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Empowering the Participant Voice (EPV) is an NCATS-funded six-CTSA collaboration to develop, demonstrate, and disseminate a low-cost infrastructure for collecting timely feedback from research participants, fostering trust, and providing data for improving clinical translational research. EPV leverages the validated Research Participant Perception Survey (RPPS) and the popular REDCap electronic data-capture platform. This report describes the development of infrastructure designed to overcome identified institutional barriers to routinely collecting participant feedback using RPPS and demonstration use cases. Sites engaged local stakeholders iteratively, incorporating feedback about anticipated value and potential concerns into project design. The team defined common standards and operations, developed software, and produced a detailed planning and implementation Guide. By May 2023, 2,575 participants diverse in age, race, ethnicity, and sex had responded to approximately 13,850 survey invitations (18.6%); 29% of responses included free-text comments. EPV infrastructure enabled sites to routinely access local and multi-site research participant experience data on an interactive analytics dashboard. The EPV learning collaborative continues to test initiatives to improve survey reach and optimize infrastructure and process. Broad uptake of EPV will expand the evidence base, enable hypothesis generation, and drive research-on-research locally and nationally to enhance the clinical research enterprise.
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INTRODUCTION: In order to tackle the challenge of efficiently meeting clinical research accrual goals, many Clinical and Translational Science Award (CTSA) recipients have developed recruitment support mechanisms and resources to help investigators successfully recruit study participants. Disseminating recruitment best practices and developing collaborations between institutions can help strengthen recruitment capabilities and methodologies currently utilized by researchers. METHODS: To discover what recruitment resources and mechanisms CTSAs are using, the CTSA Recruitment and Retention working group developed an electronic survey, which was distributed to CTSAs between May and July 2019. The survey contained over 50 multiple choice and short answer questions, with 40 of the 64 CTSA institutions completing the survey. Institutions reported on registries, feasibility assessment tools, clinical trial listings, experience recruiting special populations, program operations and evaluation, workforce education, social media use, and other recruitment resources. RESULTS: All respondents currently utilize some form of a volunteer registry; over 80% of the CTSAs provide investigators with recruitment consultations, feasibility assessments, study listings, and electronic health record (EHR) utilization; 73% assist with study materials; 47% offer social media assistance. Many institutions reported success in recruiting patients and healthy volunteers, but difficulty in recruiting special populations such as non-English-speaking persons and rural populations. Additional recruitment tools included use of the EHR to facilitate recruitment, use of registries, and use of social media to engage participants. CONCLUSIONS: Areas of opportunity or growth include the development of innovative solutions in the areas of social media advertising, identification of participants from special populations, and research volunteer engagement.
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Life course research embraces the complexity of health and disease development, tackling the extensive interactions between genetics and environment. This interdisciplinary blueprint, or theoretical framework, offers a structure for research ideas and specifies relationships between related factors. Traditionally, methodological approaches attempt to reduce the complexity of these dynamic interactions and decompose health into component parts, ignoring the complex reciprocal interaction of factors that shape health over time. New methods that match the epistemological foundation of the life course framework are needed to fully explore adaptive, multilevel, and reciprocal interactions between individuals and their environment. The focus of this article is to (1) delineate the differences between lifespan and life course research, (2) articulate the importance of complex systems science as a methodological framework in the life course research toolbox to guide our research questions, (3) raise key questions that can be asked within the clinical and translational science domain utilizing this framework, and (4) provide recommendations for life course research implementation, charting the way forward. Recent advances in computational analytics, computer science, and data collection could be used to approximate, measure, and analyze the intertwining and dynamic nature of genetic and environmental factors involved in health development.
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We identified a novel way to recruit participants into a research registry by using an Emergency Department Research Associate (EDRA) Program. Research associates working in the Emergency Department at Strong Memorial Hospital approached patients and family members to enroll into the University of Rochester Research Participant Registry and for ResearchMatch.org. We found that 73% of individuals (574/781) approached agreed to register for either one or both registries. Those who registered were more diverse than individuals who registered through other methods. Overall, using EDRAs to enroll adult patients and their family members is an effective method for growing research participant registries.
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Servicio de Urgencia en Hospital/organización & administración , Selección de Paciente , Sistema de Registros/estadística & datos numéricos , Investigadores/organización & administración , Adulto , Niño , Análisis Costo-Beneficio/estadística & datos numéricos , Servicio de Urgencia en Hospital/economía , Servicio de Urgencia en Hospital/estadística & datos numéricos , Estudios de Factibilidad , Femenino , Humanos , Masculino , Proyectos Piloto , Evaluación de Programas y Proyectos de Salud , Investigadores/economía , Voluntarios/estadística & datos numéricosRESUMEN
Efavirenz is a non-nucleoside reverse transcriptase inhibitor used for treating HIV/AIDS. We found that polymerization activity of a reverse transcriptase (RT) with the E478Q mutation that inactivates the RNase H catalytic site is much more sensitive to efavirenz than wild-type RT, indicating that a functional RNase H attenuates the effectiveness of efavirenz. Moreover, efavirenz actually stimulated wild-type RNase H binding and catalytic functions, indicating another link between efavirenz action and RNase H function. During reverse transcription in vivo, the RT that is extending the DNA primer also periodically cleaves the genomic RNA. The RNase H makes primary template cuts ~18 nucleotides from the growing DNA 3'-end, and when the RT pauses synthesis, it shifts to make secondary cuts ~9 nucleotides from the DNA 3'-end. After synthesis, RTs return to bind the remaining template RNA segments at their 5'-ends and make primary and secondary cuts, 18 and 9 nucleotides in, respectively. We found that efavirenz stimulates both 3'- and 5'-directed RNase H activity. Use of specific substrates revealed a particular acceleration of secondary cuts. Efavirenz specifically promoted binding of the RT to RNase H substrates, suggesting that it stabilizes the shifting of RTs to make the secondary cuts. We further showed that efavirenz similarly stimulates the RNase H of an RT from a patient-derived virus that is highly resistant and grows more rapidly in the presence of low concentrations of efavirenz. We suggest that for efavirenz-resistant RTs, stimulated RNase H activity contributes to increased viral fitness.
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Benzoxazinas/farmacología , Transcriptasa Inversa del VIH/metabolismo , Inhibidores de la Transcriptasa Inversa/farmacología , Ribonucleasa H/metabolismo , Alquinos , Secuencia de Bases , Ciclopropanos , Cartilla de ADN , Electroforesis en Gel de Poliacrilamida , Activación Enzimática , Proteolisis , Especificidad por SustratoRESUMEN
Previous work by our group showed that human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) containing non-nucleoside RT inhibitor (NNRTI) drug resistance mutations has defects in RNase H activity as well as reduced amounts of RT protein in virions. These deficits correlate with replication fitness in the absence of NNRTIs. Viruses with the mutant combination K101E+G190S replicated better in the presence of NNRTIs than in the absence of drug. Stimulation of virus growth by NNRTIs occurred during the early steps of the virus life cycle and was modulated by the RT backbone sequence in which the resistance mutations arose. We wanted to determine what effects RT backbone sequence would have on RT content and polymerization and RNase H activities in the absence of NNRTIs. We compared a NL4-3 RT with K101E+G190S to a patient-isolate RT sequence D10 with K101E+G190S. We show here that, unlike the NL4-3 backbone, the D10 backbone sequence decreased the RNA-dependent DNA polymerization activity of purified recombinant RT compared to WT. In contrast, RTs with the D10 backbone had increased RNase H activity compared to WT and K101E+G190S in the NL4-3 backbone. D10 virions also had increased amounts of RT compared to K101E+G190S in the NL4-3 backbone. We conclude that the backbone sequence of RT can alter the activities of the NNRTI drug-resistant mutant K101E+G190S, and that identification of the amino acids responsible will aid in understanding the mechanism by which NNRTI drug-resistant mutants alter fitness and NNRTIs stimulate HIV-1 virus replication.
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Transcriptasa Inversa del VIH/metabolismo , VIH-1/metabolismo , Ribonucleasas/metabolismo , Secuencia de Aminoácidos , Células HEK293 , Transcriptasa Inversa del VIH/genética , Humanos , Mutagénesis Sitio-Dirigida , Mutación , ADN Polimerasa Dirigida por ARN/metabolismo , Ribonucleasa H/genética , Ribonucleasa H/metabolismo , Ribonucleasas/genética , Replicación ViralRESUMEN
OBJECTIVE: Among HIV-positive patients prescribed ritonavir-boosted lopinavir, SLCO1B1 521TâC (rs4149056) is associated with increased plasma lopinavir exposure. Protease inhibitors (PIs) are also substrates for cytochrome P450 (CYP) 3A and ABCB1, which are induced by NR1I2. We characterized relationships between ABCB1, CYP3A4, CYP3A5, NR1I2, and SLCO1B1 polymorphisms and trough PI concentrations among AIDS Clinical Trials Group study A5146 participants. METHODS: At study entry, subjects with virologic failure on PI-containing regimens initiated new ritonavir-boosted PI regimens. We studied associations between week 2 PI plasma trough concentrations and 143 polymorphisms in these genes, including 4 targeted polymorphisms. RESULTS: Among 275 subjects with both drug concentrations and genetic data, allelic frequencies of SLCO1B1 521TâC were 15%, 1%, and 8% in whites, blacks, and Hispanics, respectively. Further analyses were limited to 268 white, black, or Hispanic subjects who initiated ritonavir-boosted lopinavir (n = 98), fosamprenavir (n = 69), or saquinavir (n = 99). Of targeted polymorphisms, SLCO1B1 521TâC tended to be associated with higher lopinavir concentrations, with a 1.38-fold increase in the mean per C allele (95% confidence interval, 0.97-1.96; n = 98; P = 0.07). With fosamprenavir, SLCO1B1 521TâC was associated with lower amprenavir concentrations, with a 35% decrease in the mean per C allele (geometric mean ratio 0.65; 95% confidence interval, 0.44-0.94; n = 69; adjusted P = 0.02). There was no significant association with saquinavir concentrations, and none of the remaining 139 exploratory polymorphisms were statistically significant after correcting for multiple comparisons. CONCLUSIONS: With ritonavir-boosted PIs, a SLCO1B1 polymorphism that predicts higher lopinavir trough concentrations seems to predict lower amprenavir trough concentrations. The mechanism underlying this discordant association is uncertain.
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Síndrome de Inmunodeficiencia Adquirida/genética , Estudios de Asociación Genética/métodos , Inhibidores de la Proteasa del VIH/uso terapéutico , Desequilibrio de Ligamiento/genética , Transportadores de Anión Orgánico/genética , Ritonavir/uso terapéutico , Síndrome de Inmunodeficiencia Adquirida/tratamiento farmacológico , Adulto , Femenino , Humanos , Transportador 1 de Anión Orgánico Específico del Hígado , Masculino , Persona de Mediana EdadRESUMEN
The fitness of non-nucleoside reverse transcriptase inhibitor (NNRTI) drug-resistant reverse transcriptase (RT) mutants of HIV-1 correlates with the amount of RT in the virions and the RNase H activity of the RT. We wanted to understand the mechanism by which secondary NNRTI-resistance mutations, L100I and K101E, and the nucleoside resistance mutation, L74V, alter the fitness of K103N and G190S viruses. We measured the amount of RT in virions and the polymerization and RNase H activities of mutant RTs compared to wild-type, K103N and G190S. We found that L100I, K101E and L74V did not change the polymerization or RNase H activities of K103N or G190S RTs. However, L100I and K101E reduced the amount of RT in the virions and subsequent addition of L74V restored RT levels back to those of G190S or K103N alone. We conclude that fitness changes caused by L100I, K101E and L74V derive from their effects on RT content.
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Fármacos Anti-VIH/farmacología , Farmacorresistencia Viral/genética , Transcriptasa Inversa del VIH/efectos de los fármacos , Transcriptasa Inversa del VIH/metabolismo , VIH-1/efectos de los fármacos , VIH-1/fisiología , Mutación , Inhibidores de la Transcriptasa Inversa/farmacología , Virión/enzimología , Replicación Viral , Línea Celular , Transcriptasa Inversa del VIH/genética , VIH-1/enzimología , VIH-1/genética , Humanos , Nucleósidos/farmacología , ADN Polimerasa Dirigida por ARN/metabolismo , Ribonucleasa H/metabolismo , Virión/fisiologíaRESUMEN
Potent HIV-1 specific broadly neutralizing antibodies (BNA) are uncommon in HIV infected individuals, and have proven hard to elicit by vaccination. Several, isolated monoclonal BNA are polyreactive and also recognize self-antigens, suggesting a breach of immune tolerance in persons living with HIV (PLWH). Persons with systemic lupus erythematosus (SLE) often have elevated levels of autoreactive antibodies encoded by the VH4-34 heavy chain immunoglobulin gene whose protein product can be detected by the 9G4 rat monoclonal antibody. We have recently found that levels of these "9G4+" antibodies are also elevated in PLWH. However, the putative autoreactive nature of these antibodies and the relationship of such reactivities with HIV neutralization have not been investigated. We therefore examined the autoreactivity and HIV neutralization potential of 9G4+ antibodies from PLWH. Results show that 9G4+ antibodies from PLWH bound to recombinant HIV-1 envelope (Env) and neutralized viral infectivity in vitro, whereas 9G4+ antibodies from persons with SLE did not bind to Env and failed to neutralize viral infectivity. In addition, while 9G4+ antibodies from PLWH retained the canonical anti-i reactivity that mediates B cell binding, they did not display other autoreactivities common to SLE 9G4+ antibodies, such as binding to cardiolipin and DNA and had much lower reactivity with apoptotic cells. Taken together, these data indicate that the autoreactivity of 9G4+ antibodies from PLWH is distinct from that of SLE patients, and therefore, their expansion is not due to a general breakdown of B cell tolerance but is instead determined in a more disease-specific manner by self-antigens that become immunogenic in the context of, and possibly due to HIV infection. Further studies of 9G4+ B cells may shed light on the regulation of B cell tolerance and interface between the generation of specific autoreactivities and the induction of antiviral immunity in persons living with HIV.
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Anticuerpos Neutralizantes/inmunología , Linfocitos B/inmunología , Anticuerpos Anti-VIH/inmunología , Infecciones por VIH/inmunología , Tolerancia Inmunológica , Adulto , Anciano , Animales , Anticuerpos Monoclonales/inmunología , Anticuerpos Neutralizantes/sangre , Linfocitos B/patología , Anticuerpos Anti-VIH/sangre , Infecciones por VIH/sangre , Infecciones por VIH/patología , Humanos , Células Jurkat , Lupus Eritematoso Sistémico/sangre , Lupus Eritematoso Sistémico/inmunología , Masculino , Persona de Mediana Edad , RatasRESUMEN
Infection of cells with human immunodeficiency virus type-1 (HIV-1) results in the production of both infectious and non-infectious virions. At present, several assays are available for the quantitation of virus particles based on the presence of either viral capsid protein or nucleic acid. However, the ability to detect the total number of virus particles, both infectious and non-infectious, has been an elusive goal that would advance the study of virus assembly and egress. A rapid optical detection scheme for real-time label-free quantitation of HIV-1 virus particles was developed. Virions produced in cell cultures transfected transiently were evaluated with a nanospectroscopic assay. Quantitation with the optical detection scheme correlated with routine conventional assays. Nanospectroscopy can be used for the detection of both infectious and non-infectious, wild type and mutant strains of HIV-1 in solution at concentrations as low as 7×10(10)particles/ml, requiring volumes as small as 2 µl per assay, and in significantly less time than standard techniques. This assay provides a rapid, reliable system for quantifying virus particles in solution and could be applied to the study of viral particle production in cell culture.
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VIH-1/aislamiento & purificación , Nanotecnología/métodos , Análisis Espectral/métodos , Carga Viral/métodos , Virología/métodos , Humanos , Sensibilidad y Especificidad , Factores de TiempoRESUMEN
BACKGROUND: We devised an open-label, randomized trial to evaluate whether therapeutic drug monitoring (TDM) of protease inhibitors (PIs) and dose escalation based upon a normalized inhibitory quotient (NIQ), which integrates PI trough concentration and drug resistance, could improve virologic outcome in PI-experienced patients with treatment failure. Secondary analyses through 48 weeks are presented. METHODS: Eligible HIV-infected subjects with a screening viral load of ≥ 1000 copies/mL initiated a new PI-based regimen at entry and had NIQ performed at week 2. Subjects with an NIQ ≤1 were randomized at week 4 to a standard-of-care (SOC) arm or TDM arm featuring PI dose escalation. RESULTS: One hundred and eighty-three subjects were randomized. There was no significant treatment difference in change from randomization to week 48 in HIV-1 RNA [ P = .13, median (25th, 75th percentile log10 copies/mL change): -0.03 (-0.74, 0.62) with TDM and 0.11 (-2.3, 0.82) with SOC]. In subgroup analysis, patients with ≥ 0.69 active PIs benefited from TDM compared to those with <0.69 active PIs ( P = .05). CONCLUSIONS: While the TDM strategy of PI dose escalation did not improve virologic response at week 48 overall, in subgroup analysis, TDM favorably impacted virologic outcome in subjects taking PI-based regimens with moderate antiviral activity.
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Monitoreo de Drogas , Infecciones por VIH/tratamiento farmacológico , Inhibidores de la Proteasa del VIH/uso terapéutico , VIH-1/efectos de los fármacos , Adulto , Recuento de Linfocito CD4 , Femenino , Estudios de Seguimiento , Infecciones por VIH/virología , Inhibidores de la Proteasa del VIH/efectos adversos , Humanos , Masculino , Persona de Mediana Edad , Carga ViralRESUMEN
Nonnucleoside reverse transcriptase inhibitors (NNRTIs) are potent and commonly prescribed antiviral agents used in combination therapy (CART) of human immunodeficiency virus type 1 (HIV-1) infection. The development of drug resistance is a major limitation of CART. Reverse transcriptase (RT) genotypes with the NNRTI resistance mutations K101E+G190S are highly resistant to efavirenz (EFV) and can develop during failure of EFV-containing regimens in patients. We have previously shown that virus with K101E+G190S mutations can replicate more efficiently in the presence of EFV than in its absence. In this study, we evaluated the underlying mechanism for drug-dependent stimulation, using a single-cycle cell culture assay in which EFV was added either during the infection or the virus production step. We determined that EFV stimulates K101E+G190S virus during early infection and does not affect late steps of virus replication, such as increasing the amount of active RT incorporated into virions. Additionally, we showed that another NNRTI, nevirapine (NVP), stimulated K101E+G190S virus replication during the early steps of infection similar to EFV, but that the newest NNRTI, etravirine (ETR), did not. We also showed that EFV stimulates K101E+Y188L and K101E+V106I virus, but not K101E+L100I, K101E+K103N, K101E+Y181C, or K101E+G190A virus, suggesting that the stimulation is mutation specific. Real-time PCR of reverse transcription intermediates showed that although the drug did not stimulate minus-strand transfer, it did stimulate minus-strand strong-stop DNA synthesis. Our results indicate that stimulation most likely occurs through a mechanism whereby NNRTIs stimulate priming or elongation of the tRNA.
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Benzoxazinas/farmacología , Infecciones por VIH/virología , VIH-1/efectos de los fármacos , VIH-1/crecimiento & desarrollo , Inhibidores de la Transcriptasa Inversa/farmacología , Replicación Viral/efectos de los fármacos , Alquinos , Fármacos Anti-VIH/farmacología , Ciclopropanos , Transcriptasa Inversa del VIH/genética , Humanos , Mutación Missense , Nevirapina/farmacología , Nitrilos , Piridazinas/farmacología , PirimidinasRESUMEN
A tRNA gene-like sequence has been identified near the 3' end of HIV-1. Two segments of this sequence (motif 9 and segment 1) promoted minus strand transfer in vitro. The segments are complementary to the tRNA(3)(Lys) primer, and apparently act by binding the tRNA, thereby bringing the 3' and 5' ends of viral RNA into proximity for strand transfer. In this report, we used full-length HIV-1 to demonstrate biological relevance of these segments. We constructed HIV-1 genomes capable of single cycle infection and altered in one or both of two segments. We devised a real time PCR method for quantifying the amount of (-)ssDNA that completes transfer. Results showed that depending on the mutation the efficiency of transfer decreased from 9% to 26%. Alteration of segment 1 had the greatest effect. Alteration of motif 9 or both sequences also caused a reduction, but smaller than alteration of segment 1 alone.
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VIH-1/fisiología , ARN Viral/genética , ARN Viral/metabolismo , Replicación Viral , Línea Celular , VIH-1/genética , Humanos , Mutación , ARN Complementario/genética , ARN Complementario/metabolismo , Aminoacil-ARN de Transferencia/genética , Aminoacil-ARN de Transferencia/metabolismoRESUMEN
The replication fitness of HIV-1 drug-resistant mutants has been measured using either multiple-cycle or single-cycle assays (MCAs or SCAs); these assays have not been systematically compared. We developed an MCA and an SCA that utilized either intact or env-deleted recombinant viral vectors, respectively, in which virus-infected cells were detected by flow cytometry of a reporter gene product. Fitness was measured using each assay for 11 protease mutants, 9 reverse transcriptase mutants, and two mutants with mutations in gag p6, which is important for the release of virus particles from the cell membrane. In the SCA, fitness (replication capacity [RC]) was defined as the proportion of cells infected by the mutant compared to the wild type 40 h after infection. MCA fitness (1+s) was determined by comparing the changes in the relative proportions of cells infected by the mutant and the wild type between 3 and 5 days after infection. Five protease mutants showed statistically different fitness values by the MCA versus the SCA: the D30N, G48V, I50V, I54L, and I54M mutants. When all the mutants were ranked in order from most to least fit for both assays, 4 protease mutants moved more than 5 positions in rank: the D30N, I54L, I54M, and V82A mutants. There were no significant differences in fitness for the gag p6 or reverse transcriptase mutants. We propose that discordant results in the MCA and SCA are due to alterations in late events in the virus life cycle that are not captured in an SCA, such as burst size, cell-to-cell transmission, or infected-cell life span.
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Farmacorresistencia Viral , Infecciones por VIH/virología , Inhibidores de la Proteasa del VIH/farmacología , Proteasa del VIH/genética , VIH-1/efectos de los fármacos , VIH-1/fisiología , Replicación Viral , Humanos , Virología/métodos , VirulenciaAsunto(s)
Síndrome de Inmunodeficiencia Adquirida/epidemiología , Síndrome de Inmunodeficiencia Adquirida/virología , Fármacos Anti-VIH/farmacología , Farmacorresistencia Viral , VIH/efectos de los fármacos , Síndrome de Inmunodeficiencia Adquirida/inmunología , Recuento de Linfocito CD4 , Ensayos Clínicos como Asunto , VIH/aislamiento & purificación , Humanos , Prevalencia , Carga ViralRESUMEN
In a randomized trial, AIDS Clinical Trials Group (ACTG) protocol 5146 (A5146) investigated the use of therapeutic drug monitoring (TDM) to adjust doses of HIV-1 protease inhibitors (PIs) in patients with prior virologic failure on PI-based therapy who were starting a new PI-based regimen. The overall percentage of "PI trough repeats" such as rescheduled visits or redrawn PI trough specimens increased from 2% to 5% to 10% as the process progressed from the clinical sites, the pharmacology specialty laboratory, and the study team, respectively. Cumulatively, this represents a 17% rate of failure to obtain adequate PI trough sample. While targeting a turnaround of 7 days or less from sample receipt to a drug concentration report, 12% of the received specimens required a longer period to report concentrations. The implementation of dosing changes in the TDM arm were achieved within 7 days or less for 56% of the dose change events and within 14 days or less for 77% of dose change events. This quality assurance analysis provides a valuable summary of the specific points in the TDM process that could be improved during a multicenter clinical trial including: 1) shortening the timeline of sample shipment from clinical site to the laboratory; 2) performing the collection of PI trough specimen within the targeted sampling window by careful monitoring of the last dose times and collection times by the clinicians; 3) increasing patient adherence counseling to reduce the number of samples that are redrawn due to suspecting inconsistent adherence; and 4) decreasing the time to successful TDM-based dose adjustment. The application of some of these findings may also be relevant to single-center studies or clinical TDM programs within a hospital.
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Síndrome de Inmunodeficiencia Adquirida/tratamiento farmacológico , Fármacos Anti-VIH/uso terapéutico , Terapia Antirretroviral Altamente Activa/métodos , Monitoreo de Drogas/normas , Fármacos Anti-VIH/sangre , Calibración , Inhibidores de la Proteasa del VIH/sangre , Inhibidores de la Proteasa del VIH/uso terapéutico , VIH-1 , Humanos , Laboratorios/normas , Control de Calidad , Manejo de Especímenes/normas , Resultado del TratamientoRESUMEN
Nonnucleoside reverse transcriptase (RT) inhibitors (NNRTIs) are important components of multidrug therapy for HIV-1. Understanding the effect of NNRTI-resistant mutants on virus replication and reverse transcriptase (RT) function is valuable for the development of extended-spectrum NNRTIs. We measured the fitness of six NNRTI-resistant mutants, the K103N, V106A, Y181C, G190A, G190S, and P236L viruses, using a flow cytometry-based cell culture assay. K103N and Y181C viruses had fitness similar to that of the wild type while V106A, G190A, G190S, and P236L viruses had reduced fitness. We also determined the biochemical correlates of fitness by measuring the RNase H and polymerization activities of recombinant mutant RTs and virion-associated RTs. The RNase H activities of recombinant and virion-associated RTs correlated with the relative fitness for each mutant. K103N and Y181C mutants had normal RNase H activity; V106A, G190A, and G190S mutants had moderate reductions in activity; and the P236L mutant had substantially reduced activity. With the exception of the P236L mutant, reduced fitness correlates with low virion-associated polymerization efficiency and reduced RT content. Reduced polymerase function in virions derived from low RT content rather than an intrinsic polymerization defect in each RT protein. In conclusion, severe defects in RNase H activity alone, exemplified by the P236L mutant, appear sufficient to cause a substantial reduction in fitness. For the other NNRTI mutants, reductions in RT content decreased both polymerization and RNase H activity in virions. RNase H reduction was compounded by intrinsic RNase H defects in the mutant RTs.
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Farmacorresistencia Viral , Transcriptasa Inversa del VIH/metabolismo , VIH-1/crecimiento & desarrollo , Mutación Missense , Inhibidores de la Transcriptasa Inversa/farmacología , Ribonucleasa H/metabolismo , Virión/crecimiento & desarrollo , Fármacos Anti-VIH/farmacología , Línea Celular , Transcriptasa Inversa del VIH/genética , VIH-1/enzimología , Humanos , Virión/enzimologíaRESUMEN
BACKGROUND: The replication rate (or fitness) between viral variants has been investigated in vivo and in vitro for human immunodeficiency virus (HIV). HIV fitness plays an important role in the development and persistence of drug resistance. The accurate estimation of viral fitness relies on complicated computations based on statistical methods. This calls for tools that are easy to access and intuitive to use for various experiments of viral fitness. RESULTS: Based on a mathematical model and several statistical methods (least-squares approach and measurement error models), a Web-based computing tool has been developed for improving estimation of virus fitness in growth competition assays of human immunodeficiency virus type 1 (HIV-1). CONCLUSIONS: Unlike the two-point calculation used in previous studies, the estimation here uses linear regression methods with all observed data in the competition experiment to more accurately estimate relative viral fitness parameters. The dilution factor is introduced for making the computational tool more flexible to accommodate various experimental conditions. This Web-based tool is implemented in C# language with Microsoft ASP.NET, and is publicly available on the Web at http://bis.urmc.rochester.edu/vFitness/.
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Simulación por Computador , VIH-1/crecimiento & desarrollo , Internet , Programas Informáticos , Modelos LinealesRESUMEN
The reliable detection, sizing, and sorting of viruses and nanoparticles is important for biosensing, environmental monitoring, and quality control. Here we introduce an optical detection scheme for the real-time and label-free detection and recognition of single viruses and larger proteins. The method makes use of nanofluidic channels in combination with optical interferometry. Elastically scattered light from single viruses traversing a stationary laser focus is detected with a differential heterodyne interferometer and the resulting signal allows single viruses to be characterized individually. Heterodyne detection eliminates phase variations due to different particle trajectories, thus improving the recognition accuracy as compared to standard optical interferometry. We demonstrate the practicality of our approach by resolving nanoparticles of various sizes, and detecting and recognizing different species of human viruses from a mixture. The detection system can be readily integrated into larger nanofluidic architectures for practical applications.
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Nanopartículas/análisis , Nanotecnología/instrumentación , Fenómenos Ópticos , Virus/aislamiento & purificación , Humanos , Nanopartículas/química , Tamaño de la Partícula , Procesamiento de Señales Asistido por Computador , Factores de Tiempo , Virus/clasificaciónRESUMEN
Human immunodeficiency virus type 1 (HIV-1) persists in a latent reservoir of infected resting memory CD4 cells in patients receiving antiretroviral therapy. We assessed whether multitarget therapy with enfuvirtide, 2 reverse-transcriptase inhibitors, and a ritonavir-boosted protease inhibitor leads to decay of this reservoir. Nineteen treatment-naive patients initiated this regimen; 9 experienced virologic suppression and continued enfuvirtide-containing therapy for at least 48 weeks. In enfuvirtide-treated patients with virological suppression, there was no decay of the latent reservoir (95% confidence interval for half-life, 11 months to infinity). The stability of the latent reservoir despite intensive therapy suggests that new strategies are needed to eradicate HIV-1 from this reservoir. (ClinicalTrials.gov identifier: NCT00051831 .).