RESUMO
Thirteen laboratories evaluated the reproducibility of sequencing methods to detect drug resistance mutations in HIV-1 reverse transcriptase (RT). Blinded, cultured peripheral blood mononuclear cell pellets were distributed to each laboratory. Each laboratory used its preferred method for sequencing proviral DNA. Differences in protocols included: DNA purification; number of PCR amplifications; PCR product purification; sequence/location of PCR/sequencing primers; sequencing template; sequencing reaction label; sequencing polymerase; and use of manual versus automated methods to resolve sequencing reaction products. Five unknowns were evaluated. Thirteen laboratories submitted 39043 nucleotide assignments spanning codons 10-256 of HIV-1 RT. A consensus nucleotide assignment (defined as agreement among > or = 75% of laboratories) could be made in over 99% of nucleotide positions, and was more frequent in the three laboratory isolates. The overall rate of discrepant nucleotide assignments was 0.29%. A consensus nucleotide assignment could not be made at RT codon 41 in the clinical isolate tested. Clonal analysis revealed that this was due to the presence of a mixture of wild-type and mutant genotypes. These observations suggest that sequencing methodologies currently in use in ACTG laboratories to sequence HIV-1 RT yield highly concordant results for laboratory strains; however, more discrepancies among laboratories may occur when clinical isolates are tested.
Assuntos
DNA Viral/análise , Transcriptase Reversa do HIV/genética , HIV-1/enzimologia , Laboratórios/normas , Mutação , Análise de Sequência de DNA/métodos , Códon , Resistência Microbiana a Medicamentos , Amplificação de Genes , HIV-1/efeitos dos fármacos , HIV-1/genética , Humanos , Reação em Cadeia da Polimerase , Provírus/genética , Reprodutibilidade dos Testes , Análise de Sequência de DNA/normas , Zidovudina/farmacologiaRESUMO
Antiviral susceptibilities to ganciclovir, foscarnet, and cidofovir and sequencing of UL97 and DNA polymerase were done on 23 cytomegalovirus (CMV) isolates from 10 immunocompromised persons with end-organ CMV disease who were treated with ganciclovir alone or ganciclovir followed by foscarnet. Screening of UL97 for ganciclovir resistance mutations was done by restriction digest analysis. Of 14 isolates resistant to ganciclovir, 11 (79%) contained one or more UL97 mutations at codons known to confer resistance to this compound, and 10 (91%) had a concordant mutant pattern by restriction digest analysis. Of 9 isolates containing mutations in conserved regions of the DNA polymerase, 8 were resistant to ganciclovir, and 4 were cross-resistant to cidofovir. All isolates were susceptible to foscarnet. It is concluded that ganciclovir-resistant clinical CMV isolates may contain UL97 mutations, DNA polymerase mutations, or mutations in both genes. Ganciclovir therapy may select for CMV isolates that are cross-resistant to cidofovir.
Assuntos
Infecções Oportunistas Relacionadas com a AIDS/virologia , Antivirais/farmacologia , Infecções por Citomegalovirus/virologia , Citomegalovirus/efeitos dos fármacos , Citomegalovirus/genética , DNA Polimerase Dirigida por DNA/genética , Hospedeiro Imunocomprometido , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Antivirais/uso terapêutico , Sequência de Bases , Citomegalovirus/isolamento & purificação , Infecções por Citomegalovirus/tratamento farmacológico , Primers do DNA , Foscarnet/uso terapêutico , Ganciclovir/uso terapêutico , Genótipo , Humanos , Testes de Sensibilidade Microbiana , Reação em Cadeia da PolimeraseRESUMO
Sequence-specific PCR was used in six laboratories and a ligase detection reaction was used in one laboratory to detect the zidovudine-resistance mutation at codon 215 of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase DNA. The genotypes of 27 different clinical samples, including cultured HIV-1 isolates, peripheral blood mononuclear cells, and plasma, were correctly identified by 140 of 154 (91%) assays. The sensitivity for detecting a mutation was 96% for HIV-1 reverse transcriptase DNA clone mixtures containing 30% mutant DNA and 62% for mixtures containing 6% mutant DNA.
Assuntos
DNA Ligases , Resistência Microbiana a Medicamentos/genética , HIV-1/efeitos dos fármacos , HIV-1/genética , Mutação , Reação em Cadeia da Polimerase/métodos , Antivirais/farmacologia , Sequência de Bases , Códon/genética , Primers do DNA/genética , DNA Viral/genética , Estudos de Avaliação como Assunto , Genótipo , Infecções por HIV/tratamento farmacológico , Infecções por HIV/virologia , Humanos , Laboratórios , Leucócitos Mononucleares/virologia , Plasma/virologia , Reação em Cadeia da Polimerase/estatística & dados numéricos , Sensibilidade e Especificidade , Zidovudina/farmacologiaAssuntos
Infecções por HIV/tratamento farmacológico , HIV-1/efeitos dos fármacos , Zidovudina/uso terapêutico , Adulto , Sequência de Bases , DNA Viral , Resistência a Medicamentos , Infecções por HIV/microbiologia , Infecções por HIV/transmissão , HIV-1/isolamento & purificação , Humanos , Masculino , Dados de Sequência MolecularRESUMO
The ability of an alpha CD4-pokeweed antiviral protein (PAP) immunoconjugate to inhibit replication of human immunodeficiency virus type 1 (HIV-1) was evaluated in vitro with 22 clinical HIV-1 strains obtained from four seropositive asymptomatic individuals, three patients with AIDS-related complex, and four patients with AIDS. Fifteen isolates were from zidovudine-untreated individuals, whereas seven isolates were obtained after 24 to 104 weeks of therapy with zidovudine, alone or alternating with zalcitabine. Mean zidovudine 50% inhibitory concentrations (IC50s) were 126 nM (range, 1 to 607 nM) for isolates from zidovudine-untreated individuals and 2,498 nM (range, 14 to 6,497 nM) for strains from patients treated with antiretroviral agents. Mean alpha CD4-PAP IC50s were 48 x 10(-3) nM (range, 0.02 x 10(-3) to 212 x 10(-3) nM) for isolates from zidovudine-untreated individuals, and 16 x 10(-3) nM (range, 2 x 10(-3) to 28 x 10(-3) nM) for isolates from treated patients. Overall, higher concentrations of alpha CD4-PAP were necessary to inhibit HIV-1 strains from untreated individuals at more advanced stages of disease. Seventeen isolates were susceptible to zidovudine (mean IC50, 117 nM), and five were resistant to zidovudine (mean IC50, 3,724 nM). Mean alpha CD4-PAP IC50s were 43 x 10(-3) nM for zidovudine-susceptible isolates and 19 x 10(-3) nM for isolates resistant to zidovudine. All HIV-1 strains had IC50s greater than 0.5 nM for unconjugated PAP, the alpha CD19-PAP immunoconjugate, and monoclonal antibody alpha CD4. At concentrations as high as 5,000 nM, alphaCD4-PAP did not inhibit colony formation by normal bone marrow progenitor cells(BFU-E, CFU-GM , and CFU-GEMM) or myeloid cell lines (KG-1 and HL-60) and did not decrease cell viabilities of T-cell (Jurkat) or B-cell (FL-112 and Raji) precursor lines. Overall, alphaCD4-PAP demonstrated more potent anti-HIV-1 activity than zidovudine and inhibited replication of zidovudine-susceptible and zidovudine-resistant viruses at concentrations that were not toxic to lymphohematopoietic cell populations.
Assuntos
Antivirais/farmacologia , Antígenos CD4/imunologia , HIV-1/efeitos dos fármacos , Imunotoxinas/farmacologia , N-Glicosil Hidrolases , Proteínas de Plantas/farmacologia , Complexo Relacionado com a AIDS/microbiologia , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Efeito Citopatogênico Viral , Soropositividade para HIV/microbiologia , HIV-1/imunologia , Humanos , Proteínas Inativadoras de Ribossomos Tipo 1 , Zidovudina/farmacologiaRESUMO
A study was conducted to compare our standard culture with a new microculture procedure for isolation of human immunodeficiency virus type 1 (HIV-1) from blood leukocytes. A total of 137 blood specimens from 102 HIV-1 antibody-positive individuals (52 were asymptomatic, 31 were symptomatic, and 19 had AIDS) were cultured in a microculture system in which 10(6) of the patients' peripheral blood mononuclear cells (PBMC) were cocultured with 10(6) phytohemagglutinin (PHA)-stimulated PBMC from an HIV-1 antibody-negative blood donor in 1.2 ml of culture medium. Results were compared with those of a historical control group of 139 standard HIV-1 cultures from 108 HIV-1 antibody-positive subjects (58 were asymptomatic, 36 were symptomatic, and 14 had AIDS). For standard cultures, 10 x 10(6) of the patients' PBMC were cocultured with 5 x 10(6) PHA-stimulated PBMC from an HIV-1 antibody-negative blood donor in 15 ml of culture medium. HIV-1 was isolated in 128 (93%) microcultures and 133 (96%) standard cultures. Both methods identified more than 75% of the positive cultures within 7 days and 100% of the positive cultures within 14 days. The isolation rates for HIV-1 in microcultures compared with standard cultures were 91 versus 93% (specimens from asymptomatic individuals), 93 versus 96% (specimens from symptomatic individuals), and 97 versus 100% (specimens from patients with AIDS). The median time to positivity for both culture methods was 7 days, and this correlated significantly with symptoms and CD4+ cell counts. The microculture method is a sensitive and less expensive system for isolation of HIV-1 from PBMC of HIV-1 antibody-positive individuals, and we recommend it as the culture method of choice, especially for children and patients with AIDS and severe anemia or leukopenia whose blood volume is an important consideration.
Assuntos
HIV-1/isolamento & purificação , Leucócitos/microbiologia , Virologia/métodos , Estudos de Avaliação como Assunto , Infecções por HIV/microbiologia , Humanos , Sensibilidade e Especificidade , Viremia/microbiologia , Virologia/estatística & dados numéricosRESUMO
Because morphine has been shown to alter the function of human T lymphocytes and monocytes, we postulated that morphine would promote the growth of HIV-1 in these cells. To test this hypothesis, a coculture assay was used consisting of phytohemagglutinin (PHA)-activated peripheral blood mononuclear cells (PBMC) from normal donors and PBMC which had been infected with a viral isolate from an asymptomatic patient, HIV-1AT. The growth of HIV-1AT, as reflected by the concentration of p24 antigen in coculture supernatants, was markedly increased in cocultures that contained morphine. A bell-shaped dose-response curve was observed with three- to fourfold increased growth at a morphine concentration of 10(-12) M. Augmentation of HIV-1AT growth by morphine required an interaction with the PHA-activated donor PBMC. Furthermore, potentiation of HIV-1AT growth by morphine was stereospecific and was antagonized by naloxone and beta-funaltrexamine indicating involvement of an opiate receptor mechanism. These findings provide an additional explanation of how opiates could act as a cofactor in the pathogenesis of HIV-1 in intravenous drug users.
Assuntos
HIV-1/crescimento & desenvolvimento , Leucócitos Mononucleares/microbiologia , Morfina/farmacologia , Células Cultivadas , Produtos do Gene gag/metabolismo , Proteína do Núcleo p24 do HIV , Humanos , Leucócitos Mononucleares/imunologia , Ativação Linfocitária , Naloxona/farmacologia , Naltrexona/análogos & derivados , Naltrexona/farmacologia , Antagonistas de Entorpecentes , Receptores Opioides/metabolismo , Proteínas do Core Viral/metabolismoRESUMO
Between February 1987 and October 1988, peripheral mononuclear blood cells (PBMC) from 409 adult individuals antibody positive by Western (immuno-)blot for human immunodeficiency virus type 1 (HIV-1) (56 acquired immunodeficiency syndrome [AIDS] patients, 88 patients with AIDS-related complex, and 265 asymptomatic individuals) were consecutively cultured for HIV-1 or tested for the presence of HIV-1 DNA sequences by a polymerase chain reaction assay (PCR). We isolated HIV-1 or detected HIV-1 DNA sequences from the PBMC of all 409 HIV-1 antibody-positive individuals. None of 131 healthy HIV-1 antibody-negative individuals were HIV-1 culture positive, nor were HIV-1 DNA sequences detected by PCR in the blood specimens of 43 seronegative individuals. In addition, HIV-1 PCR and HIV-1 culture were compared in testing the PBMC of 59 HIV-1 antibody-positive and 20 HIV-1 antibody-negative hemophiliacs. Both methods were found to have sensitivities and specificities of at least 97 and 100%, respectively. In contrast, the sensitivities of serum HIV-1 antigen testing in AIDS patients and asymptomatic seropositive patients were 42 and 17%, respectively. Our ability to directly demonstrate HIV-1 infection in all HIV-1 antibody-positive individuals provides definitive support that HIV-1 antibody positivity is associated with present HIV-1 infection. Moreover, the sensitivities and specificities of PCR and culture for the detection of HIV-1 appear to be equivalent, and both methods are superior to testing for HIV-1 antigen in serum for the direct detection of HIV-1.
Assuntos
Soropositividade para HIV/microbiologia , HIV-1/isolamento & purificação , Complexo Relacionado com a AIDS/imunologia , Complexo Relacionado com a AIDS/microbiologia , Síndrome da Imunodeficiência Adquirida/imunologia , Síndrome da Imunodeficiência Adquirida/microbiologia , Anticorpos Anti-HIV/isolamento & purificação , Antígenos HIV/isolamento & purificação , Soropositividade para HIV/imunologia , HIV-1/imunologia , Humanos , Reação em Cadeia da PolimeraseRESUMO
To determine whether apparently healthy persons who have had repeatedly reactive enzyme immunoassays and an indeterminate Western blot assay for antibody to the human immunodeficiency virus type 1 (HIV-1) are infected with HIV-1 or HIV-2, we studied 99 such volunteer blood donors in a low-risk area of the country. The subjects were interviewed about HIV risk factors. Coded blood specimens were tested again for HIV-1 antibody (by two different enzyme immunoassays, a Western blot assay and a radioimmunoprecipitation assay) and for HIV-2 antibody by enzyme immunoassay, for HIV-1 by the serum antigen test, for HIV-1 by culture, for human T-cell leukemia virus Type I or II antibody by enzyme immunoassay, and for sequences of HIV DNA by the polymerase chain reaction. Of the 99 blood donors, 98 reported no risk factors for HIV-1 infection; 1 donor had used intravenous drugs. After a median of 14 months (range, 1 to 30) from the time of the initial test, 65 subjects (66 percent) were still repeatedly reactive for HIV-1 antibody on at least one immunoassay. In 91 subjects (92 percent) the Western blot results were still indeterminate, whereas in 8 they were negative. No donor met the criteria for a positive Western blot assay for HIV-1, and none had evidence of HIV-1 or HIV-2 infection on culture or by any other test. We conclude that persons at low risk for HIV infection who have persistent indeterminate HIV-1 Western blots are rarely if ever infected with HIV-1 or HIV-2.
Assuntos
Doadores de Sangue , Western Blotting/normas , Anticorpos Anti-HIV/análise , Infecções por HIV/diagnóstico , HIV-1/imunologia , Adulto , Sequência de Bases , Demografia , Feminino , Humanos , Técnicas Imunoenzimáticas , Masculino , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , Ensaio de Radioimunoprecipitação , Fatores de RiscoAssuntos
Autopsia , HIV-1/isolamento & purificação , Adulto , Humanos , Masculino , Fatores de TempoRESUMO
Peripheral blood mononuclear cell samples from human immunodeficiency virus (HIV) antibody-positive subjects were inoculated into cell cultures used for routine viral isolation to determine if HIV could survive in them. HIV was recovered from 5 (19%) of 26 day 1 supernatants and from four of the five samples in all three types of cell cultures. We conclude that HIV may survive for at least 24 h in standard virology cell culture systems and therefore is a potential risk for laboratory personnel.
Assuntos
Síndrome da Imunodeficiência Adquirida/transmissão , HIV-1/isolamento & purificação , Leucócitos Mononucleares/microbiologia , Células Cultivadas , HIV-1/crescimento & desenvolvimento , HumanosRESUMO
In order to confirm the presence and determine the frequency of human immunodeficiency virus, type 1 (HIV-1) infection prior to antibody production, 23 healthy women with histories of repeated unprotected sexual exposure to HIV-1 infected hemophiliacs were tested for evidence of HIV-1 infection. Female subjects were tested for HIV-1 antibody (enzyme immunoassay [EIA] and Western blot), HIV-1 serum antigen, HIV-1 DNA gag sequences by the polymerase chain reaction, and HIV-1 virus isolation from peripheral mononuclear cells. Twenty-two of 23 (96%) women were negative by all HIV-1 assays. One woman was positive by all the HIV-1 assays including an EIA screening test for HIV-1 antibody. These preliminary results suggest that the frequency of HIV-1 infection in antibody-negative sexual partners of HIV-1 infected individuals is probably very low.
Assuntos
Síndrome da Imunodeficiência Adquirida/psicologia , Soropositividade para HIV/psicologia , HIV-1 , Hemofilia A/psicologia , Parceiros Sexuais , Síndrome da Imunodeficiência Adquirida/epidemiologia , Síndrome da Imunodeficiência Adquirida/transmissão , Adulto , Feminino , Anticorpos Anti-HIV/análise , Soropositividade para HIV/diagnóstico , Soropositividade para HIV/epidemiologia , Hemofilia A/complicações , Humanos , Pessoa de Meia-IdadeRESUMO
The detection and recruitment of HIV antigen-positive asymptomatic individuals for clinical trials is important. Two commercial enzyme-linked immunosorbent assays (ELISA) for the detection and quantitation of human immunodeficiency virus (HIV) antigens were evaluated for sensitivity by testing serum samples from 155 asymptomatic HIV Western blot positive individuals. The Abbott HIV antigen ELISA detected HIV antigen in the serum of 17 (11.0%) of 155 patients compared with 18 (11.6%) of 155 by the Coulter HIV antigen ELISA. In serial twofold dilution experiments, there was no significant difference in sensitivity between these two assays in the detection of HIV serum antigen. However, both assays are limited in their ability to detect HIV antigen in most asymptomatic HIV-infected patients. This low detection rate should be taken into account in the design of clinical trials involving asymptomatic infected patients.
Assuntos
Antígenos HIV/análise , Soropositividade para HIV/sangue , Adolescente , Adulto , Western Blotting , Ensaios Clínicos como Assunto , Ensaio de Imunoadsorção Enzimática/instrumentação , Ensaio de Imunoadsorção Enzimática/métodos , Feminino , Proteína do Núcleo p24 do HIV , Humanos , Masculino , Pessoa de Meia-Idade , Proteínas dos Retroviridae/análiseRESUMO
Cultures of peripheral blood mononuclear cells for human immunodeficiency virus type 1 (HIV-1) and assays for the p24 antigen were performed for a group of 75 unselected hemophiliacs to determine whether patients positive for HIV-1 antibody are actively infected rather than immunized by viral proteins in non-heat-treated factor VIII or IX concentrates. Fifty-six (75%) of the 75 hemophiliacs were antibody positive and 55 (98%) of the 56 with antibodies also had positive cultures. The one culture-negative individual had detectable HIV-1 proviral DNA sequences in three separate samples of peripheral blood mononuclear cell DNA, as detected by a polymerase chain reaction assay. Detection of serum p24 antigen and the time to development of a positive culture were significantly more frequent and shorter, respectively, in symptomatic vs asymptomatic patients. None of the 19 hemophiliacs negative for HIV-1 antibody had positive cultures, detectable p24 serum antigen, or symptoms of HIV-1 infection. Moreover, latent HIV-1 infection was not detected in 16 female sexual partners of hemophiliacs positive for HIV-1 antibody using Western blot testing, assays for p24 antigen, HIV-1 cultures, and polymerase chain reaction assays, despite repeated unprotected sexual exposure. We conclude that antibody-positive hemophiliacs have been actively infected by HIV-1 and that a long period of latent HIV-1 infection prior to overt seroconversion is unlikely.
Assuntos
Soropositividade para HIV/epidemiologia , Hemofilia A/imunologia , Adolescente , Adulto , Idoso , Criança , Ensaio de Imunoadsorção Enzimática , Fator IX/administração & dosagem , Fator VIII/administração & dosagem , Feminino , Amplificação de Genes , Anticorpos Anti-HIV/análise , Antígenos HIV/análise , HIV-1/genética , HIV-1/imunologia , HIV-1/isolamento & purificação , Humanos , Masculino , Pessoa de Meia-Idade , Parceiros Sexuais , Cultura de VírusRESUMO
Peripheral blood mononuclear cells from 142 consecutive patients with antibodies to human immunodeficiency virus type 1 (HIV-1) were cultured for HIV-1. All 72 patients with symptoms of HIV-1 infection were culture positive, as were 69 of 70 asymptomatic patients. Of the 142 patients, 132 (93%) were culture positive within 10 days after initiation of the culture.
Assuntos
HIV/isolamento & purificação , Leucócitos Mononucleares/microbiologia , Complexo Relacionado com a AIDS/microbiologia , Síndrome da Imunodeficiência Adquirida/microbiologia , Antígenos Virais/análise , Células Cultivadas , Feminino , HIV/imunologia , Antígenos HIV , Humanos , MasculinoRESUMO
Two commercial enzyme-linked immunosorbent assays (EIAs) for human immunodeficiency virus (HIV) antigens were evaluated for sensitivity and reproducibility by use of supernatant fluid from cell cultures of peripheral mononuclear cells of 18 patients with acquired immunodeficiency syndrome (AIDS) and 12 asymptomatic HIV antibody-positive patients. Both commercial assays detected HIV antigen in all cultures by day 21; however, the Abbott HIV antigen EIA (Abbott Laboratories, North Chicago, IL) detected HIV antigen three to seven days earlier in 15 of 48 cultures (31%), on the same day in 32 cultures (67%), and three days later in 1 culture (2%) when compared with the DuPont HIV antigen EIA (DuPont Laboratories, Wilmington, DE). In serial twofold dilution experiments, the Abbott HIV Ag EIA was found to have at least a twofold to eightfold increased sensitivity over the DuPont assay. Repeat testing of 15 initially positive supernatant fluids by both assays revealed that 13 of 15 and 12 of 15 were consistently positive by the Abbott and DuPont assays, respectively. The authors conclude that the Abbott EIA demonstrated better performance in this study than the DuPont EIA for detection of HIV in cell culture because of its shorter time-to-positivity and greater sensitivity.
Assuntos
Antígenos Virais/análise , HIV/análise , Técnicas Imunoenzimáticas , HumanosRESUMO
The reverse transcriptase (RT) assay for human immunodeficiency virus (HIV) is tedious, expensive, and nonspecific for HIV activity. Because the Retro-Tek viral capture assay (VCA) (Cellular Products, Inc., Buffalo, NY) is relatively quick, inexpensive, and specific for HIV antigens, we compared the sensitivity and specificity of the two assays in a blinded fashion to see if the VCA could replace the RT assay. Specifically, peripheral mononuclear cells were cocultured from 11 Western blot-positive acquired immunodeficiency syndrome (AIDS) patients. The culture supernatant fluids were tested every 4-7 days for at least 28 days by VCA for HIV antigens, and by RT assay for retrovirus activity. Ten seronegative healthy donors with no risk factors were also cocultured and tested. Seven (64%) of 11 AIDS patients were positive by VCA and confirmed by RT assay in each instance. Concordance between VCA and RT assay was 100% in each patient. Time-to-positive detection was 7-28 days for both assays. Once a culture became positive by either assay, subsequent culture supernatant fluids remained positive by both assays until the culture was discarded, usually at day 28. The ten healthy seronegative donors were negative by both assays. These results suggest that the VCA is preferable to the RT assay for detection of HIV.