendritic cells were profoundly more susceptible to infection with R5-tropic, but not X4-tropic, strains of HIV-1, compared with conventional CD4+ T cells. Furthermore, resting CD4+ NKT cells were also more susceptible to infection. After initial infection, HIV-1 rapidly replicated and depleted the CD4+ subset of NKT cells. In addition, peripheral blood NKT cells were markedly and selectively depleted in HIV-1 infected individuals. Although the mechanisms of this decline are not clear, low numbers or absence of NKT cells may affect the course of HIV-1 infection. Taken together, our findings indicate that CD4+ NKT cells are directly targeted by HIV-1 and may have a potential role during viral transmission and spread in vivo.

The effects of HIV protease inhibitors atazanavir and lopinavir/ritonavir on insulin sensitivity in HIV-seronegative healthy adults

Background: Therapy with some HIV protease inhibitors (PI) contributes to insulin resistance and type 2 diabetes mellitus, by inhibition of insulin-sensitive glucose transporters. Atazanavir (ATV) is a new PI with substantially less in vitro effect on glucose transport than observed with other PI, including lopinavir (LPV) or ritonavir (RTV). Methods: Randomized, double-blind, crossover study of the effect of 5 days of administering ATV, lopinavir/ritonavir (LPV/r) or placebo on insulin-stimulated glucose disposal in 30 healthy HIV-negative subjects. Each subject was studied on two of three possible treatments with a wash-out period between treatments. Results: The mean insulin-stimulated glucose disposal (mg/min per kg body weight) per unit insulin (μU/ml) (M/I) was 9.88, 9.80 and 7.52 for placebo, ATV and LPV/r, respectively (SEM, 0.84 for all). There was no significant difference between ATV and placebo. The M/I for LPV/r was 23% lower than that for ATV (P = 0.010) and 24% lower than that for placebo (P = 0.008). The mean glycogen storage rates were 3.85, 4.00 and 2.54 mg/min per kg for placebo, ATV and LPV/r, respectively (SEM, 0.39 for all). There was no significant difference between ATV and placebo. The glycogen storage rate for LPV/r was 36% lower than ATV (P = 0.003) and 34% lower than placebo (P = 0.006). Conclusions: ATV given to healthy subjects for 5 days did not affect insulin sensitivity, while LPV/r induced insulin resistance. This observation is consistent with differential in vitro effects of these PI on glucose transport. Further data are needed to assess clinical implications for body composition.

The use of vancomycin and tobramycin in acrylic bone cement: biomechanical effects and elution kinetics for use in joint arthroplasty.

We examined the effects of vancomycin on the compressive strength and fatigue life of bone cement and the pharmacokinetics and antimicrobial activity against methicillin-resistant Staphylococcus aureus of vancomycin eluted from bone cement, both alone and in combination with tobramycin. Two cements, Palacos and Simplex, were tested. Three antibiotic preparations were tested: lyophilized vancomycin (vancomycin-L), vancomycin powder (vancomycin-P), and tobramycin powder (Lilly, Indianapolis, IN). Although antibiotics did not significantly affect compressive strength, the fatigue life of bone cement was significantly decreased with vancomycin. Thus, fatigue testing revealed effects on cement strength not apparent by compression testing. Vancomycin-P had a substantially less detrimental effect on fatigue strength than vancomycin-L. Vancomycin-P elutes less efficiently than tobramycin. Although relatively little vancomycin-P eluted from bone cement, it retained biologic activity.

Mitochondrial haplogroups and peripheral neuropathy during antiretroviral therapy: an adult AIDS clinical trials group study.

Objective:HIV nucleoside reverse transcriptase inhibitors (NRTI) can cause peripheral neuropathy that is a result of mitochondrial injury. Polymorphisms in the mitochondrial genome define haplogroups that may have functional implications. The objective of this study was to determine if NRTI-associated peripheral neuropathy is associated with European mitochondrial haplogroups. Design:Case–control study of Adult AIDS Clinical Trials Group (ACTG) study 384 and ACTG Human DNA Repository participants. Methods:ACTG study 384 was a treatment strategy trial of antiretroviral therapy with didanosine (ddI) plus stavudine (d4T) or zidovudine plus lamivudine given with efavirenz, nelfinavir, or both. Subjects were followed for up to 3 years. Peripheral neuropathy was ascertained based on signs and symptoms. For this analysis, polymorphisms that define European mitochondrial haplogroups were characterized in a majority of ACTG 384 participants, and associations with peripheral neuropathy were assessed using logistic regression. Results:A total of 509 subjects were included in this analysis of whom 250 (49%) were self-identified as white, non-Hispanic. Mitochondrial haplogroup T was more frequent in subjects who developed peripheral neuropathy. Among 137 white subjects randomized to receive ddI plus d4T, 20.8% of those who developed peripheral neuropathy belonged to mitochondrial haplogroup T compared to 4.5% of control subjects (odds ratio, 5.4; 95% confidence interval, 1.4–25.1; P = 0.009). Independent predictors of peripheral neuropathy were randomization to receive ddI plus d4T, older age, and mitochondrial haplogroup T. Conclusions:A common European mitochondrial haplogroup may predict NRTI-associated peripheral neuropathy. Future studies should validate this relationship, and evaluate non-European mitochondrial haplogroups and other NRTI toxicities.

Extremely low-coverage sequencing and imputation increases power for genome-wide association studies

Genome-wide association studies (GWAS) have proven to be a powerful method to identify common genetic variants contributing to susceptibility to common diseases. Here, we show that extremely low-coverage sequencing (0.1–0.5×) captures almost as much of the common (>5%) and low-frequency (1–5%) variation across the genome as SNP arrays. As an empirical demonstration, we show that genome-wide SNP genotypes can be inferred at a mean r2 of 0.71 using off-target data (0.24× average coverage) in a whole-exome study of 909 samples. Using both simulated and real exome-sequencing data sets, we show that association statistics obtained using extremely low-coverage sequencing data attain similar P values at known associated variants as data from genotyping arrays, without an excess of false positives. Within the context of reductions in sample preparation and sequencing costs, funds invested in extremely low-coverage sequencing can yield several times the effective sample size of GWAS based on SNP array data and a commensurate increase in statistical power.