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1.  Tenofovir use and urinary biomarkers among HIV-infected women in the Women's Interagency HIV Study (WIHS) 
Background
Tenofovir has been associated with renal tubular injury. Biomarkers that signal early tubular dysfunction are needed because creatinine rise lags behind tenofovir-associated kidney dysfunction. We examined several urinary biomarkers to determine if rises accompanying tenofovir initiation preceded creatinine changes.
Methods
Three urinary biomarkers of tubular impairment- neutrophil gelatinase-associated lipocalin (NGAL), N-acetyl- β -D-glucosaminidase (NAG), and β-2-microglobulin (β2MG)-were measured across three time points (one pre-tenofovir visit and two post tenofovir visits) in one hundred and thirty two HIV-positive women from the Women's Interagency HIV Study (WIHS). Women initiating HAART containing tenofovir were propensity score matched to women initiating HAART without tenofovir and women not on HAART.
Results
There were no differences between groups for NGAL or NAG but β2MG was 19 times more likely to be elevated among tenofovir users at the 2nd post tenofovir visit compared to non-TDF users at the pre-tenofovir visit (p<0.01). History of proteinuria was associated with elevated NGAL (p <0.01). Factors associated with elevated NAG were GFR<60 ml/min, history of proteinuria, hepatitis C (p<0.01 for all) and diabetes mellitus (p=0.05). Factors associated with increased odds of elevated β2MG were HIV RNA>100,000 copies/ml, hepatitis C, boosted protease inhibitor (PI) use, and GFR<60 ml/min (p≤0.01 for all).
Conclusions
β2MG levels are elevated in women on tenofovir indicating probable early renal dysfunction. Biomarker elevation is additionally associated with baseline chronic kidney disease, uncontrolled viremia, and boosted PI use. Future studies are needed to explore urinary biomarker thresholds in identifying treated HIV-infected individuals at risk for renal dysfunction.
doi:10.1097/QAI.0b013e31828175c9
PMCID: PMC3692572  PMID: 23254151
Tenofovir; urinary biomarkers; HIV infected women
2.  Limited Nucleotide Changes in the Rev Response Element (RRE) during HIV-1 Infection Alter Overall Rev-RRE Activity and Rev Multimerization 
Journal of Virology  2013;87(20):11173-11186.
HIV-1 Rev and the Rev response element (RRE) enable a critical step in the viral replication cycle by facilitating the nuclear export of intron-containing mRNAs, yet their activities have rarely been analyzed in natural infections. This study characterized their genetic and functional variation in a small cohort of HIV-infected individuals. Multiple Rev and RRE sequences were obtained using single-genome sequencing (SGS) of plasma samples collected within 6 months after seroconversion and at a later time. This allowed the identification of cognate sequences that were linked in vivo in the same viral genome and acted together as a functional unit. Phylogenetic analyses of these sequences indicated that 4/5 infections were founded by a single transmission event. Rev and RRE variants from each time point were subjected to functional analysis as both cognate pairs and as individual components. While a range of Rev-RRE activities were seen, the activity of cognate pairs from a single time point clustered to a discrete level, which was termed the set point. In 3/5 patients, this set point changed significantly over the time period studied. In all patients, RRE activity was more sensitive to sequence variation than Rev activity and acted as the primary driver of the cognate set point. Selected patient RREs were also shown to have differences in Rev multimerization using gel shift binding assays. Thus, rather than acting as a simple on-off switch or maintaining a constant level of activity throughout infection, the Rev-RRE system can fluctuate, presumably to control replication.
doi:10.1128/JVI.01392-13
PMCID: PMC3807272  PMID: 23926352
3.  Does HIV infection promote early kidney injury in women? 
Antiviral therapy  2013;19(1):79-87.
Background
In HIV-infected women, urine concentrations of novel tubulointerstitial injury markers, interleukin-18 (IL-18) and kidney injury marker-1 (KIM-1) are associated with kidney function decline and all-cause mortality. We hypothesized that HIV-infected individuals with preserved kidney filtration function would have more extensive kidney injury, as determined by urine injury markers, compared to the uninfected controls, and that risk factors for tubulointerstitial injury would differ from risk factors for albuminuria.
Methods
In this cross-sectional study, we compared urine concentrations of IL-18, KIM-1, and ACR in 908 HIV-infected and 289 HIV-uninfected women enrolled in the Women’s Interagency HIV Study, utilizing stored urine specimens from visits between 1999 and 2000.
Results
After multivariate-adjusted linear regression analysis, mean urine concentrations were higher in HIV-infected individuals by 38% for IL-18 (p<0.0001), 12% for KIM-1 (p=0.081), and 47% for ACR (p<0.0001). Higher HIV RNA level (15% per 10-fold increase, p<0.0001), lower CD4 count (8% per doubling, p=0.0025), HCV infection (30%, p=0.00018), and lower HDL (5% per 10 mg/dL, p=0.0024) were each associated with higher IL-18 concentrations. In contrast, hypertension (81%, p<0.0001) and diabetes (47%, p=0.018) were among the strongest predictors of higher ACR, though HIV RNA level (15% per 10-fold increase, p=0.0004) was also associated with higher ACR.
Conclusions
HIV-infected women had more extensive tubulointerstitial and glomerular injury than uninfected women, but the associated factors differed among the urine biomarkers. Combinations of urinary biomarkers should be investigated to further characterize early kidney injury in HIV-infected women.
doi:10.3851/IMP2677
PMCID: PMC3933452  PMID: 23970313
4.  Underlying genetic structure impacts the association between CYP2B6 polymorphisms and response to efavirenz and nevirapine 
AIDS (London, England)  2012;26(16):2097-2106.
Objective
CYP2B6 variation predicts pharmacokinetic characteristics of its substrates. Consideration for underlying genetic structure is critical to protect against spurious associations with the highly polymorphic CYP2B6 gene.
Design
The effect of CYP2B6 variation on response to its substrates, nonnucleoside reverse transcriptase inhibitors (NNRTIs), was explored in the Women's Interagency HIV Study.
Methods
Five putative functional polymorphisms were tested for associations with virologic suppression within one year after NNRTI initiation in women naïve to antiretroviral agents (n=91). Principal components (PCs) were generated to control for population substructure. Logistic regression was used to test the joint effect of rs3745274 and rs28399499, which together indicate slow, intermediate, and extensive metabolizers.
Results
Rs3745274 was significantly associated with virologic suppression (OR=3.61, 95% CI 1.16-11.22, p trend=0.03); the remaining polymorphisms tested were not significantly associated with response. Women classified as intermediate and slow metabolizers were 2.90 (95% CI 0.79-12.28) and 13.44 (95% CI 1.66-infinity) times as likely to achieve virologic suppression compared to extensive metabolizers after adjustment for PCs (p trend=0.005). Failure to control for genetic ancestry resulted in substantial confounding of the relationship between the metabolizer phenotype and treatment response.
Conclusion
The CYP2B6 metabolizer phenotype was significantly associated with virologic response to NNRTIs; this relationship would have been masked by simple adjustment for self-reported ethnicity. Given the appreciable genetic heterogeneity that exists within self-reported ethnicity, these results exemplify the importance of characterizing underlying genetic structure in pharmacogenetic studies. Further follow-up of the CYP2B6 metabolizer phenotype is warranted given the potential clinical importance of this finding.
doi:10.1097/QAD.0b013e3283593602
PMCID: PMC3940150  PMID: 22951632
CYP2B6; population substructure; women; NNRTIs; confounding
5.  Association Of Hepatitis C With Markers Of Hemostasis In HIV-Infected and Uninfected Women in the Women’s Interagency HIV Study (WIHS) 
Coinfection with human immunodeficiency virus (HIV) and hepatitis C virus (HCV) is common. HIV infection and treatment are associated with hypercoaguability; thrombosis in HCV is under-investigated. Proposed markers of hemostasis in HIV include higher D-dimer, Factor VIII% and Plasminogen Activator Inhibitor-1 (PAI-1Ag), and lower total Protein S% (TPS), but have not been examined in HCV. We assessed the independent association of HCV with these four measures of hemostasis in a multicenter, prospective study of HIV: the Women’s Interagency HIV Study (WIHS).
We randomly selected 450 HCV-infected (anti-HCV+ with detectable plasma HCV RNA) and 450 HCV-uninfected (anti-HCV−) women. HCV was the main exposure of interest in regression models.
443 HCV+ and 425 HCV− women were included. HCV+ women had higher Factor VIII% (124.4% ±3.9 vs. 101.8% ±3.7, p <0.001) and lower TPS (75.7% ±1.1 vs. 84.3% ±1.1, <0.001) than HCV−, independent of HIV infection and viral load; there was little difference in PAI-1Ag or log10 D-dimer. After adjustment for confounders, these inferences remained. HIV infection was independently associated with higher Factor VIII% and log10 D-dimer, and lower TPS.
HCV was independently associated with higher Factor VIII% and lower TPS consistent with hypercoaguability. Higher Factor VIII % and D-dimer and lower total Protein S % were also strongly associated with HIV infection and levels of HIV viremia, independent of HCV infection. Further investigation is needed to determine if there is increased thrombotic risk from HCV. Studies examining hemostasis markers in HIV infection must also assess the contribution of HCV infection.
doi:10.1097/QAI.0b013e31827fdd61
PMCID: PMC3652915  PMID: 23221984
6.  Comparisons of creatinine and cystatin C for detection of kidney disease and prediction of all-cause mortality in HIV-infected women 
AIDS (London, England)  2013;27(14):2291-2299.
Background
Cystatin C could improve chronic kidney disease (CKD) classification in HIV-infected women relative to serum creatinine.
Design
Retrospective cohort analysis.
Methods
Cystatin C and creatinine were measured from specimens taken and stored during the 1999–2000 exam among 908 HIV-infected participants in the Women’s Interagency HIV study (WIHS). Mean follow-up was 10.2 years. The associations of baseline categories (<60, 60–90, and >90 mL/min/1.73m2) of creatinine eGFR (eGFRcr), cystatin C eGFR (eGFRcys), and combined creatinine-cystatin C eGFR (eGFRcr-cys) with all-cause mortality were evaluated using multivariable Cox regression. The net reclassification index (NRI) was calculated to evaluate the effect of cystatin C on reclassification of CKD staging.
Results
The prevalence of CKD (eGFR<60) at baseline was higher with eGFRcys (10.1%) compared to eGFRcr (6.7%, p=0.0006) and eGFRcr-cys (7.5%, p=0.011). Relative to eGFR >90, the eGFR <60 category by eGFRcys (Adjusted HR: 2.56; 95% CI: 1.63, 4.02), eGFRcr-cys (3.11; 1.94–5.00), and eGFRcr (2.34; 1.44–3.79) was associated with increased mortality risk. However, the eGFR 60–90 category was associated with increased mortality risk for eGFRcys (1.80; 1.28–2.53) and eGFRcr-cys (1.91; 1.38–2.66) but not eGFRcr (1.20; 0.85–1.67). The overall NRI for mortality was 26% when reclassifying from eGFRcr to eGFRcys (p<0.001) and was 20% when reclassifying from eGFRcr to eGFRcr-cys (p<0.001).
Conclusion
Cystatin C detected a higher prevalence of CKD relative to creatinine and improves CKD staging relative to creatinine by reclassifying individuals at the highest mortality risk to lower eGFR categories.
doi:10.1097/QAD.0b013e328362e874
PMCID: PMC3919542  PMID: 23669156
Creatinine; Cystatin C; Glomerular Filtration Rate; HIV; Mortality; Kidney; Women
7.  Risk of exposure to HIV differs according to catechol-o-methyltransferase Val158Met genotype 
AIDS (London, England)  2013;27(11):1779-1782.
The Met allele of the catechol-o-methyltransferase (COMT) Val158Met polymorphism is associated with increased cortical dopamine and risk behaviors including illicit drug use and unprotected sex. Therefore, we examined whether or not the distribution of the Val158Met genotype differed between HIV-infected and HIV-uninfected women. We conducted an Armitage-Cochran Test and logistic regression to compare genotype frequencies between 1,848 HIV-infected and 612 HIV-uninfected women from the Women’s Interagency HIV Study (WIHS). The likelihood of carrying one or two Met alleles was greater in HIV-infected women (61%) compared to HIV-uninfected women (54%), Z = −3.60, p < 0.001. We report the novel finding of an association between the Val158Met genotype and HIV serostatus that may be mediated through the impact of dopamine function on propensity for risk-taking.
doi:10.1097/QAD.0b013e328361c6a1
PMCID: PMC3897122  PMID: 23807274
8.  Association of HIV and ART with cardiometabolic traits in sub-Saharan Africa: a systematic review and meta-analysis 
Background Sub-Saharan Africa (SSA) has the highest burden of HIV in the world and a rising prevalence of cardiometabolic disease; however, the interrelationship between HIV, antiretroviral therapy (ART) and cardiometabolic traits is not well described in SSA populations.
Methods We conducted a systematic review and meta-analysis through MEDLINE and EMBASE (up to January 2012), as well as direct author contact. Eligible studies provided summary or individual-level data on one or more of the following traits in HIV+ and HIV-, or ART+ and ART- subgroups in SSA: body mass index (BMI), systolic blood pressure (SBP), diastolic blood pressure (DBP), high-density lipoprotein (HDL), low-density lipoprotein (LDL), triglycerides (TGs) and fasting blood glucose (FBG) or glycated hemoglobin (HbA1c). Information was synthesized under a random-effects model and the primary outcomes were the standardized mean differences (SMD) of the specified traits between subgroups of participants.
Results Data were obtained from 49 published and 3 unpublished studies which reported on 29 755 individuals. HIV infection was associated with higher TGs [SMD, 0.26; 95% confidence interval (CI), 0.08 to 0.44] and lower HDL (SMD, −0.59; 95% CI, −0.86 to −0.31), BMI (SMD, −0.32; 95% CI, −0.45 to −0.18), SBP (SMD, −0.40; 95% CI, −0.55 to −0.25) and DBP (SMD, −0.34; 95% CI, −0.51 to −0.17). Among HIV+ individuals, ART use was associated with higher LDL (SMD, 0.43; 95% CI, 0.14 to 0.72) and HDL (SMD, 0.39; 95% CI, 0.11 to 0.66), and lower HbA1c (SMD, −0.34; 95% CI, −0.62 to −0.06). Fully adjusted estimates from analyses of individual participant data were consistent with meta-analysis of summary estimates for most traits.
Conclusions Broadly consistent with results from populations of European descent, these results suggest differences in cardiometabolic traits between HIV-infected and uninfected individuals in SSA, which might be modified by ART use. In a region with the highest burden of HIV, it will be important to clarify these findings to reliably assess the need for monitoring and managing cardiometabolic risk in HIV-infected populations in SSA.
doi:10.1093/ije/dyt198
PMCID: PMC3887568  PMID: 24415610
HIV; ART; cardiometabolic disease; sub-Saharan Africa
9.  Relation of HLA Class I and II Supertypes with Spontaneous Clearance of Hepatitis C Virus 
Genes and immunity  2013;14(5):330-335.
Human leukocyte antigen (HLA) genotype has been associated with probability of spontaneous clearance of hepatitis C virus (HCV). However, no prior studies have examined whether this relationship may be further characterized by grouping HLA alleles according to their supertypes, defined by their binding capacities. There is debate regarding the most appropriate method to define supertypes. Therefore, previously reported HLA supertypes (46 class I and 25 class II) were assessed for their relation with HCV clearance in a population of 758 HCV-seropositive women. Two HLA class II supertypes were significant in multivariable models that included: (i) supertypes with significant or borderline associations with HCV clearance after adjustment for multiple tests, and (ii) individual HLA alleles not part of these supertypes, but associated with HCV clearance in our prior study in this population. Specifically, supertype DRB3 (prevalence ratio (PR)=0.4; p=0.004) was associated with HCV persistence while DR8 (PR=1.8; p=0.01) was associated with HCV clearance. Two individual alleles (B*57:01 and C*01:02) associated with HCV clearance in our prior study became non-significant in analysis that included supertypes while B*57:03 (PR=1.9; p=0.008) and DRB1*07:01 (PR=1.7; p=0.005) retained significance. These data provide epidemiologic support for the significance of HLA supertypes in relation to HCV clearance.
doi:10.1038/gene.2013.25
PMCID: PMC3723800  PMID: 23636221
hepatitis C virus; HLA; human leukocyte antigen; supertype
10.  Urinary Markers of Kidney Injury and Kidney Function Decline in HIV-Infected Women 
Objective
HIV-infected persons have substantially higher risk of kidney failure than persons without HIV, but serum creatinine levels are insensitive for detecting declining kidney function. We hypothesized that urine markers of kidney injury would be associated with declining kidney function among HIV-infected women.
Methods
In the Women's Interagency HIV Study (WIHS), we measured concentrations of albumin-to-creatinine ratio (ACR), interleukin-18 (IL-18), kidney injury marker-1 (KIM-1), and neutrophil gelatinase-associated lipocalin (NGAL) from stored urine among 908 HIV-infected and 289 uninfected participants. Primary analyses used cystatin C based estimated glomerular filtration rate (CKD-EPI eGFRcys) as the outcome, measured at baseline and two follow-up visits over eight years; secondary analyses used creatinine (CKD-EPI eGFRcr). Each urine biomarker was categorized into tertiles, and kidney decline was modeled with both continuous and dichotomized outcomes.
Results
Compared with the lowest tertiles, the highest tertiles of ACR (−0.15ml/min/1.73m2, p<0.0001), IL-18 (−0.09ml/min/1.73m2, p<0.0001) and KIM-1 (−0.06ml/min/1.73m2, p<0.001) were independently associated with faster eGFRcys decline after multivariate adjustment including all three biomarkers among HIV-infected women. Among these biomarkers, only IL-18 was associated with each dichotomized eGFRcys outcome: ≥3% (Relative Risk 1.40; 95%CI 1.04-1.89); ≥5% (1.88; 1.30-2.71); and ≥10% (2.16; 1.20-3.88) for the highest versus lowest tertile. In alternative models using eGFRcr, the high tertile of KIM-1 had independent associations with 5% (1.71; 1.25-2.33) and 10% (1.78; 1.07-2.96) decline, and the high IL-18 tertile with 10% decline (1.97; 1.00-3.87).
Conclusions
Among HIV-infected women in the WIHS cohort, novel urine markers of kidney injury detect risk for subsequent declines in kidney function.
doi:10.1097/QAI.0b013e3182737706
PMCID: PMC3509242  PMID: 23023103
HIV; KIM-1; NGAL; IL-18; albumin-to-creatinine ratio; cystatin C; kidney injury
11.  Associations of HIV infection with insulin and glucose levels in antiretroviral-naïve Rwandan women: a cross-sectional analysis 
BMJ Open  2013;3(12):e003879.
Objectives
The purpose of these analyses was to determine the associations of HIV infection and related immune dysfunction with a glucose homeostasis in the population of antiretroviral-naïve HIV-infected and uninfected Rwandan women. We hypothesise that insulin resistance and its consequences in the developing countries may be further elevated with HIV infection itself regardless of antiretroviral therapy.
Study design
Cross-sectional analysis of a longitudinal cohort.
Setting
Community-based women's associations.
Participants
In 2005, 710 HIV-infected (HIV positive) antiretroviral naïve and 226 HIV-uninfected (HIV negative) women were enrolled in the Rwanda Women's Interassociation Study and Assessment (RWISA). Clinical and demographic parameters, CD4 count, fasting insulin and glucose levels, anthropometric measurements and Bioelectrical Impedance Analysis (BIA) were obtained. Linear models were fit to log-transformed Homeostasis Model Assessment (HOMA) with results exponentiated back to a multiplicative effect on the original scale.
Primary outcome measures
The outcome, insulin resistance, was measured by the HOMA, calculated as fasting insulin (μU/mL)×fasting glucose (mmol/L)⁄22.5.
Results
In adjusted models, HIV-positive women were less insulin resistant than HIV-negative; an HIV-positive woman tended to have 0.728 times as much (95% CI 0.681 to 0.861) HOMA than a comparable HIV-negative woman. Among the HIV-positive women, those with CD4 <200 cells/µL tended to have 0.741 times as much HOMA (95% CI 0.601 to 0.912) as did comparable women with CD4 >350 cells/µL. The older age was independently associated with a lower HOMA insulin resistance. After adjusting for body mass index, fat and fat-free mass were not independently associated with HOMA.
Conclusions
This study found that HIV infection and more advanced HIV infection (CD4 counts <200 cells/µL) were associated with greater insulin sensitivity in antiretroviral naïve African women. These findings provide baseline information for the interpretation of future studies on the effect of antiretroviral therapy on metabolic insulin sensitivity derangements in African population.
doi:10.1136/bmjopen-2013-003879
PMCID: PMC3855496  PMID: 24319275
Diabetes & Endocrinology; Epidemiology
12.  Recombination Between Variants from Genital Tract and Plasma: Evolution of Multidrug-Resistant HIV Type 1 
AIDS Research and Human Retroviruses  2012;28(12):1766-1774.
Abstract
Multidrug-resistant (MDR) HIV-1 presents a challenge to the efficacy of antiretroviral therapy (ART). To examine mechanisms leading to MDR variants in infected individuals, we studied recombination between single viral genomes from the genital tract and plasma of a woman initiating ART. We determined HIV-1 RNA sequences and drug resistance profiles of 159 unique viral variants obtained before ART and semiannually for 4 years thereafter. Soon after initiating zidovudine, lamivudine, and nevirapine, resistant variants and intrapatient HIV-1 recombinants were detected in both compartments; the recombinants had inherited genetic material from both genital and plasma-derived viruses. Twenty-three unique recombinants were documented during 4 years of therapy, comprising ∼22% of variants. Most recombinant genomes displayed similar breakpoints and clustered phylogenetically, suggesting evolution from common ancestors. Longitudinal analysis demonstrated that MDR recombinants were common and persistent, demonstrating that recombination, in addition to point mutation, can contribute to the evolution of MDR HIV-1 in viremic individuals.
doi:10.1089/aid.2011.0383
PMCID: PMC3505048  PMID: 22364185
13.  Association of subclinical atherosclerosis with lipid levels amongst antiretroviral-treated and untreated HIV-infected women in the Women’s Interagency HIV Study 
Atherosclerosis  2012;225(2):408-411.
Objective
We examined serum lipids in association with carotid artery intima-media thickness (CIMT) in HIV-infected and HIV-uninfected women.
Methods
In 2003–4, among 1827 Women’s Interagency HIV Study participants, we measured CIMT and lipids (high-density lipoprotein cholesterol [HDL-c], low-density lipoprotein cholesterol [LDL-c], total cholesterol [TC], non-HDL-c). A subset of 520 treated HIV-infected women had pre-1997 lipid measures. We used multivariable linear regression to examine associations between lipids and CIMT.
Results
In HIV-uninfected women, higher TC, LDL-c and non-HDL-c were associated with increased CIMT. Among HIV-infected women, associations of lipids with CIMT were observed in treated but not untreated women. Among the HIV-infected women treated in 2003–4, CIMT was associated both with lipids measured a decade earlier in infection, and with late lipid measurements.
Conclusion
Among HIV-infected women, hyperlipidemia is most strongly associated with subclinical atherosclerosis in treated women. Among treated women, the association appeared strongest early in the disease course.
doi:10.1016/j.atherosclerosis.2012.09.035
PMCID: PMC3696584  PMID: 23089369
cardiovascular diseases; carotid arteries; HAART; HIV; lipids
14.  The S40 residue in HIV-1 Gag p6 impacts local and distal budding determinants, revealing additional late domain activities 
Retrovirology  2013;10:143.
Background
HIV-1 budding is directed primarily by two motifs in Gag p6 designated as late domain-1 and −2 that recruit ESCRT machinery by binding Tsg101 and Alix, respectively, and by poorly characterized determinants in the capsid (CA) domain. Here, we report that a conserved Gag p6 residue, S40, impacts budding mediated by all of these determinants.
Results
Whereas budding normally results in formation of single spherical particles ~100 nm in diameter and containing a characteristic electron-dense conical core, the substitution of Phe for S40, a change that does not alter the amino acids encoded in the overlapping pol reading frame, resulted in defective CA-SP1 cleavage, formation of strings of tethered particles or filopodia-like membrane protrusions containing Gag, and diminished infectious particle formation. The S40F-mediated release defects were exacerbated when the viral-encoded protease (PR) was inactivated or when L domain-1 function was disrupted or when budding was almost completely obliterated by the disruption of both L domain-1 and −2. S40F mutation also resulted in stronger Gag-Alix interaction, as detected by yeast 2-hybrid assay. Reducing Alix binding by mutational disruption of contact residues restored single particle release, implicating the perturbed Gag-Alix interaction in the aberrant budding events. Interestingly, introduction of S40F partially rescued the negative effects on budding of CA NTD mutations EE75,76AA and P99A, which both prevent membrane curvature and therefore block budding at an early stage.
Conclusions
The results indicate that the S40 residue is a novel determinant of HIV-1 egress that is most likely involved in regulation of a critical assembly event required for budding in the Tsg101-, Alix-, Nedd4- and CA N-terminal domain affected pathways.
doi:10.1186/1742-4690-10-143
PMCID: PMC3907034  PMID: 24257210
HIV-1; Alix; Tsg101; Nedd4; CA NTD; Viral particle maturation; Protease; Viral budding
15.  A Single-Nucleotide Polymorphism in CYP2B6 Leads to >3-Fold Increases in Efavirenz Concentrations in Plasma and Hair Among HIV-Infected Women 
The Journal of Infectious Diseases  2012;206(9):1453-1461.
Background. Efavirenz exhibits marked interindividual variability in plasma levels and toxicities. Prior pharmacogenetic studies usually measure exposure via single plasma levels, examine limited numbers of polymorphisms, and rarely model multiple contributors. We analyzed numerous genetic and nongenetic factors impacting short-term and long-term exposure in a large heterogeneous population of human immunodeficiency virus (HIV)–infected women.
Methods. We performed 24-hour intensive pharmacokinetic studies in 111 women receiving efavirenz under actual-use conditions and calculated the area-under-the-concentration-time curve (AUC) to assess short-term exposure; the efavirenz concentration in hair was measured to estimate long-term exposure. A total of 182 single-nucleotide polymorphisms (SNPs) and 45 haplotypes in 9 genes were analyzed in relationship to exposure by use of multivariate models that included a number of nongenetic factors.
Results. Efavirenz AUCs increased 1.26-fold per doubling of the alanine aminotransferase level and 1.23-fold with orange and/or orange juice consumption. Individuals with the CYP2B6 516TT genotype displayed 3.5-fold increases in AUCs and 3.2-fold increases in hair concentrations, compared with individuals with the TG/GG genotype. Another SNP in CYP2B6 (983TT) and a p-glycoprotein haplotype affected AUCs without substantially altering long-term exposure.
Conclusions. This comprehensive pharmacogenomics study showed that individuals with the CYP2B6 516TT genotype displayed >3-fold increases in both short-term and long-term efavirenz exposure, signifying durable effects. Pharmacogenetic testing combined with monitoring of hair levels may improve efavirenz outcomes and reduce toxicities.
doi:10.1093/infdis/jis508
PMCID: PMC3466997  PMID: 22927450
16.  Depressive Symptoms are Increased in the Early Perimenopausal Stage in Ethnically Diverse HIV+ and HIV− Women 
Menopause (New York, N.Y.)  2012;19(11):1215-1223.
Objective
The risk of clinically significant depressive symptoms increases during the perimenopause. With highly active antiretroviral treatment (HAART), more HIV-infected women survive to transition through the menopause. In a cross-sectional analysis, we evaluated the association of menopausal stage and vasomotor symptoms with depressive symptoms in an ethnically diverse, cohort of women with a high prevalence of HIV.
Methods
Participants included 835 HIV-infected women and 335 HIV-uninfected controls from the Women’s Interagency HIV Study (WIHS; 63% African-American). The Center for Epidemiological Studies Depression (CES-D) scale was used to screen for elevated depressive symptoms. Menopausal stages were defined according to standard definitions. Logistic regression analysis was used to identify predictors of elevated depressive symptoms.
Results
Compared to premenopausal women, early perimenopausal (OR 1.74, 95%CI 1.17–2.60), but not late perimenopausal or postmenopausal women were more likely to show elevated depressive symptoms in adjusted analyses. The odds were similar in HIV-infected and HIV-uninfected women. Persistent vasomotor symptoms also predicted elevated depressive symptoms in HIV-infected and uninfected women (OR 1.45, 95%CI 1.02–2.06). In HIV-infected women, menopausal stage interacted with antiretroviral use (p=0.02); the likelihood of elevated depressive symptoms in early perimenopause compared with premenopause was especially high in HAART-untreated women (OR 3.87, 95%CI 1.57–9.55).
Conclusions
In HIV+ and HIV− women, the odds of elevated depressive symptoms were significantly higher during the early perimenopause. Elevated depressive symptoms were associated with nonadherence to HAART, underscoring the importance of screening and treating depressive symptoms in HIV+ women who have experienced a change in the regularity of their menstrual cycles.
doi:10.1097/gme.0b013e318255434d
PMCID: PMC3483358  PMID: 22872013
HIV; Depression; Menopause; Perimenopause; African American; Vasomotor
17.  Tuberculosis in Antiretroviral Treatment Programs in Lower Income Countries: Availability and Use of Diagnostics and Screening 
PLoS ONE  2013;8(10):e77697.
Objectives
In resource-constrained settings, tuberculosis (TB) is a common opportunistic infection and cause of death in HIV-infected persons. TB may be present at the start of antiretroviral therapy (ART), but it is often under-diagnosed. We describe approaches to TB diagnosis and screening of TB in ART programs in low- and middle-income countries.
Methods and findings
We surveyed ART programs treating HIV-infected adults in sub-Saharan Africa, Asia and Latin America in 2012 using online questionnaires to collect program-level and patient-level data. Forty-seven sites from 26 countries participated. Patient-level data were collected on 987 adult TB patients from 40 sites (median age 34.7 years; 54% female). Sputum smear microscopy and chest radiograph were available in 47 (100%) sites, TB culture in 44 (94%), and Xpert MTB/RIF in 23 (49%). Xpert MTB/RIF was rarely available in Central Africa and South America. In sites with access to these diagnostics, microscopy was used in 745 (76%) patients diagnosed with TB, culture in 220 (24%), and chest X-ray in 688 (70%) patients. When free of charge culture was done in 27% of patients, compared to 21% when there was a fee (p = 0.033). Corresponding percentages for Xpert MTB/RIF were 26% and 15% of patients (p = 0.001). Screening practices for active disease before starting ART included symptom screening (46 sites, 98%), chest X-ray (38, 81%), sputum microscopy (37, 79%), culture (16, 34%), and Xpert MTB/RIF (5, 11%).
Conclusions
Mycobacterial culture was infrequently used despite its availability at most sites, while Xpert MTB/RIF was not generally available. Use of available diagnostics was higher when offered free of charge.
doi:10.1371/journal.pone.0077697
PMCID: PMC3798412  PMID: 24147059
18.  A novel denaturing heteroduplex tracking assay for genotypic prediction of HIV-1 tropism 
Journal of virological methods  2012;185(1):108-117.
Human immunodeficiency virus type 1 (HIV-1) is characterized by sequence variability. The third variable region (V3) of the HIV-1 envelope glycoprotein gp120 plays a key role in determination of viral coreceptor usage (tropism) and pathogenesis. This report describes a novel denaturing heteroduplex tracking assay (HTA) to analyze the genetic variation of HIV-1 V3 DNA. It improved upon previous non-denaturing HTA approaches to distinguish HIV-1 CCR5 and CXCR4 tropic viruses in mixed populations. The modifications included the use of a single-stranded fluorescent probe based on the consensus V3 sequence of HIV-1 CCR5 tropic viruses, Locked Nucleic Acid (LNA) “clamps” at both ends of heteroduplex DNA, and denaturing gel electrophoresis using Mutation Detection Enhancement (MDE®) as matrix. The analysis demonstrated that the LNA “clamps” increased its melting temperature (Tm) and the thermal stability of heteroduplex DNA. The partially denaturing gel used a defined concentration of formamide, and significantly induced mobility shifts of heteroduplex DNA that was dependent on the number and patterns of DNA mismatches and insertions/deletions. This new technique successfully detected tropisms of 53 HIV-1 V3 clones of known tropism, and was able to separate and detect multiple V3 DNA variants encoding tropisms for CCR5 or CXCR4 in a mixture. The assay had the sensitivity to detect 0.5% minority species. This method may be useful as a research tool for analysis of viral quasispecies and for genotypic prediction of HIV-1 tropism in clinical specimens.
doi:10.1016/j.jviromet.2012.06.013
PMCID: PMC3417067  PMID: 22728273
HIV-1 gp120 V3; denaturing heteroduplex tracking assay; Locked Nucleic Acid clamps; viral tropism
19.  Cervicovaginal HPV Infection Before and After Hysterectomy: Evidence of Different Tissue Tropism for Oncogenic and Non-Oncogenic HPV Types in a Cohort of HIV-positive and HIV-negative Women 
Human papillomavirus (HPV) is detected in nearly all cervical cancers and approximately half of vaginal cancers. However, vaginal cancer is an order of magnitude less common than cervical cancer, not only in the general population but also among women with HIV/AIDS. It is interesting therefore that recent studies found that HPV was common in both normal vaginal and cervical tissue, with higher prevalence of non-oncogenic HPV types in the vagina. In the current investigation, we prospectively examined HPV infection in 86 HIV-positive and 17 HIV-negative women who underwent hysterectomy during follow-up in a longitudinal cohort. Cervicovaginal lavage specimens were obtained semi-annually and tested for HPV DNA by PCR. To address possible selection biases associated with having a hysterectomy, subjects acted as their own comparison group – before versus after hysterectomy. The average HPV prevalence was higher in HIV-positive than HIV-negative women both before (59% versus 12%; P<0.001) and after hysterectomy (56% versus 6%; P<0.001). Multivariate random effects models (within-individual comparisons) demonstrated significantly lower HPV prevalence (odds ratio [OR]=0.71; 95% confidence interval [CI]=0.59-0.85) after hysterectomy. The association of HPV prevalence with hysterectomy was similar among HIV-positive and HIV-negative women. However, hysterectomy had greater effects on oncogenic (OR=0.48; 95%CI=0.35-0.66) than non-oncogenic HPV types (OR=0.89; 95%CI=0.71-1.11; Pinteraction=0.002). Overall, we observed greater reductions in oncogenic than non-oncogenic HPV prevalence following hysterectomy. If correct, these data could suggest that oncogenic HPV have greater tropism for cervical compared with vaginal epithelium, consistent with the lower incidence of vaginal than cervical cancer.
doi:10.1002/ijc.27363
PMCID: PMC3321069  PMID: 22120980
vaginal; HPV; hysterectomy; viral tropism; HIV
20.  Interleukin 10 Responses Are Associated With Sustained CD4 T-Cell Counts in Treated HIV Infection 
The Journal of Infectious Diseases  2012;206(5):780-789.
Background.Inflammation persists in treated human immunodeficiency virus (HIV) infection and may contribute to an increased risk for non–AIDS-related pathologies. We investigated the correlation of cytokine responses with changes in CD4 T-cell levels and coinfection with hepatitis C virus (HCV) during highly active antiretroviral treatment (HAART).
Methods.A total of 383 participants in the Women's Interagency HIV Study (212 with HIV monoinfection, 56 with HCV monoinfection, and 115 with HIV/HCV coinfection) were studied. HIV-infected women had <1000 HIV RNA copies/mL, 99.7% had >200 CD4 T cells/μL; 98% were receiving HAART at baseline. Changes in CD4 T-cell count between baseline and 2–4 years later were calculated. Peripheral blood mononuclear cells (PBMCs) obtained at baseline were used to measure interleukin 1β (IL-1β), interleukin 6 (IL-6), interleukin 10 (IL-10), interleukin 12 (IL-12), and tumor necrosis factor α (TNF-α) responses to Toll-like receptor (TLR) 3 and TLR4 stimulation.
Results.Undetectable HIV RNA (<80 copies/mL) at baseline and secretion of IL-10 by PBMCs were positively associated with gains in CD4 T-cell counts at follow-up. Inflammatory cytokines (IL-1β, IL-6, IL-12, and TNF-α) were also produced in TLR-stimulated cultures, but only IL-10 was significantly associated with sustained increases in CD4 T-cell levels. This association was significant only in women with HIV monoinfection, indicating that HCV coinfection is an important factor limiting gains in CD4 T-cell counts, possibly by contributing to unbalanced persistent inflammation.
Conclusions.Secreted IL-10 from PBMCs may balance the inflammatory environment of HIV, resulting in CD4 T-cell stability.
doi:10.1093/infdis/jis380
PMCID: PMC3491747  PMID: 22693231
21.  Evolution and Taxonomic Classification of Alphapapillomavirus 7 Complete Genomes: HPV18, HPV39, HPV45, HPV59, HPV68 and HPV70 
PLoS ONE  2013;8(8):e72565.
Background
The species Alphapapillomavirus 7 (alpha-7) contains human papillomavirus genotypes that account for 15% of invasive cervical cancers and are disproportionately associated with adenocarcinoma of the cervix. Complete genome analyses enable identification and nomenclature of variant lineages and sublineages.
Methods
The URR/E6 region was sequenced to screen for novel variants of HPV18, 39, 45, 59, 68, 70, 85 and 97 from 1147 cervical samples obtained from multiple geographic regions that had previously been shown to contain an alpha-7 HPV isolate. To study viral heterogeneity, the complete 8 kb genome of 128 isolates, including 109 sequenced for this analysis, were annotated and analyzed. Viral evolution was characterized by constructing phylogenic trees using maximum-likelihood and Bayesian algorithms. Global and pairwise alignments were used to calculate total and ORF/region nucleotide differences; lineages and sublineages were assigned using an alphanumeric system. The prototype genome was assigned to the A lineage or A1 sublineage.
Results
The genomic diversity of alpha-7 HPV types ranged from 1.1% to 6.7% nucleotide sequence differences; the extent of genome-genome pairwise intratype heterogeneity was 1.1% for HPV39, 1.3% for HPV59, 1.5% for HPV45, 1.6% for HPV70, 2.1% for HPV18, and 6.7% for HPV68. ME180 (previously a subtype of HPV68) was designated as the representative genome for HPV68 sublineage C1. Each ORF/region differed in sequence diversity, from most variable to least variable: noncoding region 1 (NCR1) / noncoding region 2 (NCR2) > upstream regulatory region (URR) > E6 / E7 > E2 / L2 > E1 / L1.
Conclusions
These data provide estimates of the maximum viral genomic heterogeneity of alpha-7 HPV type variants. The proposed taxonomic system facilitates the comparison of variants across epidemiological and molecular studies. Sequence diversity, geographic distribution and phylogenetic topology of this clinically important group of HPVs suggest an independent evolutionary history for each type.
doi:10.1371/journal.pone.0072565
PMCID: PMC3745470  PMID: 23977318
22.  Detectability and reproducibility of plasma levels of chemokines and soluble receptors☆ 
Results in Immunology  2013;3:79-84.
Background: Multiplex assays are available to measure an array of circulating chemokines, soluble cytokine receptors and growth factors. However, there is limited information regarding whether these analytes are suitable for large-scale epidemiological studies to assess their relationships with chronic diseases, including cancer.
Methods: We examined detectability, assay repeatability, and 3-year within-subject reproducibility of plasma levels of 25 chemokines and 11 soluble receptors of cytokines and growth factors selected from the Human Millipore Panels. Plasma samples were obtained from 36 men (average age 62 years) and 17 women (average age 32 years) who participated in two epidemiological studies. Inter-assay and within-subject reproducibility were assessed by intraclass correlation coefficients (ICC).
Results: All analytes, except lymphotactin (47% detectability), were detectable in >90% of plasma samples. Inter-assay reproducibility for all analytes in 36 men tested three times on separate days were good to excellent (ICCs: 0.71–1.00). Within-subject reproducibility in 17 women sampled three times in three years were excellent (ICC ≥ 0.75) for five chemokines (eotaxin, fractalkine, 6Ckine, eotaxin 3, and SDF-1α+β) and three soluble receptors (sIL-1R2, sIL-4R and sVEGFR2); ICCs were fair to good (0.4 ≤ ICC < 0.75) for 15 chemokines and eight soluble receptors. However, five chemokines (GRO, IP-10, MIP-1β, BCA-1, and MIP-3α) had ICC < 0.4, suggesting biological variability.
Conclusion: Multiplex assays for plasma levels of selected chemokines and soluble receptors showed good to excellent assay detectability and repeatability. Most analytes also had good 3-year within-subject reproducibility, indicating that a single measurement of these analytes may be used to assess biomarker-disease associations.
doi:10.1016/j.rinim.2013.07.001
PMCID: PMC3908322  PMID: 24600562
Chemokines; Soluble receptors; Within-subject variability; Biomarker; Limit of detection
23.  Potential cardiovascular disease risk markers among HIV-infected women initiating antiretroviral treatment 
Background
Inflammation and hemostasis perturbation may be involved in vascular complications of HIV infection. We examined atherogenic biomarkers and subclinical atherosclerosis in HIV-infected adults before and after beginning highly-active antiretroviral therapy (HAART).
Methods
In the Women's Interagency HIV Study (WIHS), 127 HIV-infected women studied pre- and post-HAART were matched to HIV-uninfected controls. Six semi-annual measurements of soluble CD14, tumor necrosis factor (TNF)-alpha, soluble interleukin (IL)-2 receptor, IL-6, IL-10, monocyte chemoattractant protein (MCP)-1, D-dimer, and fibrinogen were obtained. Carotid artery intima-media thickness (CIMT) was measured by B-mode ultrasound.
Results
Relative to HIV-uninfected controls, HAART-naïve HIV-infected women had elevated levels of soluble CD14 (1945 vs 1662 ng/mL, Wilcoxon signed rank P<0.0001), TNF-alpha (6.3 vs 3.4 pg/mL, P<0.0001), soluble IL-2 receptor (1587 vs 949 pg/mL, P<0.0001), IL-10 (3.3 vs 1.9 pg/mL, P<0.0001), MCP-1 (190 vs 163 pg/mL, P<0.0001) and D-dimer (0.43 vs 0.31 µg/mL, P<0.01). Elevated biomarker levels declined after HAART. While most biomarkers normalized to HIV-uninfected levels, in women on effective HAART, TNF-alpha levels remained elevated compared to HIV-uninfected women (+0.8 pg/mL, P=0.0002). Higher post-HAART levels of soluble IL-2 receptor (P=0.02), IL-6 (P=0.05), and D-dimer (P=0.03) were associated with increased CIMT.
Conclusions
Untreated HIV infection is associated with abnormal hemostasis (e.g., D-dimer), and pro-atherogenic (e.g., TNF-alpha) and anti-atherogenic (e.g., IL-10) inflammatory markers. HAART reduces most inflammatory mediators to HIV-uninfected levels. Increased inflammation and hemostasis are associated with subclinical atherosclerosis in recently treated women. These findings have potential implications for long-term risk of cardiovascular disease in HIV-infected patients, even with effective therapy.
doi:10.1097/QAI.0b013e31825b03be
PMCID: PMC3400505  PMID: 22592585
antiretroviral therapy; cardiovascular diseases; cytokines; hemostasis; HIV; inflammation
24.  Circulating Vitamin D Correlates with Serum Anti-Mullerian Hormone Levels in Late Reproductive-Aged Women: Women’s Interagency HIV Study 
Fertility and Sterility  2012;98(1):228-234.
Objective
To study the correlation between circulating 25 hydroxy-vitamin D (25OH-D) levels and serum AMH in women enrolled in the Women’s Interagency HIV Study (WIHS).
Design
A cross-sectional study.
Setting
WIHS, a multicenter prospective study.
Patient(s)
All premenopausal women (n=388) with regular menstrual cycles were included and subdivided into three groups: group 1 with age <35 (N=128), group 2 with age 35 to 39 (N=119), and group 3 with age ≥ 40 (N=141).
Intervention(s)
Serum for 25OH-D, AMH, fasting glucose and insulin, and creatinine levels.
Main Outcome Measure(s)
Correlation between 25OH-D and AMH before and after adjusting for HIV status, BMI, race, smoking, illicit drug use, glucose and insulin levels, estimated glomerular filtration rate and geographic site of participation.
Result(s)
After adjusting for all covariates, the regression slope in all participants for total 25OH-D predicting log10AMH for 25-year-olds (youngest participant) was −0.001 (SE=0.008, p=0.847); and for 45-year-olds (oldest participant), the corresponding slope was +0.011 (SE=0.005, p=0.021). Fasting insulin level was negatively correlated with serum AMH (p=0.016). The regression slope for the correlation between 25OH-D and AMH in group 1 was +0.002 (SE=0.006, p=0.764); in group 2 was +0.006 (SE=0.005, p=0.269); and in group 3 was +0.011 (SE=0.005, p=0.022). There was no association between HIV and AMH.
Conclusion(s)
A novel relationship is reported between circulating 25OH-D and AMH in women aged = 40 suggesting that 25OH-D deficiency might be associated with lower ovarian reserve in late reproductive-aged women.
doi:10.1016/j.fertnstert.2012.03.029
PMCID: PMC3389125  PMID: 22494925
Vitamin D; anti-mullerian hormonem mullerian inhibiting substance; HIV; ovarian reserve; insulin resistance; obesity
25.  Insulin Resistance and Cognition Among HIV-Infected and HIV-Uninfected Adult Women: The Women's Interagency HIV Study 
Abstract
Cognitive impairment remains prevalent in the era of combination antiretroviral therapy (cART) and may be partially due to comorbidities. We postulated that insulin resistance (IR) is negatively associated with cognitive performance. We completed a cross-sectional analysis among 1547 (1201 HIV+) women enrolled in the Women's Interagency HIV Study (WIHS). We evaluated the association of IR with cognitive measures among all WIHS women with concurrent fasting bloods and cognitive testing [Trails A, Trails B, and Symbol Digit Modalities Test (SDMT)] using multiple linear regression models. A smaller subgroup also completed the Stroop test (n=1036). IR was estimated using the Homeostasis Model Assessment (HOMA). Higher HOMA was associated with poorer performance on the SDMT, Stroop Color-Naming (SCN) trial, and Stroop interference trial, but remained statistically significant only for the SCN in models adjusting for important factors [β=3.78 s (95% CI: 0.48–7.08), p=0.025, for highest vs. lowest quartile of HOMA]. HIV status did not appear to substantially impact the relationship of HOMA with SCN. There was a small but statistically significant association of HOMA and reduced neuropsychological performance on the SCN test in this cohort of women.
doi:10.1089/aid.2011.0159
PMCID: PMC3332367  PMID: 21878059

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