RESUMEN
BACKGROUND: Critical illness induces immune disorders associated with an increased risk of hospital-acquired pneumonia (HAP) and acute respiratory distress syndrome (ARDS). Torque Teno Virus (TTV), from the Anelloviridae family, are proposed as a biomarker to measure the level of immunosuppression. Our objective was to describe the kinetics of TTV DNA loads and their association with critical-illness related complications. METHODS: We performed a longitudinal study in 115 brain-injured patients from a prospective cohort, collected endotracheal and blood samples at three time points (T1, T2, T3) during the two weeks post-admission in intensive care unit, and measured viral DNA loads using the TTV R-gene® kit (Biomerieux) and a pan-Anelloviridae in house qRT-PCR. RESULTS: TTV DNA was detected in the blood of 69, 71, and 64% of brain-injured patients at T1, T2 and T3 respectively. Time-associated variations of TTV and Anellovirus (AV) DNA loads were observed. Using a linear mixed-effects model, we found that HAP and ARDS were associated with lower blood AV DNA loads. CONCLUSION: Our results show that HAP or ARDS in critically ill patients are associated to changes in AV DNA loads, and should be evaluated further as a biomarker of immune disorders leading to these complications.
RESUMEN
The etiology of multiple sclerosis (MS) remains elusive. Among the possible causes, the increase of anti-Neu5Gc antibodies during EBV primo-infection of Infectious mononucleosis (IMN) may damage the integrity of the blood-brain barrier facilitating the transfer of EBV-infected B cells and anti-EBV T cell clones in the brain. We investigated the change in titers of anti-Neu5Gc and anti-α1,3 Galactose antibodies in 49 IMN, in 76 MS, and 73 clinically isolated syndrome (CIS) patients, as well as age/gender-matched healthy individuals. Anti-Gal and anti-Neu5Gc are significantly increased during IMN (p=0.02 and p<1.10-4 respectively), but not in acute CMV primo-infection. We show that, whereas there was no change in anti-Neu5Gc in MS/CIS, the two populations exhibit a significant decrease in anti-Gal (combined p=2.7.10-3), in contrast with patients with non-MS/CIS central nervous system pathologies. Since anti-Gal result from an immunization against α1,3 Gal, lacking in humans but produced in the gut, our data suggest that CIS and MS patients have an altered microbiota or an altered response to this microbiotic epitope.
Asunto(s)
Enfermedades Desmielinizantes/sangre , Enfermedades Desmielinizantes/inmunología , Galactosa/inmunología , Inmunoglobulina G/sangre , Adolescente , Adulto , Anciano , Niño , Preescolar , Infecciones por Virus de Epstein-Barr/sangre , Infecciones por Virus de Epstein-Barr/inmunología , Femenino , Humanos , Masculino , Persona de Mediana Edad , Embarazo , Adulto JovenRESUMEN
While enteroviruses (EV) are a well-recognized cause of aseptic meningitis in children, human parechoviruses (HPeV), especially genotype 3, have been increasingly reported as a frequent cause of sepsis-like illness and meningitis among young infants. The aim of this study was to describe the epidemiological, clinical, and laboratory characteristics of HPeV infections in infants and to compare them with those of well-known EV infections. This monocentric retrospective study was carried out at the pediatric unit of Nantes University Hospital from January 2015 to August 2018. All patients under 18 years of age with diagnosis codes referring to fever, for whom viral infection was suspected and cerebrospinal fluid (CSF) specimens were collected, were included. All CSF specimens were screened by duplex real-time polymerase chain reaction (PCR) assay that allows for the simultaneous detection of EV and HPeV in clinical samples. During the study period, 1373 CSF specimens from patients under 18 were included. A total of 312 CSF samples were positive for HPeV (n=34) or EV (n=278). Among the 34 HPeV-positive patients, 97% (33/34) were under 3 months of age, whereas the rate was 54% (149/278) for EV-positive patients (P<0.001); thus, patients under 3 months of age were defined as the study population for the rest of this work. A review of the medical records was carried out for the positive cases. In this population, the HPeV detection rate was 5.6% versus 25.3% (P<0.001) for EV. All but one of the HPeV samples available for genotyping were HPeV-3. No seasonality was observed for HPeV infections. Length of hospital stay tended to be longer for children infected with HPeV compared with those infected by EV (3 days vs. 2 days, P=0.05). Clinicians reported more severe illness presentations among HPeV-infected infants, with more frequent administration of fluid bolus (P<0.02). Regarding laboratory characteristics, a significant lack of cellular reaction in the CSF (P=0.004) as well as lower C-reactive protein (CRP) levels (P=0.006) and neutrophil counts (P<0.001) were noted for HPeV infections compared with EV infections. Our results confirm the early onset of HPeV infections (more than 95% of patients aged under 3 months). The clinical presentation and laboratory characteristics of the two infections was similar. However, some higher clinical severity criteria and a lack of CSF pleocytosis were regularly observed in patients with HPeV infections. Considering the significant proportion (5.6%; 95% CI, 3.7-7.5) of all CSF samples in our series, HPeV detection should be systematically included in the microbiological diagnosis of febrile children under 3 months of age.
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Infecciones por Enterovirus/diagnóstico , Enterovirus/aislamiento & purificación , Parechovirus/aislamiento & purificación , Infecciones por Picornaviridae/diagnóstico , Enterovirus/genética , Infecciones por Enterovirus/epidemiología , Femenino , Francia/epidemiología , Humanos , Lactante , Recién Nacido , Tiempo de Internación , Masculino , Parechovirus/genética , Infecciones por Picornaviridae/epidemiología , Reacción en Cadena de la Polimerasa , Prevalencia , Estudios Retrospectivos , Sepsis/diagnósticoRESUMEN
Human cytomegalovirus (CMV) has been shown to alter adhesion molecule expression on permissive cells such as endothelial cells. The aim of the present study was to investigate expression of receptors for these molecules on CMV infected polymorphonuclear leukocytes (PMNLs). CMV-induced variations on cellular integrin expression were examined using an in vitro system to obtain infected PMNLs. A triparametric flow cytometry approach was developed, which allows combined detection, in a single experiment, of both viral intranuclear antigen in the selected PMNLs and cellular CD11/CD18 expression. Comparison of infected PMNLs with uninfected cells showed a decrease of up to 50% in the expression of CD11b, CD11c, and CD18. This study thus demonstrates that the presence of CMV in PMNLs, which characterizes active infection, modifies the expression of integrins and may thus affect cell-to-cell interactions and immune functions.
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Citomegalovirus/fisiología , Citometría de Flujo/métodos , Integrinas/análisis , Neutrófilos/inmunología , Neutrófilos/virología , Antígenos CD11/análisis , Antígenos CD18/análisis , Humanos , InmunofenotipificaciónRESUMEN
BACKGROUND: The purpose of this prospective study was to evaluate the usefulness of quantifying DNA-cytomegalovirus (CMV) load for the diagnosis and monitoring of CMV disease among renal and pancreas transplant patients under immunosuppressive drugs. METHODS: A longitudinal study was conducted among 34 consecutive, unselected renal and pancreas/renal transplanted patients in our unit. During the first 3 posttransplant months, weekly monitoring of CMV infection and CMV disease was done, involving the determination of viremia by the shell vial assay, qualitative DNAemia by semi-nested polymerase chain reaction (PCR) and quantitative DNAemia by the hybrid capture system (HCS), a new and original hybridization method (337 samples were collected for each test). Qualitative and quantitative DNAemia results were blinded to physicians and three grades of disease were defined according to CMV related symptom occurrence. RESULTS: PCR was the most sensitive (100%) but the least specific (78%) method for the diagnosis of CMV disease. HCS was specific for CMV genome detection, sensitive and reproducible. Blood DNA levels above 60 pg/ml were predictive of severe or moderate CMV disease (sensitivity, 92%; specificity, 100%). A significant decrease in viral load was observed after ganciclovir administration, and a positive PCR or HCS result at the end of the antiviral treatment was associated with relapse of CMV infection or disease. CONCLUSIONS: It is concluded that quantitative DNAemia detection, with this new commercially available method, can predict disease and may be useful for a rational evaluation of ganciclovir preemptive therapy in such patients.
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Biomarcadores/análisis , ADN Viral/análisis , Adolescente , Adulto , Anciano , Antivirales/uso terapéutico , Infecciones por Citomegalovirus/sangre , Infecciones por Citomegalovirus/diagnóstico , Infecciones por Citomegalovirus/tratamiento farmacológico , Femenino , Estudios de Seguimiento , Ganciclovir/uso terapéutico , Humanos , Estudios Longitudinales , Masculino , Persona de Mediana Edad , Hibridación de Ácido Nucleico/métodos , Prevalencia , Estudios Prospectivos , Reproducibilidad de los ResultadosRESUMEN
The aim of this prospective study of 162 recipients of bone marrow transplantation (BMT) was to evaluate the use of DNAemia detection by semi-nested PCR for the diagnosis of human cytomegalovirus (HCMV) infection and HCMV disease. We compared the results obtained for DNAemia with those obtained for viremia, using the shell vial assay. Patients were divided in three groups, according to BMT type (allogeneic or autologous) and date of transplant; 876 DNAemia/viremia pairs were analyzed and the overall concordance between the two tests was 97.5%. Discrepancies between the two tests were essentially due to the earlier positivity of DNAemia. Among the 10 patients with positive DNAemia episodes, 9 developed HCMV disease. DNAemia was more sensitive than viremia for HCMV disease diagnosis, while viremia had a higher positive predictive value. DNAemia appeared easily adaptable to routine laboratory use, less expensive and more informative than viremia. This study shows that DNAemia is a method that can replace viremia detection, allowing HCMV infection and disease follow-up of recipients of allogeneic BMT during the first year after BMT.
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Trasplante de Médula Ósea , Infecciones por Citomegalovirus/diagnóstico , Citomegalovirus/aislamiento & purificación , ADN Viral/sangre , Reacción en Cadena de la Polimerasa/métodos , Viremia/diagnóstico , Adolescente , Adulto , Diagnóstico Diferencial , Estudios de Seguimiento , Humanos , Persona de Mediana Edad , Estudios Retrospectivos , Sensibilidad y Especificidad , Trasplante Autólogo , Trasplante HomólogoRESUMEN
Human cytomegalovirus replication was evaluated in polymorphonuclear leukocytes from ten renal transplant recipients. Three new reverse transcription polymerase chain reactions with plate hybridization suitable for automation were developed for the detection of immediate-early spliced UL123 mRNA, early-late pp65 mRNA, and late spliced UL22 mRNA. The presence of UL22mRNA was found to be significantly associated with the occurrence of cytomegalovirus (CMV) disease.
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Colorimetría/métodos , Infecciones por Citomegalovirus/diagnóstico , Trasplante de Riñón , Neutrófilos/virología , Hibridación de Ácido Nucleico/métodos , Infecciones Oportunistas/diagnóstico , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Infecciones por Citomegalovirus/complicaciones , Humanos , Infecciones Oportunistas/complicaciones , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/normas , Transcripción GenéticaRESUMEN
In order to confirm the occurrence of active Human herpesvirus-6 (HHV-6) infection, two optimal procedures were developed to detect directly replicating virus. MT4 cells and peripheral blood mononuclear cells (PBMCs) infected with two different strains (HST and a patient strain GUI) were used. The first method consisted of a one-step reverse transcription PCR amplifying a part of the late alternatively spliced U100 gene which encode the gp 82-105 viral glycoprotein. Two extraction methods and two RT-PCR kits were evaluated, leading to the selection of TaKaRa mRNA selective PCR kit. The second procedure consisted in a flow cytometry method to analyze the expression of two late viral HHV-6 antigens using 7C7 and 10G6 monoclonal antibodies. Four fixation permeabilization procedures were compared and the preparation of cells with paraformaldehyde (PFA) 4% was found to be optimal. Evaluation of these methods was then realized during a sequential culture of HST strain on MT4 cells. This kinetic study confirmed that Mabs recognized late antigens and demonstrate that the U100 gene splicing starts at a late stage of multiplication whereas unspliced forms are detectable earlier in the cycle.
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Citometría de Flujo/métodos , Herpesvirus Humano 6/genética , Herpesvirus Humano 6/fisiología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Virología/métodos , Antígenos Virales/análisis , Secuencia de Bases , Línea Celular , Cartilla de ADN/genética , Genes Virales , Herpesvirus Humano 6/inmunología , Humanos , Leucocitos Mononucleares/virología , Empalme del ARN , ARN Mensajero/análisis , ARN Mensajero/genética , ARN Viral/análisis , ARN Viral/genética , Proteínas del Envoltorio Viral/genética , Replicación ViralRESUMEN
Since detection of amplified polymerase chain reaction products is a complicated procedure, a colorimetric microtiter plate hybridization assay was developed to automate specific detection of amplified HIV targets. In this assay, hybridization is seen by an antibody reacting selectively with double-stranded DNA. One hundred and ten amplified products detected by a DNA enzyme immunoassay (DEIA) and by classical hybridization with a digoxigenin-labeling probe, detection sensitivities were less than 10 HIV targets per 10(6) cells. This study demonstrates the specificity and sensitivity of DEIA for detecting amplified HIV targets. The use of the same equipment as for ELISA and the complete automation of the procedure allow a large number of samples to be processed in the clinical laboratory.
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ADN Viral/análisis , ADN Viral/inmunología , Anticuerpos Anti-VIH/inmunología , VIH-1/aislamiento & purificación , Técnicas para Inmunoenzimas , Reacción en Cadena de la Polimerasa/métodos , Adulto , Anticuerpos Monoclonales/inmunología , Línea Celular Transformada , Colorimetría , Seropositividad para VIH/diagnóstico , Seropositividad para VIH/inmunología , VIH-1/genética , HumanosAsunto(s)
Antivirales/farmacología , Infecciones por Citomegalovirus/cirugía , Farmacorresistencia Viral , Foscarnet/farmacología , Neumonía Viral/cirugía , Trasplante de Células Madre/efectos adversos , Infecciones por Citomegalovirus/terapia , ADN Polimerasa Dirigida por ADN/genética , Femenino , Humanos , Leucemia Mielógena Crónica BCR-ABL Positiva/complicaciones , Leucemia Mielógena Crónica BCR-ABL Positiva/terapia , Persona de Mediana Edad , Neumonía Viral/terapia , Neumonía Viral/virología , Polimorfismo Genético , Trasplante Homólogo/métodos , Resultado del Tratamiento , Carga Viral , Proteínas Virales/genéticaRESUMEN
This single centre study assessed the incidence, kinetics and predictive factors of EBV reactivation and EBV-related lymphoproliferative diseases (LPD) in 33 consecutive patients who received a reduced intensity conditioning (RIC) before umbilical cord blood transplantation (UCBT). During the first 6 months after UCBT, weekly all patients were DNA-PCR screened in the peripheral blood for EBV reactivation and were clinically monitored for clinical features attributable to EBV. The cumulative incidences of EBV reactivation (defined as an EBV load >1000 EBV copies per 10(5) cells measured at least once during follow-up) at 6 months and 2 years after UCBT were 9 (95% confidence interval (CI), 2-22%) and 17% (95% CI, 6-33%), respectively. In 28 patients (85%), the EBV load remained negative at all times, and none of these patients experienced any sign of LPD. Five patients (15%) experienced at least one EBV reactivation episode. EBV reactivation was observed at a median of 132 days (range, 85-438) after UCBT. Two patients developed EBV-related LPD (cumulative incidence, 6% at 3 years). With a median follow-up of 468 days (range, 92-1277) post UCBT, the OS was 62% at 3 years. Five patients died of disease progression and seven patients died of transplant-related complications, including one case of EBV-related LPD. Univariate analysis did not identify any significant risk factor associated with EBV reactivation. We conclude that patients undergoing RIC UCBT are at risk for EBV reactivation, with the need for close EBV monitoring and the use of preemptive rituximab treatment as some cases may progress to life-threatening LPD.
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Trasplante de Células Madre de Sangre del Cordón Umbilical/efectos adversos , Infecciones por Virus de Epstein-Barr/virología , Herpesvirus Humano 4/fisiología , Acondicionamiento Pretrasplante/efectos adversos , Adolescente , Adulto , Anciano , Trasplante de Células Madre de Sangre del Cordón Umbilical/métodos , Infecciones por Virus de Epstein-Barr/tratamiento farmacológico , Infecciones por Virus de Epstein-Barr/etiología , Infecciones por Virus de Epstein-Barr/inmunología , Femenino , Neoplasias Hematológicas/cirugía , Neoplasias Hematológicas/virología , Herpesvirus Humano 4/inmunología , Humanos , Incidencia , Masculino , Persona de Mediana Edad , Estudios Retrospectivos , Acondicionamiento Pretrasplante/métodos , Resultado del Tratamiento , Activación ViralRESUMEN
This single centre study assessed the incidence, kinetics and predictive factors of Epstein-Barr Virus (EBV) reactivation and EBV-related lymphoproliferative diseases (LPDs) in 175 consecutive patients who received a reduced-intensity conditioning (RIC) before allogeneic hematopoietic stem cell transplantation (allo-HSCT). The cumulative incidence of EBV reactivation at 6 months after allo-HSCT defined as an EBV PCR load above 1000 copies of EBV DNA/10(5) cells was 15%, and none of these patients experienced any sign or symptom of LPD. A total of 17 patients, who had EBV DNA levels exceeding 1000 copies/10(5) cells on two or more occasions, were pre-emptively treated with rituximab. With a median follow-up of 655 (range, 92-1542) days post allo-HSCT, there was no statistically significant difference in term of outcome between those patients who experienced an EBV reactivation and those who did not. In multivariate analysis, the use of antithymocyte globulin as part of the RIC regimen was the only independent risk factor associated with EBV reactivation (relative risk=4.9; 95% confidence interval, 1.1-21.0; P=0.03). We conclude that patients undergoing RIC allo-HSCT using anti-thymocyte globulin as part of the preparative regimen are at higher risk for EBV reactivation. However, this did not impact on outcome, as quantitative monitoring of EBV viral load by PCR and preemptive rituximab therapy allowed for significantly reducing the risk of EBV-related LPD.
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Trasplante de Células Madre Hematopoyéticas/efectos adversos , Herpesvirus Humano 4/fisiología , Acondicionamiento Pretrasplante/efectos adversos , Activación Viral/efectos de los fármacos , Adolescente , Adulto , Anciano , Anticuerpos Monoclonales de Origen Murino/administración & dosificación , Suero Antilinfocítico/efectos adversos , Suero Antilinfocítico/uso terapéutico , Antineoplásicos , Humanos , Trastornos Linfoproliferativos/virología , Persona de Mediana Edad , Estudios Retrospectivos , Rituximab , Acondicionamiento Pretrasplante/métodos , Carga Viral/efectos de los fármacos , Adulto JovenRESUMEN
Occurrence of CMV, EBV and human herpes virus 6 (HHV6) infections and immune reconstitution were compared in 15 adult patients receiving a cord blood transplantation (CBT) and 40 patients who received an allogeneic transplantation from a matched unrelated donor (MUD). Herpes virus DNA quantifications in the blood (459 samples) were performed before and then monthly up to 9 months after transplant and the main lymphocytes populations were counted at 3, 6 and 9 months. Incidence of HHV6 infection was significantly higher in the CBT group (80 vs 42.5%; P<0.0001), with higher viral load (P<0.0001). In multivariate analysis, the use of a CBT and a myeloablative conditioning regimen were found to increase the risk of HHV6 infection (odds ratio (OR)=5.4, P=0.02 and OR=3.5, P=0.04, respectively). Incidences of CMV were similar between the two groups whereas MUD increased the risk of EBV infection, in univariate analysis only. HHV6 reactivation translated toward delayed neutrophils and plts engraftment in the two groups. MUD and CBT do not share the same immune reconstitution patterns, notably for B and CD8 lymphocytes and NK cells. There is a strong and specific relationship between HHV6 infection and the use of cord blood cells.
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Trasplante de Células Madre de Sangre del Cordón Umbilical/efectos adversos , Supervivencia de Injerto/inmunología , Hematopoyesis/inmunología , Herpesvirus Humano 6 , Agonistas Mieloablativos/toxicidad , Infecciones por Roseolovirus/etiología , Adulto , Infecciones por Citomegalovirus/etiología , Infecciones por Virus de Epstein-Barr/etiología , Femenino , Humanos , Incidencia , Cinética , Masculino , Persona de Mediana Edad , Infecciones Oportunistas/etiología , Estudios Retrospectivos , Donantes de Tejidos , Carga Viral , Activación Viral/efectos de los fármacos , Adulto JovenRESUMEN
BK virus (BKV) infection during the first year after renal transplantation was studied prospectively in 104 unselected consecutive patients. Viral DNA in urine (DNAuria) and plasma (DNAemia) samples was detected and quantified by real-time PCR. The noncoding control region (NCCR) of BKV isolates was sequenced. DNAuria and DNAemia occurred in 57% and 29% of patients, respectively. Three groups were defined, uninfected patients (group 1, n=45), patients with DNAuria (group 2, n=29) and patients with positive DNAemia (group 3, n=30). Active infection started within the first 3 months in 80% of patients. Cold ischemia duration over 24 h and the administration of tacrolimus were identified as significant risks factors for DNAuria, whereas it remains more frequently negative in patients receiving cyclosporine A. The risk for positive DNAemia was higher in patients with DNAuria (notably for viral load (VL)>4 log/mL) or treated with tacrolimus. No relationship was found with genetic variability in the NCCR sequence. Our data highlight the high frequency of active BKV infection after renal transplantation. Although high VL was detected in some patients, none developed a BKV nephropathy. A prospective follow-up of the whole population during the first year post renal transplantation is thus not useful to predict BKV disease.
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Virus BK/fisiología , Enfermedades Renales/virología , Trasplante de Riñón , Infecciones por Polyomavirus/virología , Infecciones Tumorales por Virus/virología , Secuencia de Bases , Ciclosporina/uso terapéutico , ADN Viral/análisis , Femenino , Humanos , Inmunosupresores/uso terapéutico , Enfermedades Renales/terapia , Estudios Longitudinales , Masculino , Persona de Mediana Edad , Datos de Secuencia Molecular , Estudios Prospectivos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Homología de Secuencia de Ácido Nucleico , Tacrolimus/uso terapéutico , Carga Viral , Replicación ViralRESUMEN
To quantify hepatitis A virus (HAV) in experimentally contaminated mussels, we developed an internal standard RNA with a 7-nucleotide deletion for competitive reverse transcription (RT)-PCR. Deposited directly into the sample, this standard was used both as extraction control and as quantification tool. After coextraction and competitive RT-PCR, standard and wild-type products were detected by differential hybridization with specific probes and a DNA enzyme immunoassay. The quantifiable range with this reproducible method was 10(4) to 10(7) copies of HAV/gram or 400 to 10(6) 50% tissue culture infective doses/ml.
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Bivalvos/virología , Hepatovirus/genética , Hepatovirus/aislamiento & purificación , ARN Viral/genética , ARN Viral/aislamiento & purificación , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Mariscos/virología , Animales , Secuencia de Bases , Cartilla de ADN/genética , Enfermedades Transmitidas por los Alimentos/prevención & control , Hepatitis A/prevención & control , Hepatovirus/patogenicidad , Humanos , Datos de Secuencia Molecular , ARN Viral/normas , Estándares de Referencia , Reproducibilidad de los Resultados , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/normas , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/estadística & datos numéricosRESUMEN
To determine the incidence and clinical relevance of active human herpesvirus 6 (HHV-6) infection, 92 consecutive unselected recipients of autologous or allogeneic stem cell grafts were investigated in a prospective longitudinal study. Active infection was assessed by the presence of viral deoxyribonucleic acid (DNA) in 846 peripheral blood mononuclear cell specimens and 115 plasma specimens, by means of a specially developed polymerase chain reaction designed to avoid detection of latent genome. The incidence of HHV-6 infection observed was 42.5%, irrespective of the type or source of graft, and infection was significantly associated with partial (P=.002) or total myelosuppression (P=.01) and fever (P<. 000001). Infusion of bone marrow as the source of graft, reactivation occurring before platelet or neutrophil engraftment, and presence of HHV-6 DNA in plasma were identified as risk factors for symptomatic HHV-6 infection (P<.002).
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Trasplante de Células Madre Hematopoyéticas/efectos adversos , Infecciones por Herpesviridae/etiología , Herpesvirus Humano 6 , Adolescente , Adulto , Anciano , Secuencia de Bases , Niño , Preescolar , Cartilla de ADN/genética , ADN Viral/sangre , ADN Viral/genética , Femenino , Infecciones por Herpesviridae/transmisión , Infecciones por Herpesviridae/virología , Herpesvirus Humano 6/genética , Herpesvirus Humano 6/aislamiento & purificación , Herpesvirus Humano 6/patogenicidad , Humanos , Estudios Longitudinales , Masculino , Persona de Mediana Edad , Estudios Prospectivos , Trasplante Autólogo , Trasplante HomólogoRESUMEN
Cytomegalovirus (CMV) antigenemia was directly detected in polymorphonuclear leukocytes (PMNLs) from transplant recipients by using flow cytometry (FC). Two fixation and permeabilization methods and seven anti-CMV monoclonal antibodies (MAbs) were evaluated. 1C3, SL20, and NEA-9221 MAbs were more efficacious. The antigenemia detection threshold of FC was 0.05% positive PMNLs, and percentages correlated well with DNA viral load and the appearance of clinical symptoms.
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Antígenos Virales/sangre , Infecciones por Citomegalovirus/diagnóstico , Citometría de Flujo/métodos , Neutrófilos/virología , Anticuerpos Monoclonales , Anticuerpos Antivirales , Trasplante de Médula Ósea/efectos adversos , Estudios de Evaluación como Asunto , Técnica del Anticuerpo Fluorescente , Humanos , Trasplante de Riñón/efectos adversos , Permeabilidad , Reproducibilidad de los Resultados , Factores de Tiempo , Fijación del TejidoRESUMEN
Cytotoxic T lymphocytes (CTLs) play a central role in the control of persistent human cytomegalovirus (HCMV) infection in healthy virus carriers. Previous analyses of the specificity of HCMV-reactive CD8(+) CTLs drawn from in vitro models in which antigen-presenting cells were autologous fibroblasts infected with laboratory HCMV strains have shown focusing of CTL responses against the major tegument protein, pp65. By contrast, the 72-kDa major immediate-early protein (IE1) was identified as a minor target for this response. Here we have studied the fine specificity and T-cell-receptor features of T-cell clones generated against autologous B lymphoblastoid cell lines stably transfected with HCMV cDNA coding for either pp65 or a natural variant of IE1. This strategy allowed efficient generation of T-cell clones against IE1 and pp65 and led to the identification of several new IE1 and pp65 epitopes, including some located in polymorphic regions of IE1. Such an approach may provide relevant information about the characteristics of the CTL response to IE1 and the effect of viral polymorphism on the immune response against HCMV.
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Linfocitos B/inmunología , Citomegalovirus/inmunología , Proteínas Inmediatas-Precoces/inmunología , Fosfoproteínas/inmunología , Receptores de Antígenos de Linfocitos T alfa-beta/inmunología , Linfocitos T Citotóxicos/inmunología , Proteínas de la Matriz Viral/inmunología , Proteínas Virales , Alelos , Animales , Células COS , Línea Celular Transformada , Epítopos de Linfocito T/inmunología , Antígeno HLA-A2/inmunología , Antígenos HLA-B/inmunología , Antígeno HLA-B18 , Antígenos de Histocompatibilidad Clase I/inmunología , Humanos , Proteínas Inmediatas-Precoces/genética , Fosfoproteínas/genética , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/inmunología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Linfocitos T Citotóxicos/citología , Proteínas de la Matriz Viral/genéticaRESUMEN
A technique was developed with flow cytometry to quantify the two immediate-early proteins ZEBRA and Rta, which are involved in the activation of Epstein-Barr virus replication. We evaluated four monoclonal antibodies on four cell lines (B95-8, RAJI, Namalwa, and P3HR1) with varying levels of expression of these replication-phase antigens. The Namalwa lymphoma cell line was used as a negative control. Four fixation-permeabilization procedures were compared. The preparation of cells with paraformaldehyde and methanol in sequence, and antigen detection with AZ125 and AR 5A9 monoclonal antibodies, were found to be the optimal conditions in these cell lines. Our procedure allowed ZEBRA antigen to be detected in 4.85% of peripheral blood mononuclear cells from a transplant recipient with a lymphoproliferative disease.