RESUMEN
UNLABELLED: HIV-1 protease (PR), reverse transcriptase (RT), and integrase (IN) variability presents a challenge to laboratories performing genotypic resistance testing. This challenge will grow with increased sequencing of samples enriched for proviral DNA such as dried blood spots and increased use of next-generation sequencing (NGS) to detect low-abundance HIV-1 variants. We analyzed PR and RT sequences from >100,000 individuals and IN sequences from >10,000 individuals to characterize variation at each amino acid position, identify mutations indicating APOBEC-mediated G-to-A editing, and identify mutations resulting from selective drug pressure. Forty-seven percent of PR, 37% of RT, and 34% of IN positions had one or more amino acid variants with a prevalence of ≥1%. Seventy percent of PR, 60% of RT, and 60% of IN positions had one or more variants with a prevalence of ≥0.1%. Overall 201 PR, 636 RT, and 346 IN variants had a prevalence of ≥0.1%. The median intersubtype prevalence ratios were 2.9-, 2.1-, and 1.9-fold for these PR, RT, and IN variants, respectively. Only 5.0% of PR, 3.7% of RT, and 2.0% of IN variants had a median intersubtype prevalence ratio of ≥10-fold. Variants at lower prevalences were more likely to differ biochemically and to be part of an electrophoretic mixture compared to high-prevalence variants. There were 209 mutations indicative of APOBEC-mediated G-to-A editing and 326 mutations nonpolymorphic treatment selected. Identification of viruses with a high number of APOBEC-associated mutations will facilitate the quality control of dried blood spot sequencing. Identifying sequences with a high proportion of rare mutations will facilitate the quality control of NGS. IMPORTANCE: Most antiretroviral drugs target three HIV-1 proteins: PR, RT, and IN. These proteins are highly variable: many different amino acids can be present at the same position in viruses from different individuals. Some of the amino acid variants cause drug resistance and occur mainly in individuals receiving antiretroviral drugs. Some variants result from a human cellular defense mechanism called APOBEC-mediated hypermutation. Many variants result from naturally occurring mutation. Some variants may represent technical artifacts. We studied PR and RT sequences from >100,000 individuals and IN sequences from >10,000 individuals to quantify variation at each amino acid position in these three HIV-1 proteins. We performed analyses to determine which amino acid variants resulted from antiretroviral drug selection pressure, APOBEC-mediated editing, and naturally occurring variation. Our results provide information essential to clinical, research, and public health laboratories performing genotypic resistance testing by sequencing HIV-1 PR, RT, and IN.
Asunto(s)
Desaminasas APOBEC/metabolismo , Variación Genética , Integrasa de VIH/genética , Proteasa del VIH/genética , Transcriptasa Inversa del VIH/genética , VIH-1/genética , Desaminasas APOBEC/genética , Secuencia de Aminoácidos , Fármacos Anti-VIH/uso terapéutico , Farmacorresistencia Viral/genética , Genotipo , Infecciones por VIH/tratamiento farmacológico , Infecciones por VIH/virología , Integrasa de VIH/química , Proteasa del VIH/química , Transcriptasa Inversa del VIH/química , VIH-1/enzimología , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Mutación , Inhibidores de la Transcriptasa Inversa/uso terapéuticoRESUMEN
Human pegivirus (HPgV; previously called GB virus C/hepatitis G virus) has limited pathogenicity, despite causing persistent infection, and is associated with prolonged survival in human immunodeficiency virus-infected individuals. Although HPgV RNA is found in and produced by T- and B-lymphocytes, the primary permissive cell type(s) are unknown. We quantified HPgV RNA in highly purified CD4(+) and CD8(+) T-cells, including naïve, central memory and effector memory populations, and in B-cells (CD19(+)), NK cells (CD56(+)) and monocytes (CD14(+)) using real-time reverse transcription-PCR. Single-genome sequencing was performed on viruses within individual cell types to estimate genetic diversity among cell populations. HPgV RNA was present in CD4(+) and CD8(+) T-lymphocytes (nine of nine subjects), B-lymphocytes (seven of ten subjects), NK cells and monocytes (both four of five). HPgV RNA levels were higher in naïve (CD45RA(+)) CD4(+) cells than in central memory and effector memory cells (P<0.01). HPgV sequences were highly conserved among subjects (0.117±0.02 substitutions per site; range 0.58-0.14) and within subjects (0.006±0.003 substitutions per site; range 0.006-0.010). The non-synonymous/synonymous substitution ratio was 0.07, suggesting a low selective pressure. Carboxyfluorescein succinimidyl ester (CFSE)-labelled HPgV RNA-containing particles precipitated by a commercial exosome isolation reagent delivered CSFE to uninfected monocytes, NK cells and T- and B-lymphocytes, and HPgV RNA was transferred to PBMCs with evidence of subsequent virus replication. Thus, HPgV RNA-containing serum particles including microvesicles may contribute to delivery of HPgV to PBMCs in vivo, explaining the apparent broad tropism of this persistent human RNA virus.
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Virus GB-C/aislamiento & purificación , Virus GB-C/patogenicidad , Leucocitos Mononucleares/virología , ARN Viral/sangre , Adulto , Secuencia de Aminoácidos , Linfocitos B/virología , Secuencia de Bases , Linfocitos T CD4-Positivos/virología , Linfocitos T CD8-positivos/virología , Secuencia Conservada , Femenino , Infecciones por Flaviviridae/complicaciones , Infecciones por Flaviviridae/virología , Virus GB-C/genética , Infecciones por VIH/complicaciones , Hepatitis Viral Humana/complicaciones , Hepatitis Viral Humana/virología , Humanos , Células Asesinas Naturales/virología , Masculino , Persona de Mediana Edad , Datos de Secuencia Molecular , Monocitos/virología , Filogenia , ARN Helicasas/genética , ARN Viral/genética , Serina Endopeptidasas/genética , Proteínas no Estructurales Virales/genética , Virulencia , Adulto JovenRESUMEN
BACKGROUND AND AIMS: With no report on the overall prevalence and ramifications of hepatitis Delta virus (HDV) infection in the United States for more than two decades, the characteristics of chronic hepatitis B virus (CHB) patients coinfected with HDV, including clinical presentation, rate of hepatitis C virus tri-infection, and HDV viral load, were assessed. METHODS: At California Pacific Medical Center, a retrospective chart review was conducted on all CHB patients. RESULTS: Of 1191 CHB patients, 499 had been tested for HDV, with 42 (8%) determined to be coinfected; half of these were also hepatitis C virus-infected. Cirrhosis was present in 73% of the coinfected, 80% of the tri-infected, but only 22% of the monoinfected. Twenty-nine patients (69%) were Caucasian non-Hispanic; 10 (24%) were Asians and Pacific Islanders. Of 39 patients for whom HBV-DNA quantification at time of HDV presentation was available, 22 (56%) had undetectable levels; four (10%) had levels > 100 000 IU/mL. CONCLUSIONS: HDV affects individuals of all ages and various ethnic groups. Although HBV viral loads are lower, rates of cirrhosis are higher in coinfected patients and higher still in the tri-infected. Our data support revising screening guidelines to advocate for all patients with HBV to be screened for HDV in order to both give the individual patient important information related to the possible need for treatment and to support the public health goal of reducing transmission by educating HDV-negative patients about the need for protection against superinfection and HDV-infected patients about the need to protect against transmission to others.
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Coinfección/epidemiología , Hepatitis B Crónica/epidemiología , Hepatitis D Crónica/epidemiología , Adulto , California/epidemiología , Coinfección/complicaciones , Coinfección/virología , ADN Viral/sangre , Femenino , Virus de la Hepatitis B/aislamiento & purificación , Hepatitis B Crónica/complicaciones , Hepatitis B Crónica/virología , Hepatitis D Crónica/complicaciones , Hepatitis D Crónica/virología , Virus de la Hepatitis Delta/aislamiento & purificación , Humanos , Cirrosis Hepática/epidemiología , Cirrosis Hepática/virología , Masculino , Persona de Mediana Edad , Prevalencia , Estudios Retrospectivos , Carga ViralRESUMEN
Peripheral blood mononuclear cells (PBMCs) represent an extrahepatic hepatitis C virus (HCV) reservoir, the significance of which is unclear due to limited studies and varying test methodologies. In this study, a commercial viral load assay for measuring cell-associated PBMC HCV RNA was evaluated. HCV RNA was extracted from PBMCs, sorted CD14+, and CD19+ cells and corresponding plasma samples using the Abbott m2000 and Real-Time HCV assay. Test performance and influence of HIV seropositivity on plasma and PBMC HCV RNA were studied. Among 51 patients, 67 and 62 unique patient samples had detectable plasma and PBMC HCV viral load, respectively. The median PBMC viral load was 535 IU/1 M cells (range 29-5,190). CD19+ cells had significantly higher viral load than CD14+ cells (median log(10) HCV viral load 2.63 vs. 1.50 IU/ml; P< 0.001). Stability of PBMC viral load over time was demonstrated in untreated patients; all patients with an undetectable plasma HCV viral load after HCV treatment also demonstrated undetectable PBMC viral load. Repeated testing in nine samples yielded consistent PBMC viral load, differing by only 1.3-fold (range 1.0-1.7-fold). Among samples with detectable plasma HCV RNA, the correlation between PBMC and plasma viral load was moderate (r = 0.66) and was greater among HCV mono-infected compared to HIV/HCV co-infected subjects (r = 0.80 vs. 0.52). Measurement of cell-associated PBMC HCV RNA using a commercial assay demonstrated promising test characteristics. Differences in PBMC HCV viral load based on HIV-coinfection status and the significance of greater copy number in B-cells requires further study.
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Coinfección/virología , Hepacivirus/genética , Hepatitis C/virología , Leucocitos Mononucleares/virología , ARN Viral/sangre , Carga Viral/métodos , Adulto , Anciano , Antivirales/farmacología , Antivirales/uso terapéutico , Femenino , Genotipo , Infecciones por VIH/complicaciones , Infecciones por VIH/tratamiento farmacológico , Infecciones por VIH/virología , Hepacivirus/efectos de los fármacos , Hepatitis C/complicaciones , Hepatitis C/tratamiento farmacológico , Humanos , Masculino , Persona de Mediana Edad , Reproducibilidad de los ResultadosRESUMEN
Recent studies have demonstrated that IL28B polymorphisms predict therapeutic responses in chronic hepatitis C virus (HCV)-treated patients; however, the effect on HCV viral diversity, particularly on the HCV protease gene, is not clear. This study sought to evaluate the effect of IL28B polymorphisms on HCV diversity at NS3/4 protease region, which may influence therapeutic response to an HCV protease inhibitor based regimen. Twenty-two patients co-infected with HIV and HCV genotype 1, treatment-naïve on stable HIV antiretroviral therapy initiating interferon-based treatment were evaluated. Plasma HCV NS3 gene diversity was analyzed by clonal analysis at baseline and end of treatment. IL28B (rs12979860) genotypes were tested for associations with virologic outcomes and diversity parameters. There was similar baseline NS3 diversity in patients with CC (favorable) genotype compared to those with CT/TT (unfavorable) genotypes. There was no significant association between IL28B genotype and baseline NS3 nucleotide p-distance, dS-dN, amino acid p-distance, or nucleotide changes. Among patients without a sustained virologic response, between baseline and follow-up there was a significant trend towards decreased diversity after treatment among patients with favorable genotype, which was not observed in unfavorable genotypes. In patients treated with peginterferon/ribavirin therapy, IL28B polymorphism was not associated with enhanced NS3 diversity at baseline. Among non-SVR patients with the less favorable genotype, there was no change in diversity after treatment. This suggests that IL28B genotype is unlikely to have a negative impact on subsequent HCV PI efficacy in patients co-infected with HIV and HCV patients who have previously failed HCV therapy.
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Antivirales/administración & dosificación , Infecciones por VIH/complicaciones , Hepatitis C Crónica/inmunología , Hepatitis C Crónica/virología , Interleucinas/genética , Polimorfismo Genético , Proteínas no Estructurales Virales/genética , Adulto , Femenino , Hepatitis C Crónica/tratamiento farmacológico , Hepatitis C Crónica/genética , Humanos , Interferones/administración & dosificación , Masculino , Persona de Mediana Edad , Proyectos Piloto , Estudios Prospectivos , Ribavirina/administración & dosificación , Resultado del TratamientoRESUMEN
Patients with hepatitis C virus (HCV) and human immunodeficiency virus (HIV) coinfection for whom prior treatment of HCV with interferon-ribavirin has failed may require subsequent treatment with new HCV protease inhibitors (PIs). We evaluated the diversity of HCV nonstructural protein 3 (NS3) in 26 HCV- and HIV-coinfected patients receiving stable antiretroviral therapy (ART) who were treated with interferon-ribavirin. Plasma HCV RNA clonal analysis was performed. There was greater baseline NS3 diversity in patients with nonresponse or relapse than in those with sustained virologic response. Interferon-ribavirin treatment did not result in significant changes in HCV protease gene diversity or significant HCV PI resistance mutations. The effect of prior interferon-ribavirin treatment on HCV NS3 will likely not impact HCV PI efficacy in HIV-coinfected patients receiving ART.
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Infecciones por VIH/complicaciones , Hepacivirus/clasificación , Hepatitis C Crónica/complicaciones , Hepatitis C Crónica/virología , Interferón-alfa/uso terapéutico , Polietilenglicoles/uso terapéutico , Ribavirina/uso terapéutico , Proteínas no Estructurales Virales/genética , Adulto , Anciano , Antivirales/uso terapéutico , Femenino , Genotipo , Hepacivirus/genética , Hepacivirus/aislamiento & purificación , Hepatitis C Crónica/tratamiento farmacológico , Humanos , Interferón alfa-2 , Masculino , Persona de Mediana Edad , Plasma/virología , Polimorfismo Genético , ARN Viral/genética , ARN Viral/aislamiento & purificación , Proteínas RecombinantesRESUMEN
Single-dose nevirapine (NVP) is effective in reducing mother-to-child transmission (MTCT) of HIV; however, the subsequent development of drug resistance is problematic. The pharmacokinetic profile of the HIV entry inhibitor maraviroc after a single intrapartum dose in rhesus macaques was studied to determine whether maraviroc could serve as an alternative to NVP in a single-dose strategy. Four pregnant macaques received an oral dose of maraviroc 2 h before delivery, and both infant and maternal plasma maraviroc concentrations and CCR5 receptor occupancy on CD4(+) lymphocytes were measured over time. Maximum plasma maraviroc concentrations were found at delivery (2-h-postintrapartum dose) in both the mothers and infants, with median concentrations of 974 ng/ml (range, 86 to 2,830 ng/ml) and 22 ng/ml (range, 4 to 99 ng/ml), respectively. Maraviroc was detected in the plasma of mothers up to 48 h after dosing but only as long as 3.5 h in the infants. The median fetal-maternal area under the concentration-time curve (AUC) ratio was 0.009 (range, 0.000 to 0.015). Maraviroc receptor occupancy data showed evidence of unprotected CCR5 receptors on CD4(+) cells in the mothers 24 to 48 h after dosing. Extremely low CCR5 expression on CD4(+) cells of newborn macaques prevented determination of receptor occupancy in the infants. In rhesus macaques, maraviroc was poorly transferred across the placenta and was quickly cleared from the infants' blood. The low concentrations of fetal maraviroc and short pharmacokinetic profile in infants suggest that a single maternal intrapartum dose of maraviroc would not be effective in reducing the risk of MTCT of HIV.
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Fármacos Anti-VIH/sangre , Fármacos Anti-VIH/farmacocinética , Ciclohexanos/sangre , Ciclohexanos/farmacocinética , Triazoles/sangre , Triazoles/farmacocinética , Animales , Fármacos Anti-VIH/administración & dosificación , Fármacos Anti-VIH/uso terapéutico , Linfocitos T CD4-Positivos/metabolismo , Ciclohexanos/administración & dosificación , Ciclohexanos/uso terapéutico , Femenino , Transmisión Vertical de Enfermedad Infecciosa/prevención & control , Macaca mulatta , Maraviroc , Embarazo , Receptores CCR5/metabolismo , Triazoles/administración & dosificación , Triazoles/uso terapéuticoRESUMEN
Many hepatitis C virus (HCV)-infected patients are also infected with HIV, and undergo antiretroviral (ARV) treatment for their human immunodeficiency virus (HIV) infection. Due to changes in HIV burden and immunologic status, HIV ARV treatment may have indirect effects on the HCV population, which could impact the effectiveness of subsequent HCV protease inhibitor (PI) treatment. The genetic variability of the protease-encoding HCV NS3 gene was evaluated in 10 co-infected patients initiating ARVs (both before and after ARV initiation), and compared to the genetic variability in 10 patients on stable ARV therapy. After RT-PCR of plasma-derived HCV RNA, a mean of 20 clones per patient time-point were sequenced and analyzed for changes in the HCV quasispecies population. No significant differences in sequence diversity or complexity at the nucleic acid or amino acid levels were seen at baseline between groups or between the two time points in either group. HCV protease diversity in the pre- and post-ARV treatment samples was not significantly different than samples from patients on stable ARV therapy. There was no significant development of amino acid substitutions known to confer HCV PI resistance in either group. Initiation of ARV for HIV infection does not significantly alter the genetic diversity or complexity of the HCV NS3 gene or result in increased number of HCV PI-associated amino acid changes. These results suggest ARV treatment for HIV would not affect the efficacy of HCV PI treatment.
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Fármacos Anti-VIH/uso terapéutico , Terapia Antirretroviral Altamente Activa , Variación Genética , Infecciones por VIH/tratamiento farmacológico , Hepacivirus/efectos de los fármacos , Hepatitis C Crónica/virología , Proteínas no Estructurales Virales/genética , Adulto , Clonación Molecular , Análisis por Conglomerados , Femenino , Genotipo , Infecciones por VIH/complicaciones , Hepacivirus/clasificación , Hepacivirus/genética , Hepacivirus/aislamiento & purificación , Humanos , Masculino , Persona de Mediana Edad , ARN Viral/sangre , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Análisis de Secuencia de ADNRESUMEN
OBJECTIVE: To characterize nontuberculous mycobacteria (NTM) associated with case clusters at 3 medical facilities. DESIGN: Retrospective cohort study using molecular typing of patient and water isolates. SETTING: Veterans Affairs Medical Centers (VAMCs). METHODS: Isolation and identification of NTM from clinical and water samples using culture, MALDI-TOF, and gene population sequencing to determine species and genetic relatedness. Clinical data were abstracted from electronic health records. RESULTS: An identical strain of Mycobacterium conceptionense was isolated from 41 patients at VA Medical Centers (VAMCs A, B, and D), and from VAMC A's ICU ice machine. Isolates were initially identified as other NTM species within the M. fortuitum clade. Sequencing analyses revealed that they were identical M. conceptionense strains. Overall, 7 patients (17%) met the criteria for pulmonary or nonpulmonary infection with NTM, and 13 of 41 (32%) were treated with effective antimicrobials regardless of infection or colonization status. Separately, a M. mucogenicum patient strain from VAMC A matched a strain isolated from a VAMC B ICU ice machine. VAMC C, in a different state, had a 4-patient cluster with Mycobacterium porcinum. Strains were identical to those isolated from sink-water samples at this facility. CONCLUSION: NTM from hospital water systems are found in hospitalized patients, often during workup for other infections, making attribution of NTM infection problematic. Variable NTM identification methods and changing taxonomy create challenges for epidemiologic investigation and linkage to environmental sources.
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Mycobacteriaceae/aislamiento & purificación , Infecciones por Mycobacterium no Tuberculosas/epidemiología , Infecciones por Mycobacterium no Tuberculosas/microbiología , Anciano , Anciano de 80 o más Años , Femenino , Hospitales de Veteranos , Humanos , Masculino , Persona de Mediana Edad , Mycobacteriaceae/genética , Infecciones por Mycobacterium no Tuberculosas/diagnóstico , Infecciones por Mycobacterium no Tuberculosas/prevención & control , Micobacterias no Tuberculosas/genética , Micobacterias no Tuberculosas/aislamiento & purificación , Esputo/microbiología , Estados Unidos/epidemiología , United States Department of Veterans AffairsRESUMEN
BACKGROUND: Zika virus (ZIKV) is an important flavivirus infection. Although ZIKV infection is rarely fatal, risk for severe disease in adults is not well described. Our objective was to describe the spectrum of illness in U.S. Veterans with ZIKV infection. METHODOLOGY: Case series study including patients with laboratory-confirmed or presumed positive ZIKV infection in all Veterans Health Administration (VHA) medical centers. Adjusted odds ratios of clinical variables associated with hospitalization and neurologic complications was performed. PRINCIPAL FINDINGS: Of 1,538 patients tested between 12/2015-10/2016 and observed through 3/2017, 736 (48%) were RT-PCR or confirmed IgM positive; 655 (89%) were male, and 683 (93%) from VA Caribbean Healthcare System (VACHCS). Ninety-four (13%) were hospitalized, 91 (12%) in the VACHCS. Nineteen (3%) died after ZIKV infection. Hospitalization was associated with increased Charlson co-morbidity index (adjusted odds ratio [OR] 1.2; 95% confidence interval [CI], 1.1-1.3), underlying connective tissue disease (OR, 29.5; CI, 3.6-244.7), congestive heart failure (OR, 6; CI, 2-18.5), dementia (OR, 3.6; CI, 1.1-11.2), neurologic symptom presentation (OR, 3.9; CI, 1.7-9.2), leukocytosis (OR, 11.8; CI, 4.5-31), thrombocytopenia (OR, 7.8; CI, 3.3-18.6), acute kidney injury (OR, 28.9; CI, 5.8-145.1), or using glucocorticoids within 30 days of testing (OR, 13.3; CI 1.3-133). Patients presenting with rash were less likely to be hospitalized (OR, 0.29; CI, 0.13-0.66). Risk for neurologic complications increased with hospitalization (OR, 5.9; CI 2.9-12.2), cerebrovascular disease (OR 4.9; CI 1.7-14.4), and dementia (OR 2.8; CI 1.2-6.6). CONCLUSION: Older Veterans with multiple comorbidities or presenting with neurologic symptoms were at increased risk for hospitalization and neurological complications after ZIKV infection.
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Hospitales de Veteranos/estadística & datos numéricos , Infección por el Virus Zika/epidemiología , Virus Zika/fisiología , Adulto , Anciano , Femenino , Hospitalización , Humanos , Masculino , Persona de Mediana Edad , Estados Unidos , Salud de los Veteranos/estadística & datos numéricos , Virus Zika/genética , Virus Zika/inmunología , Virus Zika/aislamiento & purificación , Infección por el Virus Zika/diagnóstico , Infección por el Virus Zika/terapia , Infección por el Virus Zika/virologíaRESUMEN
In this study, a real-time reverse transcription-polymerase chain reaction (real time RT-PCR) assay targeting 2 genetic segments was established to detect HDV RNA. Utilizing the World Health Organization International Standard for Hepatitis D Virus RNA, the lower limit of detection was 575 IU/mL, and the linearity of quantification ranged from 575,000 IU/mL to 575 IU/mL. 384 HBsAg-positive samples collected from China were tested by this method and HDV antibody detection. Eleven samples were positive for anti-HDV IgG which may persist after HDV resolution, 6 samples were HDV RNA positive, and 5 samples were positive for anti-HDV IgM. This assay showed more sensitivity than the detection of anti-HDV IgM. These data demonstrate that the real-time RT-PCR assay for HDV RNA could be implemented in the clinical detection of HDV infection in chronic HBV-infected patients in China.
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Anticuerpos Antihepatitis/inmunología , Hepatitis D/diagnóstico , Virus de la Hepatitis Delta/aislamiento & purificación , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Adulto , China , Femenino , Genotipo , Hepatitis D/virología , Virus de la Hepatitis Delta/genética , Virus de la Hepatitis Delta/inmunología , Humanos , Masculino , ARN Viral/genética , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Sensibilidad y Especificidad , Carga ViralRESUMEN
This report examines structural changes in a highly mutated, clinical multidrug-resistant HIV-1 protease, and the crystal structure has been solved to 1.3 A resolution in the absence of any inhibitor. This protease variant contains codon mutations at positions 10, 36, 46, 54, 62, 63, 71, 82, 84, and 90 that confer resistance to protease inhibitors. Major differences between the wild-type and the variant include a structural change initiated by the M36V mutation and amplified by additional mutations in the flaps of the protease, resulting in a "wide-open" structure that represents an opening that is 8 A wider than the "open" structure of the wild-type protease. A second structural change is triggered by the L90M mutation that results in reshaping the 23-32 segment. A third key structural change of the protease is due to the mutations from longer to shorter amino acid side chains at positions 82 and 84.
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Proteasa del VIH/química , Proteasa del VIH/genética , VIH-1/enzimología , Secuencia de Aminoácidos , Sitios de Unión , Codón/genética , Cristalografía , Farmacorresistencia Viral Múltiple , Inhibidores de la Proteasa del VIH/farmacología , Datos de Secuencia Molecular , Mutación , Conformación Proteica , Agua/químicaRESUMEN
A major challenge for studying authentic liver cell function and cell replacement therapies is that primary human hepatocytes rapidly lose their advanced function in conventional, 2-dimensional culture platforms. Here, we describe the fabrication of 3-dimensional hexagonally arrayed lobular human liver tissues inspired by the liver's natural architecture. The engineered liver tissues exhibit key features of advanced differentiation, such as human-specific cytochrome P450-mediated drug metabolism and the ability to support efficient infection with patient-derived inoculums of hepatitis C virus. The tissues permit the assessment of antiviral agents and maintain their advanced functions for over 5 months in culture. This extended functionality enabled the prediction of a fatal human-specific hepatotoxicity caused by fialuridine (FIAU), which had escaped detection by preclinical models and short-term clinical studies. The results obtained with the engineered human liver tissue in this study provide proof-of-concept determination of human-specific drug metabolism, demonstrate the ability to support infection with human hepatitis virus derived from an infected patient and subsequent antiviral drug testing against said infection, and facilitate detection of human-specific drug hepatotoxicity associated with late-onset liver failure. Looking forward, the scalability and biocompatibility of the scaffold are also ideal for future cell replacement therapeutic strategies.
RESUMEN
The prenylation inhibitor lonafarnib (LNF) is a potent antiviral agent providing a breakthrough for the treatment of hepatitis delta virus (HDV). The current study used a maximum likelihood approach to model LNF pharmacokinetic (PK) and pharmacodynamic (PD) parameters and predict the dose needed to achieve 99% efficacy using data from 12 patients chronically infected with HDV and treated with LNF 100 mg twice daily (bid) (group 1) or 200 mg bid (group 2) for 28 days. The LNF-PK model predicted average steady-state LNF concentrations of 860 ng/mL and 1,734 ng/mL in groups 1 and 2, respectively, with an LNF absorption rate ka = 0.43/hour and elimination rate ke = 0.045/hour. The PK/PD model identified an average delay of 0.56 hours and an LNF concentration that decreases HDV production by 50%, EC50 = 227 ng/mL, with a Hill factor h = 1.48. The HDV half-life in blood was 1.87 days, and the average steady-state LNF efficacy in blocking HDV production was É = 87.7% for group 1 and É = 95.2% for group 2. A biphasic HDV decline with an average phase 1 decline (0.9 log10 IU/mL and 1.32 log10 IU/mL) was observed in groups 1 and 2, respectively. Phase 2 was not significantly (P = 0.94) different between the two groups, with an average slope of -0.06 log IU/mL/day. The model suggests an LNF dose of â¼610 mg bid would achieve É = 99%. Conclusion: The first PK/PD modeling study in patients with chronic HDV indicates that a â¼3-fold increase in LNF dose (â¼610 mg bid) would achieve 99% antiviral efficacy. A ritonavir-boosted LNF combination may provide a means to increase LNF efficacy with minimal side effects. The modeling findings provide an important advance in understanding HDV dynamics and the basis to optimize LNF therapy for hepatitis D. (Hepatology Communications 2017;1:288-292).
RESUMEN
Nelfinavir was once one of the most commonly used protease inhibitors (PIs). To investigate the genetic mechanisms of multidrug resistance in protease isolates with the primary nelfinavir resistance mutation D30N, we analyzed patterns of protease mutations in 582 viruses with D30N from 460 persons undergoing HIV-1 genotypic resistance testing at Stanford University Hospital from 1997 to 2005. Three patterns of mutational associations were identified. First, D30N was positively associated with N88D but negatively associated with N88S. Second, D30N and L90M were negatively associated except in the presence of N88D, which facilitated the co-occurrence of D30N and L90M. Third, D30N+N88D+L90M formed a stable genetic backbone for the accumulation of additional protease inhibitor (PI) resistance mutations. In 16 patients having isolates with more than one combination of mutations at positions 30, 88, and 90, all exhibited one of the steps in the following progression: D30N-->D30N+N88D-->D30N+N88D+L90M-->D30N+N88D+L90M+(L33F+/-I84V or M46I/L+/-I54V). Although nelfinavir is now used less frequently than other PIs, the well-delineated mutational pathway we describe is likely to influence patterns of cross-resistance in viruses from persons who experience virologic failure while receiving this PI.
Asunto(s)
Farmacorresistencia Viral Múltiple/genética , Infecciones por VIH/tratamiento farmacológico , Infecciones por VIH/genética , Proteasa del VIH/genética , VIH-1/efectos de los fármacos , VIH-1/inmunología , Secuencia de Bases/genética , California , Evolución Molecular , Inhibidores de la Proteasa del VIH/farmacología , Humanos , Datos de Secuencia Molecular , Nelfinavir/farmacologíaRESUMEN
BACKGROUND: During December 2013, the first locally transmitted chikungunya virus (CHIKV) infections in the Americas were reported in the Caribbean. Although CHIKV infection is rarely fatal, risk for severe disease increases with age and medical comorbidities. Herein we describe characteristics of Veterans Health Administration (VHA) patients with CHIKV infection and, among those with infections diagnosed in Puerto Rico, investigated risk factors for hospitalization. METHODOLOGY: We queried VHA's national electronic medical records to identify patients with CHIKV testing during 2014. Demographics, clinical history, laboratory results, and outcomes were abstracted. We investigated risk factors for hospitalization among patients with laboratory-confirmed CHIKV infection in Puerto Rico. PRINCIPAL FINDINGS: We identified 180 laboratory-confirmed CHIKV infections; 148 (82.2%) were diagnosed in Puerto Rico, and 32 (17.8%) were diagnosed among returning travelers elsewhere in the United States. In Puerto Rico, where more patients were hospitalized (55.4% versus 20.0%) and died (4.1% versus 0%), risk for hospitalization increased with age (relative risk [RR]/each 10-year increase, 1.19; 95% confidence interval [CI], 1.06-1.32) and, adjusted for age, increased among patients with congestive heart failure (RR, 1.58; 95% CI, 1.25-1.99), chronic kidney disease (RR, 1.52; 95% CI, 1.19-1.94), diabetes mellitus (RR, 1.39; 95% CI, 1.06-1.84), or chronic lung disease (RR, 1.37; 95% CI, 1.03-1.82). CONCLUSIONS/SIGNIFICANCE: CHIKV infection is an emerging problem among Veterans residing in or visiting areas with CHIKV transmission. Although overall mortality rates are low, clinicians in affected areas should be aware that older patients and patients with comorbidities may be at increased risk for severe disease.
Asunto(s)
Fiebre Chikungunya/epidemiología , United States Department of Veterans Affairs , Adulto , Anciano , Anciano de 80 o más Años , Femenino , Humanos , Masculino , Persona de Mediana Edad , Puerto Rico/epidemiología , Factores de Riesgo , Viaje , Estados Unidos/epidemiologíaRESUMEN
Chronic hepatitis B virus (HBV) infection screening usually includes only HBV surface antigen (HBsAg) testing; HBV core and surface antibody (anti-HBc, anti-HBs) assays, indicating resolved infection and immunity, are not routinely performed. Yet, serum HBV DNA is measurable in approximately 10% of HBsAg-negative/anti-HBc-positive cases, representing occult HBV infection (OBI). Patient blood samples from 2 Veterans Affairs medical center look-back investigations were screened for HBV infection using HBsAg enzyme immunoassays. Supplementary testing included anti-HBc and anti-HBs enzyme immunoassays. For anti-HBc-positive samples, HBV DNA testing was performed. Background OBI prevalence was further estimated at these 2 facilities based on HBV serology testing results from 1999-2012. Finally, a literature review was performed to determine OBI prevalence in the published literature. Of 1887 HBsAg-negative cohort patients, 98 (5.2%) were anti-HBc positive/anti-HBs negative; and 175 (9.3%), anti-HBc positive/anti-HBs positive. Six of 273 were HBV DNA positive, representing 0.3% of the total tested and 2.2% who were anti-HBc positive/anti-HBs negative or anti-HBc positive/anti-HBs positive. Among 32,229 general population veterans at these 2 sites who had any HBV testing, 4/108 (3.7%) were HBV DNA positive, none of whom were part of the cohort. In 129 publications with HBsAg-negative patients, 1817/1,209,426 (0.15%) had OBI. However, excluding blood bank studies with greater than 1000 patients, the OBI rate increased to 1800/17,893 (10%). OBI is not rare and has implications for transmission and disease detection. HBsAg testing alone is insufficient for detecting all chronic HBV infections. These findings may impact blood donation, patient HBV screening, follow-up protocols for patients assumed to have cleared the infection, and initiation of immunosuppression in patients with distant or undetected HBV.
Asunto(s)
Pruebas Diagnósticas de Rutina/métodos , Investigación sobre Servicios de Salud , Hepatitis B Crónica/diagnóstico , Tamizaje Masivo/métodos , ADN Viral/sangre , Anticuerpos contra la Hepatitis B/sangre , Antígenos de Superficie de la Hepatitis B/sangre , Estudios Retrospectivos , Sensibilidad y EspecificidadRESUMEN
We have created a panel of recombinant HIV-1 infectious clones containing common patterns of reverse transcriptase (RT) mutations responsible for resistance to each of the currently available nucleoside reverse transcriptase inhibitors (NRTI), and we have submitted the panel to the National Institutes of Health AIDS Research and Reference Reagent Programme. Testing the activity of new antiretroviral compounds against this panel of drug-resistant clones will determine their relative activity against many clinically relevant NRTI-resistant viruses.
Asunto(s)
Infecciones por VIH/virología , Transcriptasa Inversa del VIH/genética , VIH-1/genética , VIH-1/aislamiento & purificación , Mutación , Células Clonales , Farmacorresistencia Viral Múltiple/genética , Genotipo , Humanos , ARN Viral/aislamiento & purificación , Inhibidores de la Transcriptasa Inversa , Cultivo de VirusRESUMEN
HIV-1 strains that possess a one or two amino acid insert between codons 102 and 103 of the reverse transcriptase (RT) gene were identified in three HIV-1-infected individuals. Each strain also had one or more known mutations associated with nucleoside RT inhibitors (NRTIs) and non-nucleoside RT inhibitors (NNRTIs). Recombinant viruses from these strains had reduced susceptibility to efavirenz and nevirapine, and homology modelling predicted a loss of binding contacts with efavirenz. Mutagenesis studies indicated that replication of insert-containing strains was dependent on RT gene mutations and polymorphisms that co-evolved with the insert. These results suggest that inserts in the NNRTI-binding pocket contribute to NNRTI resistance, but are tolerated only under specific genetic conditions.
Asunto(s)
VIH-1/genética , ADN Polimerasa Dirigida por ARN/genética , Inhibidores de la Transcriptasa Inversa/farmacología , Alquinos , Benzoxazinas , Línea Celular , Codón , Ciclopropanos , Farmacorresistencia Viral/genética , Infecciones por VIH/virología , VIH-1/efectos de los fármacos , Humanos , Modelos Moleculares , Mutación , Nevirapina/farmacología , Nucleósidos , Oxazinas/farmacología , ADN Polimerasa Dirigida por ARN/químicaRESUMEN
A two-amino acid insertion near codon 70 of the HIV-1 protease gene was found in an individual failing protease inhibitor therapy. Susceptibility of this strain to protease inhibitors was similar to that of non-insert-containing strains with comparable resistance mutations and one or more other major PI mutations.