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Find video protocols related to scientific articles indexed in Pubmed.
Single-agent tenofovir versus combination emtricitabine plus tenofovir for pre-exposure prophylaxis for HIV-1 acquisition: an update of data from a randomised, double-blind, phase 3 trial.
Lancet Infect Dis
PUBLISHED: 10-11-2014
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Antiretroviral pre-exposure prophylaxis (PrEP), with daily oral tenofovir disoproxil fumarate or tenofovir disoproxil fumarate in combination with emtricitabine, has been shown to be efficacious for HIV-1 prevention. Although the use of more than one antiretroviral agent is essential for effective HIV-1 treatment, more than one agent might not be required for effective prophylaxis. We assessed the efficacy of single-agent tenofovir disoproxil fumarate relative to combination emtricitabine plus tenofovir disoproxil fumarate as PrEP.
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Oligonucleotide Ligation Assay Detects HIV Drug Resistance Associated With Virologic Failure Among Antiretroviral-Naive Adults in Kenya.
J. Acquir. Immune Defic. Syndr.
PUBLISHED: 08-21-2014
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Transmitted drug resistance (TDR) is increasing in some areas of Africa. Detection of TDR may predict virologic failure of first-line nonnucleoside reverse transcriptase inhibitor (NNRTI)-based antiretroviral therapy (ART). We evaluated the utility of a relatively inexpensive oligonucleotide ligation assay (OLA) to detect clinically relevant TDR at the time of ART initiation.
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HIV latency. Proliferation of cells with HIV integrated into cancer genes contributes to persistent infection.
Science
PUBLISHED: 07-10-2014
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Antiretroviral treatment (ART) of HIV infection suppresses viral replication. Yet if ART is stopped, virus reemerges because of the persistence of infected cells. We evaluated the contribution of infected-cell proliferation and sites of proviral integration to HIV persistence. A total of 534 HIV integration sites (IS) and 63 adjacent HIV env sequences were derived from three study participants over 11.3 to 12.7 years of ART. Each participant had identical viral sequences integrated at the same position in multiple cells, demonstrating infected-cell proliferation. Integrations were overrepresented in genes associated with cancer and favored in 12 genes across multiple participants. Over time on ART, a greater proportion of persisting proviruses were in proliferating cells. HIV integration into specific genes may promote proliferation of HIV-infected cells, slowing viral decay during ART.
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Validation of an oligonucleotide ligation assay for quantification of human immunodeficiency virus type 1 drug-resistant mutants by use of massively parallel sequencing.
J. Clin. Microbiol.
PUBLISHED: 04-16-2014
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Global HIV treatment programs need sensitive and affordable tests to monitor HIV drug resistance. We compared mutant detection by the oligonucleotide ligation assay (OLA), an economical and simple test, to massively parallel sequencing. Nonnucleoside reverse transcriptase inhibitor (K103N, V106M, Y181C, and G190A) and lamivudine (M184V) resistance mutations were quantified in blood-derived plasma RNA and cell DNA specimens by OLA and 454 pyrosequencing. A median of 1,000 HIV DNA or RNA templates (range, 163 to 1,874 templates) from blood specimens collected in Mozambique (n = 60) and Kenya (n = 51) were analyzed at 4 codons in each sample (n = 441 codons assessed). Mutations were detected at 75 (17%) codons by OLA sensitive to 2.0%, at 71 codons (16%; P = 0.78) by pyrosequencing using a cutoff value of ? 2.0%, and at 125 codons (28%; P < 0.0001) by pyrosequencing sensitive to 0.1%. Discrepancies between the assays included 15 codons with mutant concentrations of ?2%, one at 8.8% by pyrosequencing and not detected by OLA, and one at 69% by OLA and not detected by pyrosequencing. The latter two cases were associated with genetic polymorphisms in the regions critical for ligation of the OLA probes and pyrosequencing primers, respectively. Overall, mutant concentrations quantified by the two methods correlated well across the codons tested (R(2) > 0.8). Repeat pyrosequencing of 13 specimens showed reproducible detection of 5/24 mutations at <2% and 6/6 at ? 2%. In conclusion, the OLA and pyrosequencing performed similarly in the quantification of nonnucleoside reverse transcriptase inhibitor and lamivudine mutations present at >2% of the viral population in clinical specimens. While pyrosequencing was more sensitive, detection of mutants below 2% was not reproducible.
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Lack of resistance to integrase inhibitors among antiretroviral-naive subjects with primary HIV-1 infection, 2007-2013.
Antivir. Ther. (Lond.)
PUBLISHED: 04-06-2014
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U.S. guidelines recommend genotyping for persons newly diagnosed with HIV infection to identify transmitted drug resistance mutations associated with decreased susceptibility to NRTIs, NNRTIs, and PIs. To date, testing for integrase strand transfer inhibitor (INSTI) mutations has not been routinely recommended. We aimed to evaluate the prevalence of transmitted INSTI mutations among persons with primary HIV-1 infection in Seattle, WA.
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Correlation between plasma, intracellular, and cervical tissue levels of raltegravir at steady-state dosing in healthy women.
Antimicrob. Agents Chemother.
PUBLISHED: 03-31-2014
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Raltegravir is an antiretroviral with potential value for preexposure prophylaxis (PrEP) against HIV, but the intracellular pharmacokinetics in genital tissue have not been described. In this study, healthy, HIV-uninfected nonpregnant women took 400 mg of raltegravir twice daily for 22 days. On day 8, 15, and 22, blood was collected 0, 4, 6, 8, and 12 h and cervical biopsy specimens taken 0, 6, and 12 h after raltegravir dosing. Plasma and intracellular raltegravir concentrations in peripheral blood mononuclear cells (PBMC) and cervical tissue were measured by tandem mass spectrometry. Linear mixed effects models evaluated correlations between different sample types, as well as differences in concentration between phases of the menstrual cycle. Ten women were enrolled: 9 completed all three visits and 1 completed two visits. The age (mean ± standard deviation) of participants was 30 ± 8 years. Trough plasma concentrations of raltegravir 12 h after a directly observed dose were above the HIV 95% inhibitory concentration (IC95) of 33 nM (14.6 ng/ml) in 96% of measurements, compared to 67% of PBMC and 89% of cervical tissue trough values. Across all measurements, only 2% (3/135) of plasma values fell below the IC95, compared to 10% (13/135) for PBMC and 6% (5/81) for cervical tissue. There was no impact of menstrual phase on raltegravir concentrations. In conclusion, cervical tissue raltegravir concentrations were no greater than plasma concentrations, and ?10% of all cervical tissue trough values were below the IC95, making the current twice-daily formulation of raltegravir impractical for PrEP.
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Transmission of nevirapine-resistant HIV type 1 via breast milk to infants after single-dose nevirapine in Beira, Mozambique.
J. Infect. Dis.
PUBLISHED: 03-04-2014
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Acquisition of nevirapine (NVP)-resistant human immunodeficiency virus type 1 (HIV-1) by breast-feeding infants after receipt of single-dose NVP to prevent mother-to-child transmission is not well defined. A prospective observational study of 307 infants evaluated the rate of breast milk transmission of NVP-resistant HIV and the concentrations of mutants over time. NVP resistance was detected in 9 of 24 infants (37.5%; 95% confidence interval, 18.8%-59.4%) infected via breast milk. Eight had a pure mutant HIV population at the time infection was first detected, and majority mutant populations persisted in all 6 infants with follow-up specimens. Infection of breast-feeding infants with NVP-resistant HIV resulted in mutants persisting as the dominant virus, which may indefinitely compromise treatment with NVP-based antiretroviral regimens.
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Low concentrations of HIV-1 DNA at birth delays diagnosis, complicating identification of infants for antiretroviral therapy to potentially prevent the establishment of viral reservoirs.
Clin. Infect. Dis.
PUBLISHED: 02-05-2014
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Among infants exposed to human immunodeficiency virus type 1 (HIV-1), detection of viral infection at birth was increased by 39% (95% confidence interval, 19%-47%) by increasing DNA input from dried blood spots into polymerase chain reaction. Infants with low concentrations of HIV-1 at birth may be the best target population to evaluate whether immediate antiretroviral therapy can prevent long-term infection.
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HIV-1 RNA levels and antiretroviral drug resistance in blood and non-blood compartments from HIV-1-infected men and women enrolled in AIDS clinical trials group study A5077.
PLoS ONE
PUBLISHED: 01-01-2014
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Detectable HIV-1 in body compartments can lead to transmission and antiretroviral resistance. Although sex differences in viral shedding have been demonstrated, mechanisms and magnitude are unclear. We compared RNA levels in blood, genital-secretions and saliva; and drug resistance in plasma and genital-secretions of men and women starting/changing antiretroviral therapy (ART) in the AIDS Clinical Trials Group (ACTG) 5077 study.
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Pharmacokinetics, Safety, and 48-Week Efficacy of Oral Raltegravir in HIV-1-Infected Children Aged 2 Through 18 Years.
Clin. Infect. Dis.
PUBLISHED: 10-21-2013
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Background.?IMPAACT P1066 is a phase I/II open-label multicenter trial to evaluate pharmacokinetics, safety, tolerability, and efficacy of multiple raltegravir formulations in human immunodeficiency virus (HIV)-infected youth. Methods.?Dose selection for each cohort (I: 12 to <19 years; II: 6 to <12 years; and III: 2 to <6 years) was based on review of short-term safety (4 weeks) and intensive pharmacokinetic evaluation. Safety data through weeks 24 and 48, and grade ?3 or serious adverse events (AEs) were assessed. The primary virologic endpoint was achieving HIV RNA <400 copies/mL or ?1 log10 reduction between baseline and week 24. Results.?The targeted pharmacokinetic parameters (AUC0-12h and C12h) were achieved for each cohort, allowing dose selection for 2 formulations. Of 96 final dose subjects, there were 15 subjects with grade 3 or higher clinical AEs (1 subject with drug-related [DR] psychomotor hyperactivity and insomnia); 16 subjects with grade 3 or higher laboratory AEs (1 with DR transaminase elevation); 14 subjects with serious clinical AEs (1 with DR rash); and 1 subjects with serious laboratory AEs (1 with DR transaminase increased). There were no discontinuations due to AEs and no DR deaths. Favorable virologic responses at week 48 were observed in 79.1% of patients, with a mean CD4 increase of 156 cells/µL (4.6%). Conclusions.?Raltegravir as a film-coated tablet 400 mg twice daily (6 to <19 years, and ?25 kg) and chewable tablet 6 mg/kg (maximum dose 300 mg) twice daily (2 to <12 years) was well tolerated and showed favorable virologic and immunologic responses. Clinical Trials Registration.?NCT00485264.
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Simultaneous and sensitive detection of human immunodeficiency virus type 1 (HIV) drug resistant genotypes by multiplex oligonucleotide ligation assay.
J. Virol. Methods
PUBLISHED: 05-06-2013
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Oligonucleotide ligation assay (OLA) is a highly specific and relatively simple method to detect point mutations encoding HIV-1 drug-resistance, which can detect mutants comprising ?2-5% of the viral population. Nevirapine (NVP), tenofovir (TDF) and lamivudine (3TC) are antiretroviral (ARV) drugs used worldwide for treatment of HIV infection and prevention of mother-to-child-transmission. Adapting the OLA to detect multiple mutations associated with HIV resistance to these ARV simultaneously would provide an efficient tool to monitor drug resistance in resource-limited settings. Known proportions of mutant and wild-type plasmids were used to optimize a multiplex OLA for detection of K103N, Y181C, K65R, and M184V in HIV subtypes B and C, and V106M and G190A in subtype C. Simultaneous detection of two mutations was impaired if probes annealed to overlapping regions of the viral template, but was sensitive to ?2-5% when testing codons using non-overlapping probes. PCR products from HIV-subtype B- and C-infected individuals were tested by multiplex-OLA and compared to results of single-codon OLA. Multiplex-OLA detected mutations at codon pairs 103/181, 106/190 and 65/184 reliably when compared to singleplex-OLA in clinical specimens. The multiplex-OLA is sensitive and specific and reduces the cost of screening for NVP, TDF and/or 3TC resistance.
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Associations between genital tract infections, genital tract inflammation, and cervical cytobrush HIV-1 DNA in US versus Kenyan women.
J. Acquir. Immune Defic. Syndr.
PUBLISHED: 03-09-2013
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Cervical shedding of HIV-1 DNA may influence HIV-1 sexual transmission. HIV-1 DNA was detected in 250 (80%) of 316 and 207 (79%) of 259 cervical cytobrush specimens from 56 US and 80 Kenyan women, respectively. Plasma HIV-1 RNA concentration was associated with increased HIV-1 DNA shedding among US and Kenyan women. Kenyan women had higher cervicovaginal concentrations of proinflammatory interleukins (IL)-1?, IL-6, IL-8, and anti-inflammatory secretory leukocyte protease inhibitor compared with US women (all P < 0.01). HIV-1 DNA shedding was associated with increased concentrations of IL-1? and IL-6 and lower secretory leukocyte protease inhibitor among US women but not Kenyan women.
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Development of a novel codon-specific polymerase chain reaction for the detection of CXCR4-utilizing HIV type 1 subtype B.
AIDS Res. Hum. Retroviruses
PUBLISHED: 03-06-2013
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Insight concerning the switch in HIV-1 coreceptor use will lead to a better understanding of HIV-1 pathogenesis and host-virus dynamics. Predicting CXCR4 utilization by analyzing HIV-1 envelope consensus sequences is highly specific, but minority variants in the viral population are often missed resulting in low sensitivity. Commercial phenotypic assays are costly, and the development of sensitive in-house phenotypic assays to detect CXCR4-using HIV may not be feasible for some laboratories. A sensitive, inexpensive genotyping assay was developed to detect viral sequences associated with CXCR4-utilizing virus (X4). Codon-specific primer pairs were used to detect X4-associated codons at five positions in the HIV-1 envelope V3 loop (11, 13, 24, 25, and 32). Sixty plasma samples from HIV-1-infected individuals were analyzed by consensus sequencing and codon-specific PCR (CS-PCR). Forty-six of these were also phenotyped by Trofile or Enhanced Sensitivity Trofile (ESTA). CS-PCR detected X4 variants 17% more often than 11/24/25 consensus sequencing alone (n=60), 30% more often than Trofile (n=27), and in a limited data set, 16% more often than ESTA (n=19). CS-PCR combined with consensus sequencing had approximately 80% concordance with ESTA.
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HIV drug resistance in mothers and infants following use of antiretrovirals to prevent mother-to-child transmission.
Curr. HIV Res.
PUBLISHED: 02-13-2013
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The purpose of this article is to review prominent studies on HIV drug-resistance in mothers and their infants after the use of antiretroviral drugs to prevent mother-to-child-transmission in resource-limited communities. The effects of drug-resistance on subsequent combination antiretroviral therapy are discussed, as are the probable mechanisms of acquisition and decay or persistence of drug-resistant mutants. Differences in the rates of HIV drug-resistance from interventions used to prevent mother-to-child-transmission in North America and Europe are contrasted to the simplified regimens used in resource-limited settings. Unresolved issues related to HIV drug-resistance are reviewed, including: whether maternal zidovudine monotherapy selects significant resistance; the clinical relevance of HIV drug-resistant variants selected by single-dose nevirapine that persist as minority viral variants and can affect the outcome of non-nucleoside reverse transcriptase inhibitor-based therapy; and the use of maternal combination antiretroviral therapy during breastfeeding. Finally, the current and upcoming strategies to reduce HIV drug-resistance related to use of antiretrovirals to prevent mother-to-child-transmission are discussed and contrasted with the challenges of financing and administering antiretrovirals to prevent mother-to-child-transmission in resource-limited communities.
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Human immunodeficiency viruses appear compartmentalized to the female genital tract in cross-sectional analyses but genital lineages do not persist over time.
J. Infect. Dis.
PUBLISHED: 01-11-2013
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Whether unique human immunodeficiency type 1 (HIV) genotypes occur in the genital tract is important for vaccine development and management of drug resistant viruses. Multiple cross-sectional studies suggest HIV is compartmentalized within the female genital tract. We hypothesize that bursts of HIV replication and/or proliferation of infected cells captured in cross-sectional analyses drive compartmentalization but over time genital-specific viral lineages do not form; rather viruses mix between genital tract and blood.
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Improved detection of rare HIV-1 variants using 454 pyrosequencing.
PLoS ONE
PUBLISHED: 01-01-2013
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454 pyrosequencing, a massively parallel sequencing (MPS) technology, is often used to study HIV genetic variation. However, the substantial mismatch error rate of the PCR required to prepare HIV-containing samples for pyrosequencing has limited the detection of rare variants within viral populations to those present above ~1%. To improve detection of rare variants, we varied PCR enzymes and conditions to identify those that combined high sensitivity with a low error rate. Substitution errors were found to vary up to 3-fold between the different enzymes tested. The sensitivity of each enzyme, which impacts the number of templates amplified for pyrosequencing, was shown to vary, although not consistently across genes and different samples. We also describe an amplicon-based method to improve the consistency of read coverage over stretches of the HIV-1 genome. Twenty-two primers were designed to amplify 11 overlapping amplicons in the HIV-1 clade B gag-pol and env gp120 coding regions to encompass 4.7 kb of the viral genome per sample at sensitivities as low as 0.01-0.2%.
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Healthy Neonates Possess a CD56-Negative NK Cell Population with Reduced Anti-Viral Activity.
PLoS ONE
PUBLISHED: 01-01-2013
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Neonatal Natural Killer (NK) cells show functional impairment and expansion of a CD56 negative population of uncertain significance.
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A comparison of 3 regimens to prevent nevirapine resistance mutations in HIV-infected pregnant women receiving a single intrapartum dose of nevirapine.
Clin. Infect. Dis.
PUBLISHED: 12-05-2011
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Intrapartum single-dose (SD) nevirapine (NVP) reduces perinatal transmission of human immunodeficiency virus (HIV) infection but selects for NVP-resistant virus, which compromises subsequent NVP-based therapy. A 1-week "tail" of lamivudine and zidovudine after SD-NVP decreases the risk of resistance. We hypothesized that increasing the duration or potency of the tail would further reduce this risk to <10%, using a sensitive assay to measure resistance.
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Transmitted HIV resistance to first-line antiretroviral therapy in Lima, Peru.
AIDS Res. Hum. Retroviruses
PUBLISHED: 08-05-2011
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Transmission of drug-resistant HIV (TDR) has been associated with virologic failure of "first-line," nonnucleoside reverse transcriptase inhibitor (NNRTI)-based antiretroviral therapy (ART). A national ART program began in Peru in 2004. We evaluated the prevalence of TDR in individuals initiating ART and their virologic outcome during 2 years of ART. HIV-infected, ARV-naive subjects who met criteria to start ART in Lima, Peru were enrolled in a longitudinal observational study between July 2007 and February 2009. Blood plasma and cells obtained prior to ART initiation were assessed for antiretroviral (ARV) resistance by an oligonucleotide ligation assay (OLA) sensitive to 2% mutant at reverse transcriptase (RT) codons K103N, Y181C, G190A, and M184V and a subset by consensus sequencing. A total of 112 participants were enrolled; the mean CD4 was 134 ± 89 cells/?l and the median plasma HIV RNA was 93,556 copies/ml (IQR 62,776-291,364). Drug resistance mutations conferring high-level resistance to ARV were rare, detected in one of 96 (1%) evaluable participants. This subject had the Y181C mutation detected in both plasma and peripheral blood mononuclear cells (PBMCs) at a concentration of 100% by OLA and consensus sequencing; nevertheless nevirapine-ART suppressed her viral replication. Consensus sequencing of 37 (19%) participants revealed multiple polymorphisms that occasionally have been associated with low-level reductions in ARV susceptibility. A low prevalence of TDR was detected among Peruvians initiating ART. Given the increasing availability of ART, continuing surveillance is needed to determine if TDR increases and the mutant codons associated with virologic failure.
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Prevalence and impact of minority variant drug resistance mutations in primary HIV-1 infection.
PLoS ONE
PUBLISHED: 07-21-2011
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To evaluate minority variant drug resistance mutations detected by the oligonucleotide ligation assay (OLA) but not consensus sequencing among subjects with primary HIV-1 infection.
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Selection of HIV resistance associated with antiretroviral therapy initiated due to pregnancy and suspended postpartum.
J. Acquir. Immune Defic. Syndr.
PUBLISHED: 07-19-2011
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Compare the risk of HIV drug resistance in women stopping suppressive nelfinavir (NFV)-based or Nevirapine (NVP)-based antiretroviral therapy (ART) after pregnancy.
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Postpartum viral load rebound in HIV-1-infected women treated with highly active antiretroviral therapy: AIDS Clinical Trials Group Protocol A5150.
HIV Clin Trials
PUBLISHED: 03-11-2011
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Pregnancy may lead to increases in HIV-1 RNA levels postpartum. The AIDS Clinical Trials Group (ACTG) A5150 study was designed to characterize the incidence of viral load rebound during the immediate 24 weeks postpartum and explore factors associated with viral load rebound.
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Herpes simplex virus type 2 suppressive therapy with acyclovir or valacyclovir does not select for specific HIV-1 resistance in HIV-1/HSV-2 dually infected persons.
J. Infect. Dis.
PUBLISHED: 01-12-2011
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Recent in vitro studies suggest that acyclovir may directly inhibit HIV-1 replication and can select for a specific HIV-1 reverse transcriptase mutation (V75I) with concomitant loss of an anti-HIV-1 effect. We tested for HIV-1 genotypic resistance at reverse transcriptase codon 75 in plasma from 168 HIV-1-infected persons from Botswana, Kenya, Peru, and the United States taking daily acyclovir or valacyclovir for between 8 weeks and 24 months. No V75I cases were detected (95% confidence interval, 0%-2.2%). These prospective in vivo studies suggest that standard-dose acyclovir or valacyclovir does not select for HIV-1 resistance.
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Comment on "The origins of sexually transmitted HIV among men who have sex with men".
Sci Transl Med
PUBLISHED: 09-24-2010
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Whether HIV from seminal cells or free HIV in semen is the origin of transmitted virus has important implications for the design of transmission prevention strategies. We found that a recent claim that HIV originates from seminal plasma and not from seminal cells was erroneous, because it was based on biological specimens that had been mislabeled, mixed-up, or contaminated. The origin of transmitted virus from semen therefore remains an open question.
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Genetic analyses of HIV-1 env sequences demonstrate limited compartmentalization in breast milk and suggest viral replication within the breast that increases with mastitis.
J. Virol.
PUBLISHED: 07-21-2010
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The concentration of human immunodeficiency virus type 1 (HIV-1) is generally lower in breast milk than in blood. Mastitis, or inflammation of the breast, is associated with increased levels of milk HIV-1 and risk of mother-to-child transmission through breastfeeding. We hypothesized that mastitis facilitates the passage of HIV-1 from blood into milk or stimulates virus production within the breast. HIV-1 env sequences were generated from single amplicons obtained from breast milk and blood samples in a cross-sectional study. Viral compartmentalization was evaluated using several statistical methods, including the Slatkin and Maddison (SM) test. Mastitis was defined as an elevated milk sodium (Na(+)) concentration. The association between milk Na(+) and the pairwise genetic distance between milk and blood viral sequences was modeled using linear regression. HIV-1 was compartmentalized within milk by SM testing in 6/17 (35%) specimens obtained from 9 women, but all phylogenetic clades included viral sequences from milk and blood samples. Monotypic sequences were more prevalent in milk samples than in blood samples (22% versus 13%; P = 0.012), which accounted for half of the compartmentalization observed. Mastitis was not associated with compartmentalization by SM testing (P = 0.621), but Na(+) was correlated with greater genetic distance between milk and blood HIV-1 populations (P = 0.041). In conclusion, local production of HIV-1 within the breast is suggested by compartmentalization of virus and a higher prevalence of monotypic viruses in milk specimens. However, phylogenetic trees demonstrate extensive mixing of viruses between milk and blood specimens. HIV-1 replication in breast milk appears to increase with inflammation, contributing to higher milk viral loads during mastitis.
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Nevirapine resistance by timing of HIV type 1 infection in infants treated with single-dose nevirapine.
Clin. Infect. Dis.
PUBLISHED: 04-14-2010
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In women, single-dose nevirapine for prophylaxis against mother-to-child transmission of human immunodeficiency virus type 1 (HIV-1) selects for nevirapine-resistant HIV-1, which subsequently decays rapidly. We hypothesized that the selection, acquisition, and decay of nevirapine-resistant HIV-1 differs in infants, varying by the timing of HIV-1 infection.
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Association between detection of HIV-1 DNA resistance mutations by a sensitive assay at initiation of antiretroviral therapy and virologic failure.
Clin. Infect. Dis.
PUBLISHED: 04-10-2010
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Antiretroviral therapy (ART) has become more available throughout the developing world during the past 5 years. The World Health Organization recommends nonnucleoside reverse-transcriptase inhibitor-based regimens as initial ART. However, their efficacy may be compromised by resistance mutations selected by single-dose nevirapine (sdNVP) used to prevent mother-to-child transmission of human immunodeficiency virus (HIV)-1. There is no simple and efficient method to detect such mutations at the initiation of ART.
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Detection of HIV-1 drug resistance in women following administration of a single dose of nevirapine: comparison of plasma RNA to cellular DNA by consensus sequencing and by oligonucleotide ligation assay.
J. Clin. Microbiol.
PUBLISHED: 02-24-2010
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A single dose of nevirapine (sdNVP) to prevent mother-to-child transmission of HIV-1 increases the risk of failure of subsequent NVP-containing antiretroviral therapy (ART), especially when initiated within 6 months of sdNVP administration, emphasizing the importance of understanding the decay of nevirapine-resistant mutants. Nevirapine-resistant HIV-1 genotypes (with the mutations K103N, Y181C, and/or G190A) from 21 women were evaluated 10 days and 6 weeks after sdNVP administration and at the initiation of ART. Resistance was assayed by consensus sequencing and by a more sensitive assay (oligonucleotide ligation assay [OLA]) using plasma-derived HIV-1 RNA and cell-associated HIV-1 DNA. OLA detected nevirapine resistance in more specimens than consensus sequencing did (63% versus 33%, P<0.01). When resistance was detected only by OLA (n=45), the median mutant concentration was 18%, compared to 61% when detected by both sequencing and OLA (n=51) (P<0.0001). The proportion of women whose nevirapine resistance was detected by OLA 10 days after sdNVP administration was higher when we tested their HIV-1 RNA (95%) than when we tested their HIV-1 DNA (88%), whereas at 6 weeks after sdNVP therapy, the proportion was greater with DNA (85%) than with RNA (67%) and remained higher with DNA (33%) than with RNA (11%) at the initiation of antiretroviral treatment (median, 45 weeks after sdNVP therapy). Fourteen women started NVP-ART more than 6 months after sdNVP therapy; resistance was detected by OLA in 14% of the women but only in their DNA. HIV-1 resistance to NVP following sdNVP therapy persists longer in cellular DNA than in plasma RNA, as determined by a sensitive assay using sufficient copies of virus, suggesting that DNA may be superior to RNA for detecting resistance at the initiation of ART.
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Efficacy and safety of 1-month postpartum zidovudine-didanosine to prevent HIV-resistance mutations after intrapartum single-dose nevirapine.
Clin. Infect. Dis.
PUBLISHED: 02-18-2010
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Intrapartum single-dose nevirapine plus third trimester maternal and infant zidovudine are essential components of programs to prevent mother-to-child transmission of human immunodeficiency virus (HIV) in resource-limited settings. The persistence of nevirapine in the plasma for 3 weeks postpartum risks selection of resistance mutations to nonnucleoside reverse-transcriptase inhibitors (NNRTIs). We hypothesized that a 1-month zidovudine-didanosine course initiated at the same time as single-dose nevirapine (sdNVP) would prevent the selection of nevirapine-resistance mutations.
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Intrapartum tenofovir and emtricitabine reduces low-concentration drug resistance selected by single-dose nevirapine for perinatal HIV prevention.
AIDS Res. Hum. Retroviruses
PUBLISHED: 11-06-2009
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A single dose of tenofovir/emtricitabine (TDF/FTC) during labor significantly reduces peripartum nevirapine-associated viral drug resistance when measured by consensus HIV sequencing. It is unknown whether this effect extends to HIV subpopulations of <25-50%. We conducted a randomized trial of single-dose TDF/FTC added to peripartum nevirapine to reduce drug resistance associated with nonnucleoside reverse transcriptase inhibitors (NNRTIs). To detect mutations for NNRTIs comprising > or = 2% of the viral population, we used an oligonucleotide ligation assay (OLA) at codons 103, 106, 181, and 190 of HIV reverse transcriptase. To assess development of drug resistance mutations to our study intervention, OLA was also performed at codons 65 and 184. Among the 328 women included in the 2-week analysis, those receiving TDF/FTC were less likely to have NNRTI resistance by OLA (RR = 0.40, 95% CI = 0.21-0.77). A similar trend was observed among the 315 women included in the 6-week analysis (RR = 0.45, 95% CI = 0.31-0.66). Only two (1%) specimens had detectable K65R by OLA. Both were at 6 weeks postpartum; one was detected in the intervention arm and one in the control arm (p = 0.96). M184V was not detected. The ability of single-dose TDF/FTC to protect against peripartum NVP-induced NNRTI resistance extends to minority populations. This efficacy is achieved without significant selection of TDF- or FTC-resistant viruses.
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Cross-contamination during processing of dried blood spots used for rapid diagnosis of HIV-1 infection of infants is rare and avoidable.
J. Virol. Methods
PUBLISHED: 08-28-2009
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Dried blood spot (DBS) samples are a convenient way to collect infant blood for HIV-1 diagnostic testing. Minimizing the risk of false positives is critical for diagnostic tests. A protocol for processing and testing DBS for infant HIV-1 diagnosis was evaluated to identify the rate and source of false-positive results. DBS were created on Flinders Technology Associates (FTA) filter paper with 500 copies/punch (high) or 5000 copies/punch (very high) concentrations of HIV-1 DNA. Blank discs of filter paper punched after DBS samples were tested for carry-over of HIV-1 DNA using nested PCR for the pol region. No false positives were detected in the 40 series using high concentration DBS. In series with very high concentrations of HIV-1, 8/246 (3%) reactions were falsely positive. When tubes were spun prior to opening, contact with caps minimized, and spaces left between lanes of the gel, repeat second-round PCR of five false positives resulted in only one repeat false-positive PCR. This study outlines procedures that minimize false-positive results for nested PCR of HIV-1 DNA from DBS.
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Assessment of mitochondrial toxicity by analysis of mitochondrial protein expression in mononuclear cells.
Cytometry B Clin Cytom
PUBLISHED: 07-21-2009
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Real-time PCR has quantified decreased mitochondrial DNA levels in association with nucleoside reverse transcriptase inhibitor (NRTI) therapy of HIV-infected populations. However, real-time PCR is best suited to distinguish log differences in an analyte. In an effort to monitor individuals in more detail, we developed a flow cytometric assay to gauge mitochondrial function.
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Compartmentalization of HIV-1 within the female genital tract is due to monotypic and low-diversity variants not distinct viral populations.
PLoS ONE
PUBLISHED: 06-04-2009
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Compartmentalization of HIV-1 between the genital tract and blood was noted in half of 57 women included in 12 studies primarily using cell-free virus. To further understand differences between genital tract and blood viruses of women with chronic HIV-1 infection cell-free and cell-associated virus populations were sequenced from these tissues, reasoning that integrated viral DNA includes variants archived from earlier in infection, and provides a greater array of genotypes for comparisons.
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Increased detection of HIV-1 drug resistance at time of diagnosis by testing viral DNA with a sensitive assay.
J. Acquir. Immune Defic. Syndr.
PUBLISHED: 04-29-2009
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HIV-1 drug resistance has been detected in 8%-24% of recently infected North Americans when assessed by consensus sequencing of plasma. We hypothesized that rates were likely higher but not detected because drug-resistant mutants are transmitted or regressed to levels below the limit of detection by consensus sequencing of HIV-1 RNA.
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Increased mutations in Env and Pol suggest greater HIV-1 replication in sputum-derived viruses compared with blood-derived viruses.
AIDS
PUBLISHED: 04-08-2009
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Low-level HIV-1 replication may occur during antiretroviral therapy (ART) that suppresses plasma HIV-1 RNA to less than 50 copies/ml (suppressive ART). Antiretroviral drugs appear less effective in macrophages and monocytes compared with lymphocytes, both in vitro and as implied in vivo by greater viral evolution observed during suppressive ART. Our objective was to examine sputum, which is rich in macrophages, for evidence of increased HIV-1 replication compared with that in the blood during suppressive ART.
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Monotypic human immunodeficiency virus type 1 genotypes across the uterine cervix and in blood suggest proliferation of cells with provirus.
J. Virol.
PUBLISHED: 04-01-2009
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Understanding the dynamics and spread of human immunodeficiency virus type 1 (HIV-1) within the body, including within the female genital tract with its central role in heterosexual and peripartum transmission, has important implications for treatment and vaccine development. To study HIV-1 populations within tissues, we compared viruses from across the cervix to those in peripheral blood mononuclear cells (PBMC) during effective and failing antiretroviral therapy (ART) and in patients not receiving ART. Single-genome sequences of the C2-V5 region of HIV-1 env were derived from PBMC and three cervical biopsies per subject. Maximum-likelihood phylogenies were evaluated for differences in genetic diversity and compartmentalization within and between cervical biopsies and PBMC. All subjects had one or more clades with genetically identical HIV-1 env sequences derived from single-genome sequencing. These sequences were from noncontiguous cervical biopsies or from the cervix and circulating PBMC in seven of eight subjects. Compartmentalization of virus between genital tract and blood was observed by statistical methods and tree topologies in six of eight subjects, and potential genital lineages were observed in two of eight subjects. The detection of monotypic sequences across the cervix and blood, especially during effective ART, suggests that cells with provirus undergo clonal expansion. Compartmentalization of viruses within the cervix appears in part due to viruses homing to and/or expanding within the cervix and is rarely due to unique viruses evolving within the genital tract. Further studies are warranted to investigate mechanisms producing monotypic viruses across tissues and, importantly, to determine whether the proliferation of cells with provirus sustain HIV-1 persistence in spite of effective ART.
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Nevirapine-Resistant HIV-1 DNA in Breast Milk After Single-Dose Nevirapine With or Without Zidovudine for Prevention of Mother-to-Child Transmission.
J Pediatric Infect Dis Soc
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Among 30 human immunodeficiency virus type 1 (HIV-1)-infected women who received single-dose nevirapine (NVP), 17 (57%) had NVP-resistant HIV-1 detected in breast milk. NVP resistance in breast milk persisted for at least 8 months postpartum and was apparently transmitted to at least 1 infant. NVP resistance was detected less often in women who also received zidovudine.
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An increasing proportion of monotypic HIV-1 DNA sequences during antiretroviral treatment suggests proliferation of HIV-infected cells.
J. Virol.
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Understanding how HIV-1 persists during effective antiretroviral therapy (ART) should inform strategies to cure HIV-1 infection. We hypothesize that proliferation of HIV-1-infected cells contributes to persistence of HIV-1 infection during suppressive ART. This predicts that identical or monotypic HIV-1 DNA sequences will increase over time during ART. We analyzed 1,656 env and pol sequences generated following single-genome amplification from the blood and sputum of six individuals during long-term suppressive ART. The median proportion of monotypic sequences increased from 25.0% prior to ART to 43.2% after a median of 9.8 years of suppressive ART. The proportion of monotypic sequences was estimated to increase 3.3% per year (95% confidence interval, 2.3 to 4.4%; P < 0.001). Drug resistance mutations were not more common in the monotypic sequences, arguing against viral replication during times with lower antiretroviral concentrations. Bioinformatic analysis found equivalent genetic distances of monotypic and nonmonotypic sequences from the predicted founder virus sequence, suggesting that the relative increase in monotypic variants over time is not due to selective survival of cells with viruses from the time of acute infection or from just prior to ART initiation. Furthermore, while the total HIV-1 DNA load decreased during ART, the calculated concentration of monotypic sequences was stable in children, despite growth over nearly a decade of observation, consistent with proliferation of infected CD4(+) T cells and slower decay of monotypic sequences. Our findings suggest that proliferation of cells with proviruses is a likely mechanism of HIV-1 DNA persistence, which should be considered when designing strategies to eradicate HIV-1 infection.
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Quantification of mitochondrial toxicity in HIV-infected individuals by quantitative PCR compared to flow cytometry.
Cytometry B Clin Cytom
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Non-invasive diagnostic assays to evaluate mitochondrial toxicity could have significant clinical utility for HIV-infected individuals on antiretroviral therapy (ART).
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Interaction between lactobacilli, bacterial vaginosis-associated bacteria, and HIV Type 1 RNA and DNA Genital shedding in U.S. and Kenyan women.
AIDS Res. Hum. Retroviruses
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Bacterial vaginosis has been associated with genital HIV-1 shedding; however, the effect of specific vaginal bacterial species has not been assessed. We tested cervicovaginal lavage from HIV-1-seropositive women for common Lactobacillus species: L. crispatus, L. jensenii, and seven BV-associated species: BVAB1, BVAB2, BVAB3, Leptotrichia, Sneathia, Megasphaera, and Atopobium spp. using quantitative PCR. We used linear and Poisson regression to evaluate associations between vaginal bacteria and genital HIV-1 RNA and DNA. Specimens from 54 U.S. (310 visits) and 50 Kenyan women (137 visits) were evaluated. Controlling for plasma viral load, U.S. and Kenyan women had similar rates of HIV-1 RNA (19% of visits vs. 24%; IRR=0.95; 95% CI 0.61, 1.49) and DNA shedding (79% vs. 76%; IRR=0.90; 0.78, 1.05). At visits during antiretroviral therapy (ART), the likelihood of detection of HIV-1 RNA shedding was greater with BVAB3 (IRR=3.16; 95% CI 1.36, 7.32), Leptotrichia, or Sneathia (IRR=2.13; 1.02, 4.72), and less with L. jensenii (IRR=0.39; 0.18, 0.84). At visits without ART, only L. crispatus was associated with a lower likelihood of HIV-1 RNA detection (IRR=0.6; 0.40, 0.91). Vaginal Lactobacillus species were associated with lower risk of genital HIV-1 shedding, while the presence of certain BV-associated species may increase that risk.
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Effects of short-course zidovudine on the selection of nevirapine-resistant HIV-1 in women taking single-dose nevirapine.
J. Infect. Dis.
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Single-dose nevirapine (sdNVP) given to prevent mother-to-child-transmission of HIV-1 selects NVP-resistance. Short-course zidovudine (ZDV) was hypothesized to lower rates of NVP-resistance. HIV-1 infected pregnant women administered sdNVP with or without short-course ZDV were assessed for HIV-1 mutations (K103N, Y181C, G190A, and V106M) prior to delivery and postpartum. Postpartum NVP-resistance was lower among 31 taking ZDV+sdNVP compared to 33 taking only sdNVP (35.5% vs. 72.7%; ?2 P = .003). NVP mutants decayed to <2% in 24/35 (68.6%) at a median 6 months postpartum, with no differences based on ZDV use (logrank P = .99). Short-course ZDV was associated with reduced NVP-resistance mutations among women taking sdNVP.
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