RESUMO
A novel method for the collection and transportation of dried-blood-plasma samples, SampleTanker (ST), was developed and compared to standard shipping protocols for frozen-plasma specimens containing human immunodeficiency virus type 1 (HIV-1) and/or hepatitis C virus (HCV). Matched frozen and dried 1-ml EDTA-containing plasma samples were collected and analyzed by several molecular-based virologic assays. After addition of 1.175 ml of reconstitution buffer, 1.035 ml of dried plasma was recovered. Mean intra-assay variances were 0.05, 0.05, and 0.06 log(10) copies/ml for the Versant, Amplicor, and NucliSens QT HIV-1 load assays, respectively (P, not significant). However, mean HIV-1 viral load was consistently reduced in dried samples by 0.32 to 0.51 log(10) copies/ml, depending on assay type (P < 0.05). Infectious HIV-1 was not recovered from dried ST plasma. There was no significant difference in HIV-1 viral load results obtained using ST after 8 weeks of storage at ambient temperature. Compared to frozen plasma, HIV-1 genotypic results were >99% concordant at the nucleotide and amino acid levels, as well as for resistance-associated mutations. We further demonstrated successful detection of multiple analytes, including HIV-1 viral load, HIV-1 antiretroviral resistance genotype, and HCV genotype, from a single ST unit. Dried plasma collected with ST yielded comparable results to frozen samples for multiple-analyte clinical testing. As such, ST could be a useful alternative for virologic tests and clinical trials worldwide by significantly diminishing transportation cost and the sample volume restrictions associated with dried-blood-spot technology.
Assuntos
Dessecação , Infecções por HIV/diagnóstico , HIV/isolamento & purificação , Hepacivirus/isolamento & purificação , Hepatite C/diagnóstico , Plasma/virologia , Manejo de Espécimes/métodos , Genótipo , Humanos , Testes de Sensibilidade Microbiana , Reprodutibilidade dos Testes , Carga ViralRESUMO
Nucleic acid extraction and human immunodeficiency virus type 1 (HIV-1) genotyping using the NucliSens miniMAG platform and the TruGene HIV-1 genotyping kit gave HIV-1 sequence data from HIV-1-negative plasma spiked with 100 copies/ml reference HIV-1 RNA and from low-viremia clinical samples (<500 copies/ml) without the need for ultracentrifugation or nested second-round PCR.
Assuntos
Infecções por HIV/virologia , HIV-1/classificação , Magnetismo , RNA Viral/isolamento & purificação , Dióxido de Silício , Viremia/virologia , Genótipo , HIV-1/genética , HIV-1/isolamento & purificação , Humanos , RNA Viral/sangue , RNA Viral/genética , Kit de Reagentes para Diagnóstico , Carga ViralRESUMO
We evaluated the performance characteristics of a new, real-time nucleic acid sequence-based amplification (NASBA) assay that incorporates molecular beacon technology for detection of human immunodeficiency virus type 1 (HIV-1). The quantitative results were comparable to those obtained with three leading commercially available assays. The analytical sensitivity was 37 IU/ml. The NASBA assay detected clinically relevant recombinant viruses and all group M HIV-1 subtypes.
Assuntos
Infecções por HIV/virologia , HIV-1/isolamento & purificação , Sondas Moleculares , Replicação de Sequência Autossustentável/métodos , HIV-1/genética , Humanos , RNA Viral/análise , RNA Viral/isolamento & purificação , Kit de Reagentes para Diagnóstico , Recombinação Genética , Replicação de Sequência Autossustentável/instrumentação , Sensibilidade e EspecificidadeRESUMO
BACKGROUND: HIV infection is associated with a painful distal sensory polyneuropathy (DSP) that can severely limit the quality of life of affected subjects. The pathogenesis of DSP is unknown, although both HIV proteins and products of immune activation triggered by HIV infection have been implicated. OBJECTIVE: To assess the association between baseline markers of immune activation and HIV RNA levels (viral load) and time to symptomatic DSP (SDSP). METHODS: A cohort of 376 subjects, most receiving highly active antiretroviral therapy (HAART), were followed semiannually for up to 48 months. Blood and CSF levels of HIV viral load, monocyte chemotactic protein-1, macrophage colony-stimulating factor (M-CSF), matrix metalloproteinase-2, and tumor necrosis factor-alpha were measured in addition to CD4 lymphocyte cell count. RESULTS: In subjects without SDSP at baseline (62.5% of the cohort), among the virologic and immunologic markers, only baseline CSF M-CSF levels were associated with time to SDSP (hazard ratio = 2.97, p = 0.05). The Kaplan-Meier estimate of the 1-year incidence of SDSP was 21%, a 15% decrease from that observed in the Dana cohort, a pre-HAART cohort enrolled with the same inclusion/exclusion criteria. CONCLUSION: Highly active retroviral therapy (HAART) has changed the natural history of HIV-associated symptomatic distal sensory polyneuropathy (SDSP), which may explain, in contrast with studies from the pre-HAART era, the lack of association between SDSP and baseline HIV viral load and CD4 cell count.
Assuntos
Infecções por HIV/complicações , Sistema Imunitário/imunologia , Neurônios Aferentes/imunologia , Doenças do Sistema Nervoso Periférico/imunologia , RNA Viral/metabolismo , Carga Viral , Adulto , Terapia Antirretroviral de Alta Atividade/estatística & dados numéricos , Biomarcadores/análise , Biomarcadores/metabolismo , Contagem de Linfócito CD4 , Quimiocina CCL2/sangue , Quimiocina CCL2/imunologia , Estudos de Coortes , Feminino , Gânglios Espinais/imunologia , Gânglios Espinais/virologia , Infecções por HIV/tratamento farmacológico , Infecções por HIV/virologia , Humanos , Sistema Imunitário/virologia , Estudos Longitudinais , Fator Estimulador de Colônias de Macrófagos/sangue , Fator Estimulador de Colônias de Macrófagos/imunologia , Masculino , Metaloproteinase 2 da Matriz/sangue , Metaloproteinase 2 da Matriz/imunologia , Pessoa de Meia-Idade , Neurônios Aferentes/virologia , Nervos Periféricos/imunologia , Nervos Periféricos/fisiopatologia , Nervos Periféricos/virologia , Doenças do Sistema Nervoso Periférico/fisiopatologia , Doenças do Sistema Nervoso Periférico/virologia , RNA Viral/análise , RNA Viral/genética , Fator de Necrose Tumoral alfa/imunologiaRESUMO
OBJECTIVE: To evaluate whether baseline levels of plasma and CSF HIV RNA, tumor necrosis factor alpha (TNFalpha), monocyte chemoattractant protein-1 (MCP-1), matrix metalloproteinase-2 (MMP-2), or macrophage colony stimulating factor (M-CSF) are predictors of incident HIV-associated dementia (HIVD) in a cohort with advanced HIV infection. METHODS: A total of 203 nondemented subjects with CD4 lymphocyte counts less than 200/muL, or <300/microL but with cognitive impairment, underwent semiannual neurologic, cognitive, functional, and laboratory assessments. HIVD and minor cognitive motor disorder (MCMD) were defined using American Academy of Neurology criteria. The cumulative incidence of HIVD was estimated using Kaplan-Meier curves. Cox proportional hazards regression models were used to examine the associations between biologic variables and time to HIVD, adjusting for age, sex, years of education, duration of HIV infection, type of antiretroviral use, premorbid IQ score, and presence of MCMD. RESULTS: After a median follow-up time of 20.7 months, 74 (36%) subjects reached the HIVD endpoint. The dementia was mild in 70% of cases. The cumulative incidence of HIVD was 20% at 1 year and 33% at 2 years. Highly active antiretroviral therapy (HAART) was used by 73% of subjects at baseline. A plasma HIV RNA level was undetectable in 23% of subjects and a CSF HIV RNA level was undetectable in 48% of subjects. In adjusted analyses, neither plasma nor CSF HIV RNA levels (log10) were associated with time to HIVD; log10 levels of plasma TNFalpha (HR 3.07, p = 0.03) and CSF MCP-1 (HR = 3.36, p = 0.06) tended to be associated with time to HIVD. CONCLUSION: The lack of association between baseline plasma and CSF HIV RNA levels and incident dementia suggests highly active antiretroviral therapy may be affecting CNS viral dynamics, leading to lower HIV RNA levels, and therefore weakening the utility of baseline HIV RNA levels as predictors of HIV-associated dementia.
Assuntos
Complexo AIDS Demência/epidemiologia , Terapia Antirretroviral de Alta Atividade , Citocinas/sangue , HIV-1/isolamento & purificação , RNA Viral/análise , Carga Viral , Complexo AIDS Demência/sangue , Complexo AIDS Demência/líquido cefalorraquidiano , Complexo AIDS Demência/imunologia , Adulto , Afeto , Fármacos Anti-HIV/uso terapêutico , Biomarcadores , Contagem de Linfócito CD4 , Quimiocina CCL2/análise , Quimiocina CCL2/sangue , Quimiocina CCL2/líquido cefalorraquidiano , Cognição , Estudos de Coortes , Feminino , Infecções por HIV/sangue , Infecções por HIV/líquido cefalorraquidiano , Infecções por HIV/tratamento farmacológico , Infecções por HIV/imunologia , Infecções por HIV/psicologia , Humanos , Incidência , Testes de Inteligência , Avaliação de Estado de Karnofsky , Tábuas de Vida , Fator Estimulador de Colônias de Macrófagos/análise , Fator Estimulador de Colônias de Macrófagos/sangue , Fator Estimulador de Colônias de Macrófagos/líquido cefalorraquidiano , Masculino , Metaloproteinase 2 da Matriz/análise , Metaloproteinase 2 da Matriz/sangue , Metaloproteinase 2 da Matriz/líquido cefalorraquidiano , Pessoa de Meia-Idade , Modelos Imunológicos , Exame Neurológico , Testes Neuropsicológicos , Valor Preditivo dos Testes , Modelos de Riscos Proporcionais , RNA Viral/sangue , RNA Viral/líquido cefalorraquidiano , Fator de Necrose Tumoral alfa/análise , Fator de Necrose Tumoral alfa/líquido cefalorraquidianoRESUMO
OBJECTIVE: To examine the association between HIV RNA levels, patterns of antiretroviral resistance, and neurologic status. METHODS: Autopsy samples from 13 HIV-infected subjects were examined for HIV-1 viral RNA (vRNA), and viral reverse transcriptase (RT) genotype was determined. All subjects had been clinically characterized using standard instruments before death. RESULTS: The median HIV-1 vRNA level in brain samples from subjects with moderate dementia was 7.79 log(10) copies/g (range 5.56 to 9.75 log(10) copies/g) compared with 5.44 log(10) copies/g (range 3.51 to 9.32 log(10) copies/g) for mildly demented subjects and 4.87 log(10) copies/g (3.51 to 6.86 log(10) copies/g) for those obtained from nondemented individuals. There were differences between subjects with moderate dementia and nondemented subjects (p = 0.0002) and between subjects with moderate and mild dementia (p = 0.0128). No significant differences among the groups were observed for vRNA levels in peripheral tissues. Some demented subjects had relatively low levels of HIV-1 vRNA, and paradoxically some nondemented subjects had high vRNA brain levels. Little subject effect in vRNA was noted in peripheral regions, but high regional variation in vRNA was noted within the brain. Patterns of the major zidovudine (ZDV) RT mutations in brain and peripheral tissues were concordant in most subjects. Subjects with longer duration of exposure to ZDV tended to have lower brain vRNA levels and a greater number of RT mutations than those with limited to no exposure. CONCLUSIONS: The presence and severity of HIV dementia correlates with the levels of productive HIV replication within the brain. Other pathophysiologic events (including macrophage activation) probably also contribute to neurologic dysfunction.
Assuntos
Complexo AIDS Demência/tratamento farmacológico , Fármacos Anti-HIV/uso terapêutico , HIV-1/genética , Zidovudina/uso terapêutico , Complexo AIDS Demência/virologia , Encéfalo/virologia , Contagem de Linfócito CD4 , Transcriptase Reversa do HIV/genética , HIV-1/isolamento & purificação , Humanos , Imunidade Inata , Mutação , RNA Viral/análise , Índice de Gravidade de Doença , Replicação ViralRESUMO
The use is described of a commercially available, silica-based extraction procedure for HIV-1 RNA that can be substituted for the extraction procedure supplied with a commercially available, PCR-based genotyping kit for HIV-1. The advantages of using this alternative, commercially available extraction procedure include the following: (1) reduced safety concerns, due to inactivation of virus in the initial step of the extraction procedure; (2) enhanced sensitivity, allowing the use of plasma with HIV-1 RNA levels of less than 2000 copies/ml; (3) improved yield, allowing a 60% reduction in plasma volume; and (4) convenience and improved reproducibility, with a single extraction providing RNA suitable for PCR-based sequencing and for quantitation of HIV-1 RNA levels.
Assuntos
HIV-1/classificação , HIV-1/genética , Reação em Cadeia da Polimerase/métodos , RNA Viral/isolamento & purificação , Genótipo , Infecções por HIV/virologia , Humanos , Kit de Reagentes para Diagnóstico , Sensibilidade e EspecificidadeRESUMO
Development of anti-retroviral regimens with enhanced efficacy against brain HIV-1 is essential if viral eradication is to be achieved. To address this, a severe combined immune deficiency mouse model of HIV-1 encephalitis was used to assay the effect of protease-containing and protease-sparing drug regimens on viral replication in brain macrophages. Here, HIV-1-infected human monocyte-derived macrophages (MDM) are inoculated into basal ganglia, causing a multinucleated giant cell encephalitis reminiscent of human disease. Drugs were administered at the time of MDM inoculation and continued until sacrifice. Immunohistochemical tests evaluated ongoing viral replication, glial immunity, and neuronal survival. Treatment with ddI/d4T decreased the numbers of infected cells by 75%, while ddI/d4T/amprenavir or ZDV/3TC/ABC diminished infection by 98%. Triple drug regimens decreased astrogliosis by > or = 25%. This small-animal model may be used to screen drug regimens that affect ongoing HIV-1 replication within its brain sanctuary.
Assuntos
Fármacos Anti-HIV/uso terapêutico , Terapia Antirretroviral de Alta Atividade , Modelos Animais de Doenças , Encefalite Viral/complicações , Encefalite Viral/tratamento farmacológico , Infecções por HIV/complicações , Infecções por HIV/tratamento farmacológico , Animais , Fármacos Anti-HIV/administração & dosagem , Fármacos Anti-HIV/farmacologia , Gânglios da Base/patologia , Gânglios da Base/virologia , Barreira Hematoencefálica , Sobrevivência Celular/efeitos dos fármacos , Encefalite Viral/patologia , Encefalite Viral/virologia , Infecções por HIV/patologia , Infecções por HIV/virologia , HIV-1/efeitos dos fármacos , HIV-1/fisiologia , Humanos , Imuno-Histoquímica , Injeções Intraventriculares , Macrófagos/transplante , Macrófagos/ultraestrutura , Macrófagos/virologia , Camundongos , Camundongos SCID , Microscopia Eletrônica , Neuroglia/efeitos dos fármacos , Neuroglia/patologia , Neuroglia/ultraestrutura , Neuroglia/virologia , Neurônios/diagnóstico por imagem , Neurônios/efeitos dos fármacos , Neurônios/patologia , Neurônios/virologia , Ultrassonografia , Replicação Viral/efeitos dos fármacosAssuntos
Infecções por HIV/tratamento farmacológico , Inibidores da Protease de HIV/uso terapêutico , HIV-1/genética , Nelfinavir/uso terapêutico , Terapia de Salvação , Fármacos Anti-HIV/uso terapêutico , Criança , Quimioterapia Combinada , Infecções por HIV/imunologia , Infecções por HIV/virologia , Transcriptase Reversa do HIV/genética , HIV-1/classificação , HIV-1/isolamento & purificação , Humanos , Subpopulações de Linfócitos/imunologia , RNA Viral/sangue , Inibidores da Transcriptase Reversa/uso terapêutico , Fatores de Tempo , Carga ViralRESUMO
Accurate assessment of plasma HIV RNA levels at low concentrations is clinically important. We evaluated a second-generation quantitative HIV RNA assay (NucliSens HIV-1 QT), and three simple adaptations of the NucliSens standard protocol to lower the lower cutoff level. The assays were evaluated in constructed panels with known HIV RNA concentrations and in clinical samples. Results were compared with those obtained with the first generation (NASBA HIV-1 QT) and with two other commercially available assays: the Amplicor HIV Monitor test and the Quantiplex assay. In a constructed panel, results obtained by NASBA QT were on average 0.13 log(10) copies/ml (SD 0.15) higher than those of NucliSens. The NucliSens assay could quantify HIV RNA in at least 50% of the samples down to 518 (2.71 log(10)) copies/ml and NASBA QT to 5.80 x 10(3) (3.76 log(10)) copies/ml). Both assays correlated well with the known input (R NucliSens = 0.99; R NASBA QT = 0.996), but results were more variable at lower input levels. With the three different ultrasensitive NucliSens adaptations, HIV RNA could be quantified in at least 50% of the samples down to 100 (2.00 log(10)), 46 (1.66 log(10)), and 10 (1.00 log(10)) copies/ml, respectively. In patient samples, Amplicor results were on average 0.11 (SD 0.20) log(10) copies/ml above, NucliSens 0.02 (SD 0.29) copies/ml above, and Quantiplex 0.13 (SD 0.19) copies/ml below the mean of the three assay results per sample. The variation remained the same over the range of RNA levels with all three assays. The NucliSens assay can quantify HIV RNA at lower levels than the NASBA QT and is comparable to other commercially available assays. The lower cutoff of the NucliSens can be lowered down to 10 copies/ml.
Assuntos
Infecções por HIV/virologia , HIV-1/genética , RNA Viral/sangue , Protocolos Clínicos , Infecções por HIV/sangue , Infecções por HIV/tratamento farmacológico , Humanos , Kit de Reagentes para Diagnóstico , Sensibilidade e EspecificidadeRESUMO
OBJECTIVES: To compare the efficacy of the nucleoside reverse transcriptase inhibitors (NRTIs) abacavir, zidovudine (AZT), lamivudine (3TC), didanosine (ddI), and stavudine (d4T) to inhibit viral replication in brain macrophages. A severe combined immunodeficiency (SCID) mouse model of HIV-1 encephalitis (HIVE) was used to monitor spreading viral infection in the CNS. BACKGROUND: The development of antiretroviral therapies with CNS efficacy against neuroinvasive virus is important if eradication of HIV-1 can be achieved within critical "hidden reservoirs." METHODS: HIV-1-infected human monocyte-derived macrophages (MDMs) (after a single round of viral replication) were inoculated into the caudate and putamen of SCID mice. This resulted in the spreading of viral infection with a concomitant multinucleated giant cell encephalitis (astrogliosis, microglial activation, and neuronal injury). NRTIs were administered to animals at the time of intracerebral MDM inoculations and continued until the time of sacrifice. Antiretroviral effects were assessed by viral load and percentages of infected MDMs. RESULTS: In brains of SCID mice with HIVE, abacavir and lamivudine reduced HIV-1 p24 antigen-positive cells by 80% and 95%, respectively, whereas both decreased viral load by approximately 1 log. Zidovudine, didanosine, and stavudine showed variable effects. CONCLUSION: Abacavir and lamivudine showed significant antiretroviral activity in SCID mice with HIVE when compared with other NRTIs. The extrapolation of these results to humans with HIV-1 dementia awaits future investigations.
Assuntos
Complexo AIDS Demência/tratamento farmacológico , Didesoxinucleosídeos/farmacologia , Modelos Animais de Doenças , HIV-1 , Camundongos SCID , Inibidores da Transcriptase Reversa/farmacologia , Complexo AIDS Demência/patologia , Animais , Células Cultivadas , Didanosina/farmacologia , Humanos , Lamivudina/farmacologia , Masculino , Camundongos , Monócitos/citologia , Monócitos/virologia , Estavudina/farmacologia , Replicação Viral/efeitos dos fármacos , Zidovudina/farmacologiaRESUMO
Biphasic plasma viral decays were modeled in 48 patients treated with ritonavir, zidovudine, and lamivudine. Estimated first- and second-phase decay rates were d1 as 0.47/day and d2 as 0.04/day. Interpatient differences in both decay rates were significant. The d1 was directly correlated with baseline CD4+, CD4+CD28+, and CD8+CD28+ T lymphocyte counts (P<.05) and inversely correlated with baseline virus load (P=.044) and the magnitude of CD4+ and CD8+ T lymphocyte recovery (P<.01). The d2 was directly correlated with baseline percentage of CD8+ T lymphocytes (P=.023), the CD8+CD38+ cell number (P=.024), and the level of IgG that binds to human immunodeficiency virus (HIV) type 1 gp120 (P=.02). Viral decay rates were not predictive of treatment failure or durability of viral suppression. These exploratory findings are consistent with a model in which immunologic factors contribute to elimination of HIV-infected cells and suggest a dynamic interplay between regulation of HIV expression and lymphocyte activation and recovery.
Assuntos
Síndrome da Imunodeficiência Adquirida/tratamento farmacológico , Fármacos Anti-HIV/administração & dosagem , Antígenos CD , HIV-1 , ADP-Ribosil Ciclase , ADP-Ribosil Ciclase 1 , Síndrome da Imunodeficiência Adquirida/virologia , Antígenos de Diferenciação/análise , Contagem de Linfócito CD4 , Quimioterapia Combinada , Humanos , Imunoglobulina G/sangue , Glicoproteínas de Membrana , NAD+ Nucleosidase/análise , RNA Viral/sangueRESUMO
Cerebrospinal fluid (CSF) human immunodeficiency virus (HIV) RNA levels were measured with the Nucleic Acid Sequence-Based Amplification (NASBA) assay to determine the relationship with neurological status; 37 subjects with HIV dementia (HIV-D) were compared with 77 with HIV with minor neurological signs (HIV-MCMD) and 93 neurologically normal HIV-seropositive individuals (HIV-NML). The NASBA assay had a lower limit of detection of 100 copies per milliliter. Mean CSF log HIV RNA levels were significantly higher in those with dementia after adjusting for CD4 count and were correlated with dementia severity. Plasma levels did not distinguish comparably immunosuppressed subjects with or without dementia. CSF and plasma RNA levels were significantly intercorrelated for subjects with CD4 counts <200/mm3 and also correlated inversely with CSF beta2-microglobulin. CSF RNA levels were independent of CSF pleocytosis or antiretroviral exposure. Brain RNA levels were consistently higher than CSF but correlated with CSF values for dementia subjects. The NASBA assay can be used reliably to determine HIV RNA levels in CSF, brain, and plasma samples. CSF HIV RNA may be a surrogate marker for brain infection, based on the observed correlation with brain levels. The association between plasma HIV RNA and CSF levels of HIV and beta2-microglobulin suggests that both viral load and CNS immune activation are important determinants of neurological disease.
Assuntos
Complexo AIDS Demência/virologia , Encéfalo/virologia , Carga Viral , Complexo AIDS Demência/líquido cefalorraquidiano , Complexo AIDS Demência/imunologia , Biomarcadores , Contagem de Linfócito CD4 , Estudos de Coortes , Estudos Transversais , Progressão da Doença , HIV-1/genética , HIV-1/imunologia , HIV-1/isolamento & purificação , Humanos , Testes Neuropsicológicos , RNA Viral/análise , RNA Viral/sangue , RNA Viral/líquido cefalorraquidiano , Reprodutibilidade dos Testes , Carga Viral/normasRESUMO
Type I and II topoisomerase activities were partially purified from Pneumocystis carinii. The catalytic (strand-passing) activities of both enzymes were selectively inhibited by members of a series of dicationic-substituted bis-benzimidazoles compared with those of topoisomerases of mammalian (calf thymus) origin. The most active inhibitors of the parasite enzymes were also highly effective in an in vivo animal model of P. carinii pneumonia. Selected dicationic-substituted bis-benzimidazoles also strongly inhibited the induction of the topoisomerase I- and II-mediated cleavable complex, suggesting that the biologically active DNA minor groove-binding molecules inhibit the enzyme-DNA binding step of the topoisomerase reaction sequence. The apparent selectivities for the parasite enzymes and the low levels of toxicity to mammalian cells for the biologically active bis-benzimidazoles suggest that these compounds hold promise as effective therapeutic agents in the treatment of a life-threatening AIDS-related disease, P. carinii pneumonia.