RESUMEN
Education and diagnostic tests capable of early detection represent our most effective means of preventing transmission of human immunodeficiency virus (HIV). The importance of early detection is underlined by studies demonstrating increased life expectancy following early initiation of antiviral treatment. The Elecsys(®) HIV combi PT assay is a fourth-generation antigen-antibody combination assay developed to allow earlier detection of seroconversion, and to have increased sensitivity and improved specificity. We aimed to determine how early the assay could detect infection compared with existing assays; whether all HIV variants could be detected; and the assay's specificity using samples from blood donors, routine specimens, and patients with potential cross-reacting factors. Samples were identified as positive by the Elecsys(®) assay 4.9 days after a positive polymerase chain reaction result (as determined by the panel supplier), which was earlier than the 5.3-7.1 days observed with comparators. The analytical sensitivity of the Elecsys(®) HIV combi PT assay for the HIV-1 p24 antigen was 1.05 IU/mL, which compares favorably with the comparator assays. In addition, the Elecsys(®) assay identified all screened HIV subtypes and displayed greater sensitivity to HIV-2 homologous antigen and antibodies to HIV-1 E and O and HIV-2 than the other assays. Overall, the specificity of the Elecsys(®) assay was 99.88 % using samples from blood donors and 99.81 % when analyzing unselected samples. Potential cross-reacting factors did not interfere with assay performance. The Elecsys(®) HIV combi PT assay is a sensitive and specific assay that has been granted the CE mark according to Directive 2009/886/EC.
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Técnicas de Laboratorio Clínico/métodos , Pruebas Diagnósticas de Rutina/métodos , Anticuerpos Anti-VIH/sangre , Proteína p24 del Núcleo del VIH/sangre , Infecciones por VIH/diagnóstico , VIH-1/aislamiento & purificación , VIH-2/aislamiento & purificación , VIH-1/inmunología , VIH-2/inmunología , Humanos , Inmunoensayo/métodos , Sensibilidad y EspecificidadRESUMEN
BACKGROUND: Serologic testing algorithms for recent HIV seroconversion (STARHS) provide important information for HIV surveillance. We have shown that a patient's antibody reaction in a confirmatory line immunoassay (INNO-LIA HIV I/II Score, Innogenetics) provides information on the duration of infection. Here, we sought to further investigate the diagnostic specificity of various Inno-Lia algorithms and to identify factors affecting it. METHODS: Plasma samples of 714 selected patients of the Swiss HIV Cohort Study infected for longer than 12 months and representing all viral clades and stages of chronic HIV-1 infection were tested blindly by Inno-Lia and classified as either incident (up to 12 m) or older infection by 24 different algorithms. Of the total, 524 patients received HAART, 308 had HIV-1 RNA below 50 copies/mL, and 620 were infected by a HIV-1 non-B clade. Using logistic regression analysis we evaluated factors that might affect the specificity of these algorithms. RESULTS: HIV-1 RNA < 50 copies/mL was associated with significantly lower reactivity to all five HIV-1 antigens of the Inno-Lia and impaired specificity of most algorithms. Among 412 patients either untreated or with HIV-1 RNA ≥ 50 copies/mL despite HAART, the median specificity of the algorithms was 96.5% (range 92.0-100%). The only factor that significantly promoted false-incident results in this group was age, with false-incident results increasing by a few percent per additional year. HIV-1 clade, HIV-1 RNA, CD4 percentage, sex, disease stage, and testing modalities exhibited no significance. Results were similar among 190 untreated patients. CONCLUSIONS: The specificity of most Inno-Lia algorithms was high and not affected by HIV-1 variability, advanced disease and other factors promoting false-recent results in other STARHS. Specificity should be good in any group of untreated HIV-1 patients.
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Técnicas de Laboratorio Clínico/métodos , Infecciones por VIH/diagnóstico , Virología/métodos , Adulto , Algoritmos , Femenino , VIH-1/clasificación , VIH-1/genética , VIH-1/inmunología , Humanos , Inmunoensayo , Masculino , ARN Viral/sangre , Sensibilidad y EspecificidadRESUMEN
HIV-1 diagnosis is usually based on the detection of specific antibodies, appearing in a time-determined pattern following the infection. We describe a prolonged HIV-1 seroconversion in an elite controller (defined as having HIV-1 RNA persistently <50copies/ml while untreated). HIV-1 diagnosis was delayed and complicated by this atypical evolution.
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Seropositividad para VIH/diagnóstico , VIH-1/inmunología , Serodiagnóstico del SIDA , Adulto , Anticuerpos Antivirales/sangre , Seropositividad para VIH/inmunología , Seropositividad para VIH/virología , Humanos , Leucocitos Mononucleares/inmunología , Leucocitos Mononucleares/virología , Masculino , ARN Viral/sangreRESUMEN
OBJECTIVES: Etravirine (ETV) is a novel nonnucleoside reverse transcriptase inhibitor (NNRTI) with reduced cross-resistance to first-generation NNRTIs, which has been primarily studied in randomized clinical trials and not in routine clinical settings. METHODS: ETV resistance-associated mutations (RAMs) were investigated by analysing 6072 genotypic tests. The antiviral activity of ETV was predicted using different interpretation systems: International AIDS Society-USA (IAS-USA), Stanford, Rega and Agence Nationale de Recherches sur le Sida et les hépatites virales (ANRS). RESULTS: The prevalence of ETV RAMs was higher in NNRTI-exposed patients [44.9%, 95% confidence interval (CI) 41.0-48.9%] than in treatment-naïve patients (9.6%, 95% CI 8.5-10.7%). ETV RAMs in treatment-naïve patients mainly represent polymorphism, as prevalence estimates in genotypic tests for treatment-naïve patients with documented recent (<1 year) infection, who had acquired HIV before the introduction of NNRTIs, were almost identical (9.8%, 95% CI 3.3-21.4). Discontinuation of NNRTI treatment led to a marked drop in the detection of ETV RAMs, from 51.7% (95% CI 40.8-62.6%) to 34.5% (95% CI 24.6-45.4%, P=0.032). Differences in prevalence among subtypes were found for V90I and V179T (P<0.001). Estimates of restricted virological response to ETV varied among algorithms in patients with exposure to efavirenz (EFV)/nevirapine (NVP), ranging from 3.8% (95% CI 2.5-5.6%) for ANRS to 56.2% (95% CI 52.2-60.1%) for Stanford. The predicted activity of ETV decreased as the sensitivity of potential optimized background regimens decreased. The presence of major IAS-USA mutations (L100I, K101E/H/P and Y181C/I/V) reduced the treatment response at week 24. CONCLUSIONS: Most ETV RAMs in drug-naïve patients are polymorphisms rather than transmitted RAMs. Uncertainty regarding predictions of antiviral activity for ETV in NNRTI-treated patients remains high. The lowest activity was predicted for patients harbouring extensive multidrug-resistant viruses, thus limiting ETV use in those who are most in need.
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Fármacos Anti-VIH/uso terapéutico , Farmacorresistencia Viral Múltiple/genética , Infecciones por VIH/tratamiento farmacológico , VIH-1/genética , Mutación , Piridazinas/uso terapéutico , Algoritmos , Estudios de Cohortes , Frecuencia de los Genes , Genotipo , Infecciones por VIH/virología , VIH-1/efectos de los fármacos , Humanos , Pruebas de Sensibilidad Microbiana/métodos , Mutación/genética , Nitrilos , Polimorfismo Genético , Valor Predictivo de las Pruebas , Pirimidinas , Inhibidores de la Transcriptasa Inversa/uso terapéutico , Suiza , Insuficiencia del Tratamiento , Carga ViralRESUMEN
In a multicenter study a new, fully automated Roche Diagnostics Elecsys HBsAg II screening assay with improved sensitivity to HBsAg mutant detection was compared to well-established HBsAg tests: AxSYM HBsAg V2 (Abbott), Architect HBsAg (Abbott), Advia Centaur HBsAg (Bayer) Enzygnost HBsAg 5.0 (Dade-Behring), and Vitros Eci HBsAg (Ortho). A total of 16 seroconversion panels, samples of 60 HBsAg native mutants, and 31 HBsAg recombinant mutants, dilution series of NIBSC and PEI standards, 156 HBV positive samples comprising genotypes A to G, 686 preselected HBsAg positive samples from different stages of infection, 3,593 samples from daily routine, and 6,360 unselected blood donations were tested to evaluate the analytical and clinical sensitivity, the detection of mutants, and the specificity of the new assay. Elecsys HBsAg II showed a statistically significant better sensitivity in seroconversion panels to the compared tests. Fifty-seven out of 60 native mutants and all recombinant mutants were found positive. Among 156 HBV samples with different genotypes and 696 preselected HBsAg positive samples Elecsys HBsAg II achieved a sensitivity of 100%. The lower detection limit for NIBSC standard was calculated to be 0.025 IU/ml and for the PEI standards ad and ay it was <0.001 and <0.005 U/ml, respectively. Within 2,724 daily routine specimens and 6.360 unselected blood donations Elecsys HBsAg II showed a specificity of 99.97 and 99.88%, respectively. In conclusion the new Elecsys HBsAg II shows a high sensitivity for the detection of all stages of HBV infection and HBsAg mutants paired together with a high specificity in blood donors, daily routine samples, and potentially interfering sera.
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Donantes de Sangre , Antígenos de Superficie de la Hepatitis B/sangre , Virus de la Hepatitis B/genética , Hepatitis B Crónica/diagnóstico , Automatización , Genotipo , Humanos , Técnicas de Inmunoadsorción/normas , Sensibilidad y EspecificidadRESUMEN
Fourth-generation screening assays which permit a simultaneous detection of human immunodeficiency virus (HIV) antigen and antibody reduce the diagnostic window on average by four days in comparison to third-generation antibody assays. Recently, the new automated Elecsys HIV combi was compared in a multicenter study to alternative fourth- and third-generation assays, p24 antigen test and HIV-1 RNA RT-PCR. A total of 104 serocon-version panels, samples of the acute phase of infection after seroconversion (n = 33), anti-HIV-1 positive specimens (n = 572) from patients in different stages of the disease, 535 subtyped samples from different geographical locations, including group M (subtypes A-J) and group O, anti-HIV-2 positive sera (n = 364), dilutions of cell culture supernatants (n = 60) infected with different HIV-1 subtypes, selected performance panels, 8406 unselected samples from blood donors originating from different blood transfusion centers, 3810 unselected sera from daily routine and from hospitalized patients, 9927 unselected samples from South Africa and 1943 potentially interfering samples were tested with the Elecsys HIV combi. Elecsys HIV combi showed a comparable sensitivity to HIV-1 Ag stand-alone assays for early detection of HIV infection in seroconversion panels. The mean time delay of Elecsys HIV combi (last negative sample + 1 day) in comparison to HIV-1 RT-PCR for 92 panels tested with both methods was 3.23 days. The diagnostic window was reduced with Elecsys HIV combi between 1.56 and 5.32 days in comparison to third-generation assays. The specificity of Elecsys HIV combi in blood donors was 99.80% after repeated testing. Our results show that a fourth-generation assay with improved specificity and sensitivity like the Elecsys HIV combi is suitable for blood donor screening due to its low number of false positives and since it detects HIV p24 antigen with a comparable sensitivity to single antigen assays.
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Anticuerpos Anti-VIH/sangre , Proteína p24 del Núcleo del VIH/sangre , Infecciones por VIH/diagnóstico , VIH-1/aislamiento & purificación , VIH-2/aislamiento & purificación , Inmunoensayo , Diagnóstico Precoz , Humanos , ARN Viral/análisis , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Sensibilidad y EspecificidadRESUMEN
In resource-limited countries, nosocomial transmission of bloodborne pathogens is a major public health concern. After a major outbreak of human immunodeficiency virus (HIV) infection in approximately 400 children in 1998 in Libya, we tested HIV, hepatitis C virus (HCV), and hepatitis B virus (HBV) markers in 148 children and collected epidemiological data in a subgroup of 37 children and 46 parents. HIV infection was detected in all children but one, with HCV or HBV coinfection in 47% and 33%, respectively. Vertical transmission was ruled out by analysis of parents' serology. The children visited the same hospital 1-6 times; at each visit, invasive procedures with potential blood transmission of virus were performed. HIV and HCV genotypic analyses identified a HIV monophyletic group, whereas 4 clusters of HCV sequences were identified. To our knowledge, this is the largest documented outbreak of nosocomial HIV transmission.
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Patógenos Transmitidos por la Sangre , Infección Hospitalaria/epidemiología , Brotes de Enfermedades , Infecciones por VIH/epidemiología , VIH-1/aislamiento & purificación , Hepatitis B/epidemiología , Hepatitis C/epidemiología , Adulto , Niño , Genotipo , Infecciones por VIH/sangre , Infecciones por VIH/diagnóstico , VIH-1/clasificación , VIH-1/genética , Hepacivirus/genética , Hepacivirus/aislamiento & purificación , Hepatitis B/sangre , Hepatitis B/diagnóstico , Anticuerpos contra la Hepatitis B/sangre , Hepatitis C/sangre , Hepatitis C/diagnóstico , Anticuerpos contra la Hepatitis C/sangre , Humanos , Libia/epidemiología , Libia/etnología , Padres , Filogenia , ARN Viral/sangre , ARN Viral/aislamiento & purificación , Suiza , Viremia/diagnóstico , Viremia/epidemiologíaRESUMEN
After occupational exposures, immediate HIV testing of source patients may avoid the unnecessary use of post-exposure prophylaxis (PEP). Two time periods were compared. Before the availability of 24 h a day immediate testing, PEP was initiated after 12.6% of exposures, compared with 3.7% during the second period. The adjusted relative odds ratio of PEP during the second compared with the first period, was 0.23. The availability of immediate HIV testing limits unnecessary occupational PEP.
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Serodiagnóstico del SIDA , Infecciones por VIH/prevención & control , Transmisión de Enfermedad Infecciosa de Paciente a Profesional , Exposición Profesional , Pacientes , Anticuerpos Anti-VIH/sangre , Infecciones por VIH/diagnóstico , Infecciones por VIH/transmisión , Personal de Salud , Humanos , Factores de TiempoRESUMEN
BACKGROUND: Hepatitis C virus (HCV) infection is highly prevalent among HIV-1-infected individuals, but its contribution to the morbidity and mortality of coinfected patients who receive potent antiretroviral therapy is controversial. We used data from the ongoing Swiss HIV Cohort Study to analyse clinical progression of HIV-1, and the virological and immunological response to potent antiretroviral therapy in HIV-1-infected patients with or without concurrent HCV infection. METHODS: We analysed prospective data on survival, clinical disease progression, suppression of HIV-1 replication, CD4-cell recovery, and frequency of changes in antiretroviral therapy according to HCV status in 3111 patients starting potent antiretroviral therapy. RESULTS: 1157 patients (37.2%) were coinfected with HCV, 1015 of whom (87.7%) had a history of intravenous drug use. In multivariate Cox's regression, the probability of progression to a new AIDS-defining clinical event or to death was independently associated with HCV seropositivity (hazard ratio 1.7 [95% CI 1.26-2.30]), and with active intravenous drug use (1.38 [1.02-1.88]). Virological response to antiretroviral therapy and the probability of treatment change were not associated with HCV serostatus. In contrast, HCV seropositivity was associated with a smaller CD4-cell recovery (hazard ratio for a CD4-cell count increase of at least 50 cells/microL=0.79 [0.72-0.87]). INTERPRETATION: HCV and active intravenous drug use could be important factors in the morbidity and mortality among HIV-1-infected patients, possibly through impaired CD4-cell recovery in HCV seropositive patients receiving potent antiretroviral therapy. These findings are relevant for decisions about optimum timing for HCV treatment in the setting of HIV infection.
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Infecciones Oportunistas Relacionadas con el SIDA/tratamiento farmacológico , Fármacos Anti-VIH/uso terapéutico , Hepatitis C/tratamiento farmacológico , Adolescente , Adulto , Recuento de Linfocito CD4 , Estudios de Cohortes , Progresión de la Enfermedad , Femenino , Hepatitis C/complicaciones , Humanos , Masculino , Persona de Mediana Edad , Estudios Prospectivos , Análisis de Supervivencia , Suiza , Carga Viral , Viremia/etiologíaRESUMEN
Five methods for the assessment of plasma viral load (VL) were evaluated in 103 seropositive patients infected with various subtypes of HIV-1. The methods included three RNA-based assays (Amplicor Monitor 1.5, Quantiplex version 2.0, NucliSens), one ultrasensitive reverse transcriptase (PERT) assay and one "boosted" p24 antigen (Ag) enzyme immunoassay (EIA). Subtyping was based on sequencing in env. The sensitivities were, in decreasing order, Amplicor > PERT > p24 Ag > NucliSens > Quantiplex. The low sensitivity of NucliSens was related to the missing of several non-B (A, E, F, G) or recombinant strains, whereas that of Quantiplex did not depend on subtype. In the 1 group O sample and 4 group M samples, only PERT assay or p24Ag EIA produced a positive result. In the quantitative range, correlation was best between Amplicor and Quantiplex (r = 0.8848), fair between Amplicor and NucliSens (r = 0.7064) or PERT assay (r = 0.7266), lowest between Amplicor and p24Ag EIA (r = 0.3989). Amplicor underestimated VL in 1 subtype E sample. Thus, Amplicor performed best in terms of sensitivity (compared with all other assays) and accuracy (compared with NucliSens, PERT assay, and p24Ag) for non-B subtypes in group M samples. PERT assay appears useful for VL assessment in infections by group O or other highly divergent viruses.
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Infecciones por VIH/sangre , VIH-1/clasificación , Carga Viral , Infecciones por VIH/virología , Humanos , Técnicas para Inmunoenzimas , Reacción en Cadena de la Polimerasa , ARN Viral/análisis , ADN Polimerasa Dirigida por ARN/análisisRESUMEN
The relationship between blood CD8+ T lymphocyte subsets, as defined by CD28 and CD38 expression, and plasma viraemia and CD4+ T cells in HIV-1 infection was investigated. In a cross-sectional study of 46 patients with either no or stable anti-retroviral treatment, there was a strong negative correlation between the percentage of CD8+CD28- and the percentage of CD4+ T cells (r = -0.75, P < 0.0001), and a positive correlation between absolute numbers of CD8+CD28+ and CD4+ T cells (r = 0.56, P < 0.0001). In contrast, the expression of CD38 by CD8+ T lymphocytes correlated primarily with plasma viraemia (e.g. the percentage of CD38+ in CD8bright cells, r = 0.76, P < 0.0001). In the 6 months following triple therapy initiation in 32 subjects, there was a close correlation between changes (delta) in CD8+CD28+ or CD8+CD28- and in CD4+ T cells (e.g. delta % CD8+CD28+ versus delta % CD4+, r = 0.37, P = 0.0002; delta % CD8+CD28- versus delta % CD4+, r = -0.66, P < 0.0001). A marked decline of the number of CD8+ T cells expressing CD38 was also observed. These results suggest the existence of a T cell homeostasis mechanism operating in blood with CD4+ and CD8+CD28+ cells on the one hand, and with CD8+CD28- cells on the other. In addition, the percentage of CD38+ cells in CD8+ cells, generally considered an independent prognostic factor, could merely reflect plasma viral load.
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Antígenos CD , Antígenos de Diferenciación/metabolismo , Antígenos CD28/metabolismo , Linfocitos T CD8-positivos/inmunología , Infecciones por VIH/inmunología , VIH-1 , NAD+ Nucleosidasa/metabolismo , ADP-Ribosil Ciclasa , ADP-Ribosil Ciclasa 1 , Fármacos Anti-VIH/uso terapéutico , Biomarcadores , Linfocitos T CD4-Positivos/inmunología , Estudios de Cohortes , Estudios Transversales , Infecciones por VIH/tratamiento farmacológico , Homeostasis , Humanos , Glicoproteínas de Membrana , Pronóstico , Suiza , Subgrupos de Linfocitos T/inmunología , Viremia/inmunologíaRESUMEN
HIV-1 subtypes were determined in newly diagnosed residents of Switzerland. Blood was anonymously collected from patients with a first confirmed positive HIV-1 test result. Viral DNA from the env V3-V5 region was amplified by nested polymerase chain reaction (PCR) and screened for subtype B by heteroduplex mobility assay. All amplicons not identified as B were sequenced. From November 1996 to February 1998, 206 samples were analyzed. Main transmission risks were unprotected heterosexual (55.7%) or homosexual (27.1%) sexual contact or intravenous drug use (12.9%). Subtype B dominated in patients of Swiss, other European, American, or Asian citizenship; particularly high frequencies were found in homosexuals (97%) and drug users (94%). Non-B subtypes including A, C, D, E, F, G, H, a possible B/F recombinant, and a sequence related to J were present in 28.2% (95% confidence interval [CI], 22.9%-35.0%). Non-B were frequent in African citizens (95%), heterosexually infected individuals (44%), and women (43%). Heterosexually infected Swiss males harbored non-B strains in 18% and females in 33%. The results document a change in the epidemiology of newly diagnosed HIV-1 infections in Switzerland: predominance of heterosexual transmission and a high frequency of non-B subtypes.
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Infecciones por VIH/virología , VIH-1/clasificación , Adulto , Femenino , Productos del Gen env/análisis , Infecciones por VIH/epidemiología , Heterosexualidad , Homosexualidad , Humanos , Masculino , Prevalencia , Factores de Riesgo , Abuso de Sustancias por Vía Intravenosa/complicaciones , Suiza/epidemiologíaAsunto(s)
Inmunodeficiencia Variable Común/diagnóstico , Antígenos VIH/sangre , Proteína p24 del Núcleo del VIH/inmunología , Seropositividad para VIH/diagnóstico , VIH-1/inmunología , Pruebas de Neutralización/métodos , Serodiagnóstico del SIDA/métodos , Serodiagnóstico del SIDA/normas , Adulto , Inmunodeficiencia Variable Común/sangre , Inmunodeficiencia Variable Común/inmunología , Inmunodeficiencia Variable Común/terapia , Errores Diagnósticos , Reacciones Falso Positivas , Seropositividad para VIH/sangre , Seropositividad para VIH/inmunología , Humanos , Inmunoglobulinas Intravenosas/uso terapéutico , Masculino , Pruebas de Neutralización/normas , Factor Reumatoide/sangre , Factor Reumatoide/inmunologíaRESUMEN
A total of 234 pools were prepared from 10,692 consecutive serum samples negative for antibodies to human immunodeficiency virus type 1 (HIV-1) and HIV-2 collected at five virological laboratories (average pool size, 45 serum samples). Pools were screened for the presence of HIV-1 RNA by a modified commercial assay (Amplicor HIV-1 Monitor test) which included an additional polyethylene glycol (PEG) precipitation step prior to purification of viral RNA (PEG Amplicor assay). The sensitivity of this assay for HIV-1 RNA detection in individual serum samples within pools matches that of standard commercial assays for individual serum samples, i.e., 500 HIV-1 RNA copies per ml. Five pools were identified as positive, and each one contained one antibody-negative, HIV-1 RNA-positive serum sample, corresponding to an average of 1 infected sample per 2,138 serum samples. Retrospective analysis revealed that the five HIV-1 RNA-positive specimens originated from individuals who had symptomatic primary HIV-1 infection at the time of sample collection and who were also positive for p24 antigenemia. We next assessed the possibility of performing the prepurification step by high-speed centrifugation (50,000 x g for 80 min) of 1.5-ml pools containing 25 microl of 60 individual serum samples, of which only 1 contained HIV-1 RNA (centrifugation Amplicor assay). The sensitivity of this assay also matches the sensitivities of standard commercial assays for HIV-1 RNA detection in individual serum samples. The results demonstrate that both assays with pooled sera can be applied to the screening of large numbers of serum samples in a time- and cost-efficient manner.
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Infecciones por VIH/diagnóstico , Seronegatividad para VIH , VIH-1/aislamiento & purificación , ARN Viral/sangre , Centrifugación , Precipitación Química , Estudios de Evaluación como Asunto , Anticuerpos Anti-VIH/sangre , Infecciones por VIH/sangre , VIH-1/genética , VIH-1/inmunología , VIH-2/inmunología , Humanos , Polietilenglicoles , Reacción en Cadena de la Polimerasa/métodos , Juego de Reactivos para Diagnóstico , Sensibilidad y EspecificidadRESUMEN
The clinical evaluation of the Cobas Core beta 2-Microglobulin EIA was performed on the random access analyzer Cobas Core. The coefficients of variations for the intra-assay and inter-assay precisions ranged between 3% and 6%. In comparison to healthy persons (0.7-2.7 mg/l), significantly elevated serum levels of beta 2-m were found in patients with lymphoproliferative disorders like monoclonal gammopathies of the IgG (1.98-78 mg/l; p = 0.0001), IgA (1.3-7.1 mg/l; p = 0.0002) and of the IgM (2.1-8.7 mg/l; p = 0.0001) type, in malignant lymphoma (1.5-33.5 mg/l; p = 0.0001) and in chronic lymphatic leukemia (1.5-22.4 mg/l; p = 0.0001). In cases of HIV-infection, increasing levels of beta 2-m exhibited an inverse correlation to the CD4+ T-lymphocyte count (p = 0.0001) and indicated disease progression. In patients having renal transplantation a rejection of the graft was accompanied by a rise of the beta 2-m serum level. In summary, the data clearly indicate the reliability of the measuring system as well as the clinical relevance of beta-2-M determinations for such patient groups.
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Microglobulina beta-2/análisis , Recuento de Linfocito CD4 , Humanos , Técnicas para InmunoenzimasRESUMEN
Fine-needle aspiration was used to collect lymph node cells (LNC) from 9 antiretroviral-naive patients entering a double-blind single- or combined-drug study of zidovudine, zalcitabine, and saquinavir. LNC were obtained twice before and 1 and 6 months after initiation of treatment. The effect of antiretroviral treatment on virus load ranged from no response to a dramatic decrease in plasma and LNC human immunodeficiency virus (HIV) RNA levels. The decrease in unspliced or spliced (or both) HIV RNAs in LNC was correlated with but consistently smaller than the decrease in plasma viremia. When present, the increase in blood CD4 T cells was, in general, moderate and transient. However, a striking rise in blood CD4 T cell count and in LNC CD4:CD8 ratio was observed in the 1 patient with the deepest sustained decrease in HIV RNA level in both plasma and lymph nodes.
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Síndrome de Inmunodeficiencia Adquirida/tratamiento farmacológico , Fármacos Anti-VIH/uso terapéutico , VIH-1 , Ganglios Linfáticos/virología , Saquinavir/uso terapéutico , Zalcitabina/uso terapéutico , Zidovudina/uso terapéutico , Síndrome de Inmunodeficiencia Adquirida/inmunología , Síndrome de Inmunodeficiencia Adquirida/patología , Biopsia con Aguja , Recuento de Linfocito CD4 , Relación CD4-CD8 , Método Doble Ciego , Monitoreo de Drogas/métodos , Quimioterapia Combinada , VIH-1/genética , VIH-1/aislamiento & purificación , Humanos , Ganglios Linfáticos/patología , Reacción en Cadena de la Polimerasa , Empalme del ARN , ARN Viral/genética , ARN Viral/aislamiento & purificación , Análisis de Regresión , Método Simple Ciego , Viremia/sangreRESUMEN
We have developed a sensitive and reproducible one-step competitive reverse transcriptase (RT) PCR assay, which allows hepatitis C virus (HCV) RNA quantitation in plasma over a broad range of values. The RNA samples and a constant amount of an internal standard were reverse transcribed and coamplified with the same primers in the same tube. A standard curve was obtained from an additional series of tubes containing both the internal standard and known amounts of a wild-type HCV RNA transcript, thus eliminating the need for titrating samples with the competitor. Eighty-eight anti-HCV-positive samples were tested by RT-PCR and a branched-DNA (bDNA) assay which has a detection limit of 3.5 x 10(5) copies per ml. Fifty-five samples were quantifiable by both methods (correlation coefficient, 0.72), the ranges of values found by the RT-PCR and bDNA assays being, respectively, 0.127 x 10(6) to 18.4 x 10(6) and 0.44 x10(6) to 38 x 10(6) copies per ml. Six samples that had indeterminate values by the bDNA assay had RT-PCR values between 0.37 x 10(5) and 9.6 x 10(5) copies per ml. Twenty-two samples that had values below the cutoff value by the bDNA assay had RT-PCR values between 2.5 x 10(3) and 10.4 x 10(5) (18 less than and 4 more than the limit of 3.5 x 10(5) copies per ml). The remaining five samples were negative by both assays. The level of RT-PCR interassay reproducibility was high (correlation coefficient between duplicate values, 0.94). Our method, with a detection limit of 2,500 copies per ml, was markedly more sensitive than the bDNA assay. This method is convenient for following up patients with low viremia, a common situation with alpha interferon treatment.
Asunto(s)
ADN Viral/genética , Hepacivirus/genética , Hepacivirus/aislamiento & purificación , Reacción en Cadena de la Polimerasa/métodos , ARN Viral/sangre , ARN Viral/genética , Virología/métodos , Estudios de Evaluación como Asunto , Hepatitis C/terapia , Hepatitis C/virología , Hepatitis Crónica/terapia , Hepatitis Crónica/virología , Humanos , Interferón-alfa/uso terapéutico , Reacción en Cadena de la Polimerasa/normas , Reacción en Cadena de la Polimerasa/estadística & datos numéricos , ARN Viral/normas , Estándares de Referencia , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , Transcripción Genética , Viremia/terapia , Viremia/virología , Virología/normas , Virología/estadística & datos numéricosRESUMEN
We wished to establish the feasibility of fine needle aspiration of lymph nodes as a noninvasive method for measuring subsets of immune cells and viral load in HIV-infected patients. Twenty-five patients (CD4+ T cell range 4-760/microl, median 362) were selected. Lymph node aspiration was attempted in 21 patients. Lymph node cells (LNC), ranging from 6 x 10(3) to 2 x 10(6) (median 6 x 10(5)) were obtained in 17 subjects, and compared with peripheral blood mononuclear cells (PBMC) obtained simultaneously. Immunophenotype could be determined by flow cytometry in 9 patients. Mean percent of CD4+ CD3+ T cells in LNC and PBMC and 23.2 and 14.6. Mean percent of CD8+ CD3+ T cells in LNC and PBMC was 23.1 and 45.0, respectively. Therefore, CD4+/CD8+ ratios were much higher in LNC (mean +/- SD: 1.06 +/- 0.31) than in PBMC (0.35 +/- 0.13). The amount of HIV DNA (11 patients) and RNA (8 patients) was determined in the plasma, LNC, and PBMC by competitive reverse transcriptase-polymerase chain reaction (RT-PCR). The number of copies of viral DNA/10(5) cells was higher in LNC than in PBMC (LNC/PBMC ratio ranger: 0.54-25, median 3.4). The number of copies of unspliced viral RNA/10(5) cells was much higher in LNC than in PBMC (LNC/PBMC ratio range 65-1,159, median 435). The plasma RNA copy number, a measure of circulating cell-free virus, was correlated with the RNA copy number in PBMC, but not in LNC. A RT-PCR system specific for spliced transcripts was also used to assess the level of transcripts independent of genomic RNA. This assay also detected more signal in LNC than in PBMC. The level of spliced transcripts in LNC and PBMC correlated with the amount of full-length RNA detected by competitive PCR. A semiquantitative coculture assay with lymphoblasts from healthy donors was used to assess the infectivity of LNC as compared with PBMC in 14 patients. The minimum number of LNC necessary to cause a positive coculture ranged from 10(3) to > 10(5) (median 10(4)); the corresponding number for PBMC ranged from 10(3) to > 10(6) (median 5 x 10(5)). In most patients selected for palpable lymph nodes, LNC could be obtained by fine needle aspiration, thus allowing noninvasive monitoring of viral burden in lymphoid tissue. The present study also suggests that both T-cell subsets and viral load differ in the blood and lymphoid tissue, which raises the question of whether the study of the lymphoid tissue would yield better prognostic markers of disease course.
Asunto(s)
Biopsia con Aguja , Infecciones por VIH/inmunología , Infecciones por VIH/virología , Ganglios Linfáticos/inmunología , Ganglios Linfáticos/virología , Adulto , Anciano , Complejo CD3/análisis , Antígenos CD4/análisis , Antígenos CD8/análisis , ADN Viral/análisis , Estudios de Evaluación como Asunto , Femenino , Infecciones por VIH/sangre , Humanos , Inmunofenotipificación/métodos , Masculino , Persona de Mediana Edad , Reacción en Cadena de la Polimerasa , ARN Viral/análisis , Subgrupos de Linfocitos T/inmunología , Subgrupos de Linfocitos T/virologíaRESUMEN
CD28 is a transmembrane glycoprotein that provides T cells with an essential co-stimulatory signal during antigen presentation. Using flow cytometry, we document here an expansion of CD28- T cells in HIV infection. Whereas the percentage of CD4+CD28+ T cells among total lymphocytes was decreased, a small increase of the percentage of CD4+CD28- T cells was observed. In the CD8+ subset, there was a marked expansion of CD8+CD28- T cells. An increased percentage of CD8+ T cells positive for HLA-DR was found in both CD28+ and CD28- cells. Results were similar for CD38 expression. HIV infection was also distinguished by a shift from LFA-1lowCD28low to LFA-1highCD28high and LFA-1high-CD28neg expression pattern on CD8+ T cells. Negative correlations were found between percentage and absolute number of CD8+CD28+ T cells and several serum parameters usually associated with poor prognosis (IgA, IgE, beta 2-microglobulin and HIV-1 p24 antigen). Thus, HIV infection is characterized by a marked expansion of CD28- T cells with an abnormal expression of activation markers and cell adhesion molecules. In addition, CD8+CD28+, but not CD8+CD28- or total CD8+ T cell numbers, correlated with the levels of established serological markers of disease severity or progression and may, therefore, have predictive value.
Asunto(s)
Antígenos CD28/biosíntesis , Linfocitos T CD4-Positivos/metabolismo , Linfocitos T CD8-positivos/metabolismo , Infecciones por VIH/inmunología , Recuento de Linfocito CD4 , Moléculas de Adhesión Celular/biosíntesis , Humanos , Inmunoglobulina A/análisis , Inmunoglobulina E/análisis , Recuento de Linfocitos , Pronóstico , Linfocitos T/metabolismo , Microglobulina beta-2/análisisRESUMEN
A new enzyme immunoassay (EIA), the Cobas Core Anti-HIV-1/HIV-2 EIA DAGS (also referred to as Roche DAGS), for the detection of antibodies to human immunodeficiency virus type 1 (HIV-1) and HIV-2 was evaluated in four centers. The assay is based on the double-antigen sandwich (DAGS) format, which enables the detection of all classes of antibodies. The antigens consist of recombinant proteins in their native conformation and of synthetic peptides. Of a total of 5,836 negative serum samples, including 95 samples likely to produce false reactivities, 6 were false positive, resulting in a specificity of 99.9%. None of 35 sera that were from noninfected individuals but contained p24-cross-reacting antibodies as revealed by Western blot (immunoblot) analysis were reactive by the Roche DAGS assay. In samples from individuals infected with HIV-1 group M (n = 499) and HIV-2 (n = 200), the sensitivity of the assay was 100%. Although containing antigens with sequences from subtype B only, the assay was also able to correctly identify with high optical density/cutoff ratios samples from subjects infected with HIV-1 subtype O (n = 10). In 17 of 19 seroconversion panels tested, the assay detected the presence of HIV-1 antibodies as early as another sandwich EIA. Eight of these panels were also analyzed by an indirect second-generation assay, which detected antibodies 2 to 10 days later than did the DAGS assay under evaluation. The excellent specificity and sensitivity of the new Cobas Core Anti-HIV-1/HIV-2 EIA DAGS are the result of the DAGS format as well as of the native, naturally folded form of the recombinant protein used as the gag antigen.