RESUMO
Good markers for monitoring the efficacy of antiretroviral therapy in children do not currently exist. This study examined the effect of antiretroviral therapy on human immunodeficiency virus (HIV-1) unintegrated DNA (uDNA), integrated DNA (iDNA), percent uDNA, immune complex dissociated (ICD) p24 antigenemia, and plasma viral titer. Seven children were followed at therapy initiation and at approximately 3- and 10-month intervals. HIV-1 uDNA was detected in all children prior to start of therapy (average percent uDNA, 43%). At 3 months, the percent HIV uDNA decreased in all patients to an average of 18% (p = 0.01) and at 10 months decreased to an average of 1%. In contrast, the amount of HIV iDNA was relatively constant after initiation of therapy. ICD HIV p24 antigen was detected in all patients prior to therapy (average, 538 pg/ml). Over the study period, the ICD p24 antigen level decreased in three patients and remained relatively unchanged in four patients. Plasma cultures of HIV-1 were positive in only one of the seven patients prior to therapy. Among the methods evaluated, measurement of uDNA was the only parameter which reliable decreased after initiation of nucleoside therapy.
Assuntos
DNA Viral/sangue , Didanosina/uso terapêutico , Infecções por HIV/tratamento farmacológico , HIV-1/genética , Zidovudina/uso terapêutico , Contagem de Linfócito CD4 , Pré-Escolar , Quimioterapia Combinada , Feminino , Proteína do Núcleo p24 do HIV/sangue , Infecções por HIV/virologia , HIV-1/efeitos dos fármacos , HIV-1/imunologia , Humanos , Lactente , Leucócitos Mononucleares/virologia , Masculino , Reação em Cadeia da Polimerase , Viremia/virologia , Zidovudina/farmacologiaRESUMO
The objective of this work was to determine the amount of unintegrated human immunodeficiency virus (HIV) DNA (HIV uDNA) in asymptomatic individuals in the presence or absence of antiretroviral therapy. Twenty-one healthy seropositive individuals with no history of any opportunistic infection or previous use of nucleoside antiretrovirals, and 9 similarly asymptomatic individuals who had initiated nucleoside antiretroviral therapy within the last 24 months were studied. All patients had CD4 lymphocyte counts above 400/microliters. All subjects administered antiretrovirals received 400-600 mg of zidovudine daily for 2-24 months. Two individuals additionally received 400 mg of dideoxyinosine (ddI) daily for 4 and 5 months. Patient peripheral blood mononuclear cells (PBMCs) were examined for integrated and unintegrated HIV DNA by a quantitative PCR assay. In addition, CD4 counts were measured, and free and immune complex dissociated p24 antigen was detected in plasma by ELISA. The mean percentage of HIV uDNA in asymptomatic individuals not on therapy was 59%, with 95% confidence limits from 50 to 69%. In contrast, patients on therapy had a mean of only 13% HIV uDNA, with confidence limits from 2 to 25% (p < 0.001). These findings indicate that a significant amount of HIV DNA in infected, healthy patients not on therapy is in the unintegrated form, and that the amount of HIV uDNA in asymptomatic patients on nucleoside therapy is much less. The amount of HIV uDNA in PBMCs deserves further study as a new marker of the efficacy of antiretroviral therapy.
Assuntos
DNA Viral/sangue , Infecções por HIV/microbiologia , HIV-1/isolamento & purificação , Adulto , Antivirais/uso terapêutico , Biomarcadores , Linfócitos T CD4-Positivos , Didanosina/uso terapêutico , Feminino , Proteína do Núcleo p24 do HIV/sangue , Infecções por HIV/sangue , Infecções por HIV/tratamento farmacológico , Humanos , Contagem de Leucócitos , Masculino , Pessoa de Meia-Idade , Zidovudina/uso terapêuticoRESUMO
Better surrogate markers need to be developed to evaluate therapy in HIV-infected children. This study evaluated plasma RNA, immune complex-dissociated p24 antigenemia, and unintegrated DNA (uDNA) in HIV-infected pediatric patients. Ten children were followed from initiation of nucleoside antiretroviral therapy at intervals up to 24 months. Prior to initiation of therapy, HIV RNA was detected in 10 of 10 patients (median, 76,000 Eq/ml), p24 antigen was detected in 8 of 10 patients (median, 193 pg/ml), and uDNA was detected in 6 of 7 patients (median, 10% uDNA). After 12 months the RNA decreased in all patients and became undetectable in six. In contrast, p24 antigenemia decreased in 6 of 10 patients, remained undetectable in 1, and increased in 3. HIV uDNA decreased in six of six patients and became undetectable in three. There was no overall change in CD4 cell count. Plasma RNA and uDNA levels are both sensitive markers of nucleoside therapy in children; however, they do not covary strongly.
Assuntos
DNA Viral/sangue , Proteína do Núcleo p24 do HIV/sangue , Infecções por HIV/virologia , HIV-1/genética , HIV-1/imunologia , RNA Viral/sangue , Biomarcadores , Contagem de Linfócito CD4 , Criança , Pré-Escolar , Feminino , Seguimentos , Proteína do Núcleo p24 do HIV/imunologia , Infecções por HIV/sangue , Infecções por HIV/imunologia , HIV-1/isolamento & purificação , Humanos , Lactente , Masculino , Resultado do Tratamento , Viremia , Integração Viral , Zidovudina/uso terapêuticoRESUMO
The immunologic relationship between bovine herpesvirus-1 and pseudorabies virus was examined by 80% serum cross-neutralization test, enzyme-linked immunosorbent assays, and Western immunoblotting procedures. Immunogenic cross reactivity between the 2 viruses was observed with both the serum-neutralization test and the enzyme-linked immunosorbent assay. A probing of viral Western immunoblots with rabbit hyperimmune antiserum showed that there were a number of viral-specific cross-reactive proteins between bovine herpesvirus-1 and pseudorabies virus.
Assuntos
Herpesvirus Bovino 1/análise , Herpesvirus Suídeo 1/análise , Proteínas Virais/análise , Animais , Antígenos Virais/análise , Bovinos , Linhagem Celular , Reações Cruzadas , Eletroforese em Gel de Poliacrilamida , Ensaio de Imunoadsorção Enzimática , Herpesvirus Bovino 1/imunologia , Herpesvirus Suídeo 1/imunologia , Soros Imunes , Rim , Peso Molecular , Testes de Neutralização , Especificidade da EspécieRESUMO
The DNA of pseudorabies virus (PRV) strains from the United States and Taiwan and attenuated vaccine strains from Romania were compared by restriction endonuclease analysis. Electrophoretically separated PRV DNA fragments of KpnI and BamHI digests demonstrated cleavage pattern variations which clearly distinguished the 3 Taiwan isolates from all other strains, as well as from each other. One type of variation involved the loss or gain of restriction endonuclease cleavage sites. Examples of this type of variation were clearly observed in fragment patterns of the Taiwan isolates. Another type of variation that occurred at higher frequency for fragments mapping in the repeat and repeat-unique joint regions of the PRV genome involved sequence additions or deletions from existing fragments. This second type of variation occurred in most of the strains analyzed.
Assuntos
DNA Viral/análise , Herpesvirus Suídeo 1/análise , Animais , Linhagem Celular , Enzimas de Restrição do DNA/metabolismo , Variação Genética , Herpesvirus Suídeo 1/genética , Herpesvirus Suídeo 1/patogenicidade , Hungria , Rim , Conformação de Ácido Nucleico , Coelhos , Romênia , Taiwan , Estados Unidos , VirulênciaAssuntos
Vacina contra Difteria, Tétano e Coqueluche/administração & dosagem , Infecções por HIV/imunologia , HIV/imunologia , Vacinas contra Influenza/administração & dosagem , Contagem de Linfócito CD4 , Criança , Estudos de Coortes , Feminino , HIV/genética , HIV/crescimento & desenvolvimento , Infecções por HIV/sangue , Humanos , Masculino , RNA Viral/sangue , Vacinação , Carga ViralRESUMO
A variety of magnetohydrodynamic (MHD) phenomena have been observed on NSTX. Many of these affect fast particle losses, which are of major concern for future burning plasma experiments. Usual diagnostics for studying these phenomena are arrays of Mirnov coils for magnetic oscillations and p-i-n diode arrays for soft x-ray emission from the plasma core. Data reported here are from a unique fast soft x-ray imaging camera (FSXIC) with a wide-angle (pinhole) tangential view of the entire plasma minor cross section. The camera provides a 64x64 pixel image, on a charge coupled device chip, of light resulting from conversion of soft x rays incident on a phosphor to the visible. We have acquired plasma images at frame rates of 1-500 kHz (300 frames/shot) and have observed a variety of MHD phenomena: disruptions, sawteeth, fishbones, tearing modes, and edge localized modes (ELMs). New data including modes with frequency >90 kHz are also presented. Data analysis and modeling techniques used to interpret the FSXIC data are described and compared, and FSXIC results are compared to Mirnov and p-i-n diode array results.
RESUMO
A linear array of four small biased electrodes was installed in NSTX in an attempt to control the width of the scrape-off layer by creating a strong local poloidal electric field. The set of electrodes was separated poloidally by a 1 cm gap between electrodes and were located slightly below the midplane of NSTX, 1 cm behind the rf antenna, and oriented so that each electrode is facing approximately normal to the magnetic field. Each electrode can be independently biased to +/-100 V. Present power supplies limit the current on two electrodes to 30 A and the other two to 10 A each. The effect of local biasing was measured with a set of Langmuir probes placed between the electrodes and another set extending radially outward from the electrodes, and also by the gas puff imaging diagnostic located 1 m away along the magnetic field lines intersecting the electrodes. Two fast cameras were also aimed directly at the electrode array. The hardware and controls of the biasing experiment will be presented and the initial effects on local plasma parameters will be discussed.
RESUMO
The DNA sequence homology between bovine herpesvirus type 1 (BHV-1) and pseudorabies virus (PRV) was examined. Reciprocal cross-hybridization of viral DNA labelled by nick translation to Southern blots of restriction endonuclease-digested DNA detected homologous sequences dispersed throughout the genomes of the two viruses. The DNA-DNA hybrids formed were stable under high-stringency wash conditions. Sequences of a 32P-labelled PRV DNA fragment probe were found to hybridize only to a specific region of the BHV-1 genome, suggesting that the detected sequence homology was not due to fortuitous hybridization of G + C-rich sequences. As measured by liquid reassociation kinetics the homology between these two viruses was approximately 8%.
Assuntos
Genes Virais , Herpesvirus Bovino 1/genética , Herpesvirus Suídeo 1/genética , Animais , Sequência de Bases , Bovinos , Linhagem Celular , Enzimas de Restrição do DNA , DNA Viral/isolamento & purificação , Rim , Cinética , Hibridização de Ácido Nucleico , Biossíntese de Proteínas , Coelhos , Especificidade da EspécieRESUMO
New erythrocyte lysis and leukocyte stabilization reagents (Streck Laboratories, Inc.) were tested in the cytomegalovirus pp65 antigenemia assay, to determine if whole-blood processing time could be delayed to 24 h postdraw. The combination of these reagents gave results comparable to those for patient samples processed immediately after blood draw.
Assuntos
Antígenos Virais/sangue , Infecções por Citomegalovirus/diagnóstico , Infecções por Citomegalovirus/virologia , Citomegalovirus/imunologia , Citomegalovirus/isolamento & purificação , Indicadores e Reagentes , Fosfoproteínas/sangue , Fosfoproteínas/imunologia , Viremia/diagnóstico , Viremia/virologia , Virologia/métodos , Infecções Oportunistas Relacionadas com a AIDS/diagnóstico , Infecções Oportunistas Relacionadas com a AIDS/virologia , Infecções por Citomegalovirus/complicações , Estudos de Avaliação como Assunto , Humanos , Leucócitos/virologia , Fatores de TempoRESUMO
We examined the effect of bacterial pneumonia on the magnitude of circulating plasma HIV RNA in HIV-infected patients. Serum samples from 13 adult HIV-infected patients (median CD4 count = 83 cells/microl) were assayed for HIV RNA using the reverse transcriptase polymerase chain reaction assay (a) before bacterial pneumonia, (b) during the acute phase, and (c) after the recovery from the disease. Patients remained on constant antiretroviral therapy: HIV RNA was detected in all samples tested. The medians before, during, and after bacterial pneumonia were 60,000 copies per ml, 245,000 copies per ml, and 84,000 copies per ml, respectively. All 13 patients had increased HIV RNA levels on developing pneumonia. There was a decline in the level of HIV RNA with recovery from pneumonia in 12 of 13 patients. The difference between the HIV RNA levels before and after pneumonia was not significant, nor was there significant difference in the CD4 counts before and after pneumonia. In conclusion, bacterial pneumonia is associated with a consistent, transient increase in HIV RNA of variable magnitude in AIDS patients. Interpretation of HIV RNA changes for clinical management of AIDS patients must take into account this reversible elevation during infections.
Assuntos
Síndrome da Imunodeficiência Adquirida/complicações , Síndrome da Imunodeficiência Adquirida/virologia , HIV/genética , Pneumonia Bacteriana/complicações , RNA Viral/análise , Adulto , Contagem de Linfócito CD4 , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase , RNA Viral/sangue , Fatores de Tempo , Carga ViralRESUMO
The polymerase chain reaction was used to measure the DNA copy number of human immunodeficiency virus (HIV). Differences in polymerase chain reaction amplification efficiency were controlled by amplifying known amounts of HIV DNA in parallel with samples. This technique is a sensitive, accurate, and reproducible method for the quantitation of HIV DNA.
Assuntos
DNA Viral/análise , HIV/análise , Reação em Cadeia da Polimerase/métodos , Síndrome da Imunodeficiência Adquirida/microbiologia , DNA Viral/genética , DNA Viral/normas , Estudos de Avaliação como Assunto , Amplificação de Genes , HIV/genética , Humanos , Padrões de ReferênciaRESUMO
Better markers for determining therapeutic efficacy of antiretroviral drugs are needed for human immunodeficiency virus (HIV) infection. The amounts of unintegrated HIV DNA (uDNA) were sequentially determined in peripheral blood mononuclear cells (PBMC) from 20 HIV-infected patients starting nucleoside therapy. HIV copy number was determined using a quantitative polymerase chain reaction assay. Before therapy, 19 of 20 patients had detectable HIV uDNA. The average percentage of uDNA was 42%. After 1, 4, and 8 weeks of nucleoside therapy the average decreased to 23% (P < .001), 7%, and 3%, respectively. The amount of HIV uDNA decreased in all 19 patients during the first week and was undetectable in 14 by 8 weeks. Thus, measurement of HIV uDNA has many characteristics needed for a good marker of therapeutic efficacy of antiretroviral drugs, including detectability in a high proportion of patients, large and rapid response to initiation of therapy, and a biologically plausible mechanism.
Assuntos
DNA Viral/metabolismo , Infecções por HIV/tratamento farmacológico , HIV-1/genética , Nucleosídeos/uso terapêutico , Adulto , Marcadores Genéticos , Infecções por HIV/microbiologia , Humanos , Reação em Cadeia da Polimerase , Estudos Prospectivos , Integração ViralRESUMO
A new assay system for the detection of polymerase chain reaction (PCR) amplification products is presented. This single-pot sandwich assay system employs solid-support oligonucleotide-coated capture beads, a rare earth metal chelate-labeled probe, and a time-resolved fluorescence detection. The new assay system was evaluated for various reaction conditions including, DNA denaturation time, hybridization salt concentration, probe concentration, and hybridization time, all of which are important in designing an assay with a high level of sensitivity for the detection of duplex DNA. This nonisotopic assay system was applied to the detection of purified human immunodeficiency virus (HIV) DNA and sensitivity was compared with agarose gel electrophoresis and slot blot hybridization using a 32P-labeled probe. We were able to detect the amplified product from one copy of HIV DNA after 35 cycles of PCR amplification in less than 30 min using this assay, which compared with one copy by gel electrophoresis after 40 cycles of PCR amplification and one copy by slot blot hybridization after 35 cycles of PCR amplification and an overnight exposure of the autoradiogram. Thus, this assay is rapid, sensitive, and easy to use.
Assuntos
DNA Viral/análise , HIV/genética , Hibridização de Ácido Nucleico , Reação em Cadeia da Polimerase , Sequência de Bases , Sondas de DNA , Eletroforese em Gel de Ágar , Fluorescência , Humanos , Dados de Sequência Molecular , Oligonucleotídeos/química , Espectrometria de FluorescênciaRESUMO
We investigated gender as a factor in viral load measurements for human immunodeficiency virus-infected patients. Forty antiretroviral-therapy-naive, age- and CD4-matched women and men were tested for serum RNA and p24 antigen levels prior to antiretroviral therapy and at approximately 12 weeks after therapy. No gender differences were observed for these two markers of viral load.
Assuntos
Infecções por HIV/virologia , HIV-1/isolamento & purificação , RNA Viral/sangue , Viremia/virologia , Adolescente , Adulto , Idoso , Antivirais/uso terapêutico , Biomarcadores , Feminino , Proteína do Núcleo p24 do HIV/sangue , Infecções por HIV/tratamento farmacológico , Humanos , Masculino , Pessoa de Meia-Idade , Caracteres Sexuais , Viremia/tratamento farmacológicoRESUMO
Human immunodeficiency virus (HIV) load markers are being used increasingly to monitor disease progression and evaluate antiretroviral therapy. This study examined plasma HIV RNA and p24 antigen levels before, during, and after 15 AIDS-associated opportunistic disease events in patients with AIDS (median CD4 cell count = 65/microL). Plasma HIV RNA was detected during 13 of the 15 events (median level before an event = 21,000 copies/mL). There was an increase in the level of plasma HIV RNA with the onset of an AIDS-associated opportunistic disease during 11 of 13 events for which HIV RNA was detectable (median level during an event = 145,000 copies/mL). There was a decline in the level of HIV RNA with the recovery from disease (median level after an event = 29,700 copies/mL). In contrast, there was no consistent or significant change in p24 antigen levels or CD4 cell counts with either the onset of or recovery from an event. Clinical interpretation of plasma HIV RNA changes must take into account this reversible elevation during AIDS-associated opportunistic disease.
Assuntos
Infecções Oportunistas Relacionadas com a AIDS/virologia , Síndrome da Imunodeficiência Adquirida/virologia , Proteína do Núcleo p24 do HIV/sangue , RNA Viral/sangue , Infecções Oportunistas Relacionadas com a AIDS/complicações , Síndrome da Imunodeficiência Adquirida/complicações , Adulto , Biomarcadores , Feminino , Proteína do Núcleo p24 do HIV/genética , Humanos , Estudos Longitudinais , Masculino , Pessoa de Meia-Idade , RNA Viral/genéticaRESUMO
The development of technology to increase the sensitivity and speed of detection of bacterial pathogens in samples is important for diagnosis and monitoring of illness. We have developed a sensitive and rapid method for the detection of bacteria, using Escherichia coli as a model, which combines transcription-based target amplification with a bead-based sandwich hybridization assay using rare earth metal chelate labelled probes and time-resolved fluorescence detection. Using these methods as little as 100 copies (0.00016 attomoles) of purified native Escherichia coli rRNA or just one bacterial cell in a spiked sample could be detected. These results demonstrate that amplification of rRNA by transcription-based amplification and detection by time-resolved fluorescence provide a sensitive technology for the direct detection of micro-organisms without the requirement for prior cultivation.
Assuntos
Escherichia coli/isolamento & purificação , Técnicas de Amplificação de Ácido Nucleico , RNA Bacteriano/isolamento & purificação , RNA Ribossômico/isolamento & purificação , Sequência de Bases , Corantes Fluorescentes , Humanos , Metais Terras Raras , Microesferas , Dados de Sequência Molecular , Sondas de Oligonucleotídeos , Poliestirenos , RNA Bacteriano/genética , RNA Ribossômico/genética , Sensibilidade e EspecificidadeRESUMO
Simple and sensitive methods to directly detect the human immunodeficiency virus (HIV) are needed for routine use in the clinical laboratory. In this study, we compared DNA probes prepared by: (1) nick translation with biotinylated dATP; (2) direct covalent biotinylation with photobiotin; (3) direct covalent reaction with 2-acetylaminofluorene (AAF); and (4) a standard radioactive (32P) nick translation procedure. These four DNA probes were hybridized with dilutions of purified target HIV DNA blotted onto nitrocellulose strips. Hybridization was detected using a complex of strepavidin-alkaline phosphatase [for (1) and (2)], alkaline phosphatase-tagged antibodies [for (3)] and by autoradiography [for (4)]. Alkaline phosphatase was detected colorimetrically using nitroblue tetrazolium and 5-bromo-4-chloro-3-indolyl phosphate. After 1 h, AAF probes were most sensitive (amount detected less than 5 pg), followed by biotin (10 pg), photobiotinylated probes (20 pg) and the radioactive probe (10 pg). The AAF probes were then used to detect HIV DNA in infected CEM cells. We conclude that non-radioactive DNA labelling methods can be used to directly detect HIV DNA under conditions compatible with present clinical laboratory procedures.
Assuntos
DNA Viral/análise , HIV/genética , Hibridização de Ácido Nucleico , 2-Acetilaminofluoreno , Biotina , HIV/isolamento & purificação , Humanos , Radioisótopos de FósforoRESUMO
Simian acquired immune deficiency syndrome (SAIDS) in rhesus macaques (Macaca mulatta) at the California Primate Research Center is caused by a type D retrovirus designated SAIDS retrovirus serotype 1 (SRV-1). This syndrome is characterized by profound immunosuppression and death associated with opportunistic infections. Neurologic signs and lesions have not been described as part of this syndrome. The distribution of SRV-1 in the salivary glands, lymph nodes, spleens, thymuses, and brains of eight virus-infected rhesus macaques was examined by immunohistochemistry. Electron microscopy, in situ RNA hybridization, and Southern blot hybridization were also performed on selected tissues to detect viral particles, RNA, and DNA, respectively. In seven of eight SRV-1-infected animals, the transmembrane envelope glycoprotein (gp20) of SRV-1 was present in three or more tissues, but never in the brain. In the remaining animal, no viral antigen was detected in any tissue. In this same group of animals, viral nucleic acid was detected in the lymph nodes of six of six animals by Southern blot hybridization, in the salivary glands of two of five animals by both Southern blot and in situ hybridizations, and, surprisingly, in the brains of three of three animals by Southern blot and of three of five animals by in situ hybridization, including the one animal in which viral gp20 was undetectable. None of these animals had neurologic signs or lesions. The detection of viral nucleic acid in the absence of viral antigen in the brain suggests latent SRV-1 infection of the central nervous system.
Assuntos
Síndrome da Imunodeficiência Adquirida/veterinária , Retroviridae/crescimento & desenvolvimento , Síndrome da Imunodeficiência Adquirida/microbiologia , Síndrome da Imunodeficiência Adquirida/patologia , Animais , Anticorpos Monoclonais , Encéfalo/microbiologia , Técnicas Imunoenzimáticas , Tecido Linfoide/microbiologia , Macaca mulatta , Microscopia Eletrônica , Hibridização de Ácido Nucleico , RNA Viral/metabolismo , Glândulas Salivares/microbiologia , Distribuição Tecidual , Proteínas do Envelope Viral/metabolismoRESUMO
There is an urgent need for rapid and sensitive methods to assess human immunodeficiency virus (HIV) infection in infants and children. We evaluated an approach by using the self-sustained sequence replication reaction (3SR) to amplify HIV type 1 (HIV-1) RNA directly. The amplified RNA product was then detected by bead-based sandwich oligonucleotide capture hybridization and rare earth metal chelate time-resolved fluorescence. The sensitivity of this technology was determined to be less than 12 HIV-1 RNA copies with an amplification level of 10(10)-fold with purified HIV-1 RNA. Plasma samples from 19 high-risk pediatric patients younger than 5 years of age were examined, and results were compared with viral culture of patient plasma. Results from plasma culture and 3SR amplification agreed for 14 of these patients and disagreed for 5. Of the five samples which did not agree, four were positive by 3SR and negative by culture and one was positive by culture and negative by 3SR but became positive by 3SR at a subsequent testing. We conclude that 3SR amplification coupled with time-resolved fluorescence is a promising technology for investigating the relationship between the presence of HIV-1 RNA in plasma and progression of disease in HIV-infected pediatric patients. This technology should be important in the assessment of HIV-1 infection, in evaluating drug therapies, and in understanding the pathogenesis and transmission of the virus.