RESUMEN
The aims of the study were to assess the risk of HHV8 transmission resulting from organ transplantation, and related morbidity in liver, heart and kidney transplant recipients. Donor and recipient serologies were screened between January 1, 2004 and January 1, 2005 using HHV8 indirect immunofluorescence latent assay (latent IFA) and indirect immunofluorescent lytic assay (lytic IFA). Recipients negative for latent IFA with a donor positive for at least one test were sequentially monitored for HHV8 viremia and underwent serological tests over a period of 2 years. The results showed that among 2354 donors, HHV8 seroprevalence was 9.9% (lytic IFA) and 4.4% (latent IFA). A total of 454 organ recipients (281 renal, 116 liver and 57 heart) were monitored over a 2-year period. Seroconversion was observed in 12 patients (cumulative incidence 28%) whose donor had positive latent IFA and in 36 patients (cumulative incidence 29%) whose donors were positive only for lytic IFA, without differences across types of transplants. Positive HHV8 viremia was detected in only 4 out of 89 liver transplant recipients during follow-up and not in recipients of other types of transplant. Two liver transplant recipients and one kidney transplant recipient developed KS. In conclusion, although HHV8 transmission is a frequent event after organ transplantation, HHV8-related morbidity is rather rare but can be life threatening. Donor screening is advisable for monitoring HHV8 seronegative liver transplant recipients.
Asunto(s)
Infecciones por Herpesviridae/transmisión , Herpesvirus Humano 8/aislamiento & purificación , Trasplante de Órganos , Adulto , Femenino , Técnica del Anticuerpo Fluorescente , Infecciones por Herpesviridae/fisiopatología , Infecciones por Herpesviridae/virología , Humanos , Masculino , Persona de Mediana Edad , Donantes de Tejidos , ViremiaRESUMEN
PURPOSE OF THE STUDY: Very few is known on genotype II hepatitis A virus (HAV) since it is rarely isolated. From 2002 to 2007, the French observatory of HAV identified six sub-genotype IIA strains of which one from a patient having travelled to West Africa. To investigate the possible African origin of sub-genotype IIA, we determined its prevalence among French travellers in 2008 and characterised its genetic variability. PATIENTS AND METHODS: The 2008 mandatory notification records were screened for travel to Africa. Viral genotype was determined on the nucleotide sequencing of the VP1/2A junction region. The P1 region coding for capsid proteins was used to compare the genetic diversity of IIA isolates to those of other genotypes. RESULTS: In 2008, five out of 54 patients returning from West Africa were infected by IIA strains and an additional "autochthonous" case was identified. Two more African cases were identified in 2009. A total of 14 IIA isolates (eight African and six "autochthonous") were analysed. Nucleotide and amino-acid variability of IIA sequences was lower than that of the other genotypes. Phylogenetic analysis revealed the clustering of two "autochthonous" cases with African isolates whereas the other ones belonged to a different lineage. CONCLUSION: Most IIA strains isolated in France are imported by travellers returning from West Africa. However, the unexplained contamination mode of some "autochthonous" cases suggests another geographical origin to discover or a French reservoir to explore.
Asunto(s)
Virus de la Hepatitis A/genética , Hepatitis A/virología , ARN Viral/genética , Adolescente , Adulto , África Occidental , Secuencia de Aminoácidos , Niño , Femenino , Francia , Variación Genética , Genotipo , Virus de la Hepatitis A/clasificación , Humanos , Masculino , Persona de Mediana Edad , Datos de Secuencia Molecular , Filogenia , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Viaje , Proteínas Estructurales Virales/genética , Adulto JovenRESUMEN
PURPOSE OF THE STUDY: Hepatitis C virus genotyping is needed for treatment decision and monitoring. The results of a genotyping assay based on real-time PCR and TaqMan chemistry were compared with the results of NS5B region sequencing. MATERIALS AND METHODS: One hundred and two sera (genotypes 1-6) were tested. Amplification and detection of viral RNA were performed with the Abbott RealTime HCV Genotype II assay targeting 5'non-coded region (5'NC) for the identification of genotypes 1 to 6 and NS5B, for 1a and 1b subtypes detection. Sequencing of 5'NC fragment was used to resolve discrepant results. RESULTS: No indeterminate results were obtained. Concordance with NS5B sequencing was 93% (95 on 102), 96% at the genotype level (98 on 102) and 93% for genotype 1 subtyping (40 on 43). Discordant genotyping results were a 2f subtype identified as 5, a 6a typed as 1, a 3a identified as a 1-3 co-infection and a 4r identified as a 1-4 co-infection. Discordant subtyping results were 2 1b subtypes only typed as 1 and a 1e identified as 1a. CONCLUSION: Abbott RealTime HCV Genotype II assay is a rapid, automated and simple to interpret method for HCV genotyping. It allows the detection of possible mixed infections which might have a negative impact on therapeutic response. However, the discrepant results found in this small series underline the need for assay optimization.
Asunto(s)
Sistemas de Computación , Genotipo , Hepacivirus/genética , Reacción en Cadena de la Polimerasa/métodos , ARN Viral/genética , Juego de Reactivos para Diagnóstico , Análisis de Secuencia de ARN/métodos , Hepacivirus/clasificación , Hepacivirus/aislamiento & purificación , Hepatitis C Crónica/sangre , Hepatitis C Crónica/virología , Humanos , Estudios Prospectivos , ARN Viral/sangre , ARN Viral/aislamiento & purificación , Polimerasa Taq , Factores de Tiempo , Viremia/virologíaRESUMEN
We report the successful control of an outbreak caused by imipenem-resistant VIM-1-producing Klebsiella pneumoniae (IR-Kp) in France. This outbreak occurred in a care centre for abdominal surgery that includes a 15-bed liver intensive care unit and performs more than 130 liver transplantations per year. The index case was a patient with acute liver failure transferred from a hospital in Greece for urgent liver transplantation who was carrying IR-Kp at admission as revealed by routine culture of a rectal swab. Infection control measures were undertaken and included contact isolation and promotion of hand hygiene with alcohol-based hand rub solution. Nevertheless, secondary IR-Kp cases were identified during the six following months from 3 December 2003 to 2 June 2004. From 2 June to 21 October, extended infection control measures were set up, such as cohorting IR-Kp carriers, contact patients and new patients in distinct sections with dedicated staff, limiting ward admission, and strict control of patient transfer. They led to a rapid control of the outbreak. The global attack rate of the IR-Kp outbreak was 2.5%, 13% in liver transplant patients and 0.4% in the other patients in the care centre (p<0.005). Systematic screening for IR-Kp of all patients admitted to the care centre is still maintained to date and no secondary IR-Kp case has been detected since 2 June 2004.
Asunto(s)
Infección Hospitalaria/prevención & control , Brotes de Enfermedades/prevención & control , Control de Infecciones/métodos , Infecciones por Klebsiella/epidemiología , Klebsiella pneumoniae/aislamiento & purificación , Antibacterianos/farmacología , Antibacterianos/uso terapéutico , Infección Hospitalaria/epidemiología , Infección Hospitalaria/microbiología , Farmacorresistencia Bacteriana , Femenino , Francia/epidemiología , Hospitales con más de 500 Camas , Humanos , Imipenem/farmacología , Imipenem/uso terapéutico , Unidades de Cuidados Intensivos , Infecciones por Klebsiella/tratamiento farmacológico , Infecciones por Klebsiella/microbiología , Infecciones por Klebsiella/prevención & control , Klebsiella pneumoniae/clasificación , Klebsiella pneumoniae/enzimología , Trasplante de Hígado , Masculino , Pruebas de Sensibilidad Microbiana , beta-Lactamasas/metabolismoRESUMEN
The impact of preexisting or acquired Kaposi sarcoma herpesvirus (KSHV) infection in kidney transplant recipients was evaluated in a prospective study. Serum collected from kidney donors and recipients before transplantation were tested for antibodies against KSHV latent nuclear antigen. Three groups of recipients were defined: group A (KSHV+), group B (KSHV-, KSHV+ donor) and group C (donor and recipient KSHV-). Blood was collected from recipients, every 3 months for 3 years, for KSHV viremia (groups A and B), quantitative (group A) and qualitative serology (group B). Data of group C recipients were extracted from a French database. The prevalence of KSHV antibodies was 1.1% in donors and 3.2% in recipients. There were respectively 161, 64 and 4744 recipients in groups A, B and C. In group A, 13% developed Kaposi's sarcoma (KS). Age >53.5 years (p = 0.025) and black skin (p = 0.0054) were associated with KS development. In group B, three recipients developed clinical manifestations related to KSHV infection. There was no difference in terms of survival and graft loss between the three groups. In conclusion, although kidney recipients should be aware of the additional risk of KSHV morbidity, KSHV+ recipients should not be systematically excluded from kidney transplantation.
Asunto(s)
Anticuerpos Antivirales/sangre , Infecciones por Herpesviridae/mortalidad , Trasplante de Riñón/mortalidad , Población Negra/estadística & datos numéricos , Femenino , Estudios de Seguimiento , Francia/epidemiología , Supervivencia de Injerto , Infecciones por Herpesviridae/etnología , Herpesvirus Humano 8/inmunología , Humanos , Trasplante de Riñón/etnología , Masculino , Persona de Mediana Edad , Morbilidad , Estudios Prospectivos , Factores de Riesgo , Estudios Seroepidemiológicos , Análisis de Supervivencia , Población Blanca/estadística & datos numéricosRESUMEN
Since September 2008, 26 cases of hepatitis A with a history of travel to Egypt have been reported in France. Investigations indicate that a common source of contamination linked to Nile river cruises is the most likely explanation of the increase in the number of cases reported in France as well as in several other European Union countries.
Asunto(s)
Brotes de Enfermedades/estadística & datos numéricos , Hepatitis A/epidemiología , Vigilancia de la Población , Medición de Riesgo/métodos , Viaje/estadística & datos numéricos , Análisis por Conglomerados , Francia/epidemiología , Humanos , Incidencia , Factores de RiesgoRESUMEN
Following the notification of nine hepatitis A cases clustered in the Cotes d Armor district in northwestern France, epidemiological, environmental and microbiological investigations were set up in order to identify the source and vehicle of contamination and implement control measures. In total, 111 cases were identified in the outbreak, all of whom lived or had stayed as tourists in the Cotes d Armor district. Of the cases, 87% had eaten raw shellfish, and 81% specifically oysters. Traceback investigations carried out on raw shellfish consumed by the cases showed that the raw shellfish originated from a single shellfish farm. The shellfish were probably contaminated either in the submersible tanks or in a depuration land-based tank where they were stored. The source of contamination was not identified but shellfish could have been tainted by sewage overflows or by wastewater releases from a polluted storm sewer close to the shellfish farm or from on-site sanitation facilities. To prevent future hepatitis A outbreaks due to shellfish consumption from this area, hazards specific to each farm should be analysed. Timely information on sewage overflows should also be part of communities efforts regarding sewage collection and treatment.
Asunto(s)
Brotes de Enfermedades/estadística & datos numéricos , Contaminación de Alimentos/estadística & datos numéricos , Enfermedades Transmitidas por los Alimentos/epidemiología , Hepatitis A/epidemiología , Vigilancia de la Población , Mariscos/virología , Enfermedades Transmitidas por los Alimentos/virología , Francia/epidemiología , Virus de la Hepatitis A/aislamiento & purificación , Humanos , Incidencia , Medición de Riesgo/métodos , Factores de RiesgoRESUMEN
The incidence of Kaposi sarcoma (KS) related to Kaposi sarcoma-associated herpesvirus (KSHV/HHV-8) after organ transplantation is 500-1000 times greater than in the general population, and its occurrence is associated with immunosuppressive therapy. The reported incidence of posttransplant KS ranges from 0.5% to 5%, depending on the patient's country of origin and the type of organ received, mainly after renal transplantation. Posttransplant KS is caused by two possible mechanisms: KSHV reactivation in patients who were infected before the graft and KSHV contamination from the infected organ's donor to the recipient. KSHV reactivation appears to play a greater role in the risk of KS than incident infections. However, some studies, with findings based not only on serological data but also on molecular tracing of the viral infection, have shown that organ-related transmission of KSHV could be more common than previously thought and associated in some cases with severe KSHV-related disease. Precise estimates of KSHV seroprevalence in the organ donor and recipient populations in different countries are lacking. However, studies have reported seroprevalences among donors and recipients that are similar to those among the general population of the country considered. Many studies have suggested the potential utility of screening of KSHV antibodies among organ donors and recipients. However, to date the results of these studies have argued in favor of KSHV screening, even in low-KSHV infection prevalence countries, not to exclude the graft but to have the KSHV status information in order to have the opportunity to monitor, clinically and biologically, patients at risk for KSHV-related disease development. The detection of KSHV antibodies could be done in the days after the transplantation and the results transmitted to the physicians retrospectively. In conclusion, the question of screening donors and recipients for KSHV, even in low-KSHV infection prevalence countries, is still debated, and prospective studies are needed to evaluate the benefit of pre- and posttransplantation strategies.
Asunto(s)
Anticuerpos Antivirales/sangre , Herpesvirus Humano 8/inmunología , Trasplante de Órganos/efectos adversos , Sarcoma de Kaposi/etiología , Donantes de Tejidos , Animales , Humanos , Trasplante de Riñón/efectos adversos , Factores de Riesgo , Sarcoma de Kaposi/epidemiología , Sarcoma de Kaposi/prevención & controlRESUMEN
In November 2006, six symptomatic cases of hepatitis A in pupils of a secondary school in Upper Normandy, France, were reported to the district health service. This paper describes the outbreak investigation undertaken with the aim to identify the vehicle and source of infection, implement control measures and estimate the size of the outbreak. A primary case at the secondary school was defined as a pupil or a member of the staff with IgM anti-HAV detected in the serum and with onset of symptoms between 12 and 21 November 2006; a secondary case was defined as a contact to a primary case and who developed symptoms and had IgM anti-HAV two to seven weeks later. We performed a case control study of primary cases, controls being pupils visiting the same school (cases/controls 1:4) and inspected the canteen facilities. All 13 canteen employees were examined for anti-HAV IgM antibodies. A phylogenetic analysis of HAV of cases was performed. We identified 10 primary and 5 secondary cases. Among primary cases 90% reported eating liver pate at the canteen compared to 62% among controls (OR 5.5, 95% CI 0.62-256.9). One liver pate sample contained markers of faecal contamination. HAV genotypes were of one identical type. All 13 canteen employees were negative for IgM anti-HAV while four had anti-HAV total antibodies. We found deficiencies regarding food preparing procedures and insufficient hand washing facilities. The vehicle of the outbreak was believed to be the liver pate but the source of HAV could not be identified. Insufficient facilities in the canteen hindered staff from maintaining a high hygiene standard and were subsequently improved.
Asunto(s)
Brotes de Enfermedades/estadística & datos numéricos , Contaminación de Alimentos/estadística & datos numéricos , Hepatitis A/epidemiología , Vigilancia de la Población , Medición de Riesgo/métodos , Instituciones Académicas/estadística & datos numéricos , Adolescente , Adulto , Femenino , Francia/epidemiología , Humanos , Incidencia , Masculino , Factores de RiesgoRESUMEN
UNLABELLED: This study aimed at describing the evolution of the epidemiological pattern of hepatitis A in New Caledonia since 1986 and the recent epidemic which occurred in 2005-2006, regarding particularly its demographic and virological aspects and the public health response implemented. The annual or monthly activity records for Hepatitis A sero-diagnostic performed at the Pasteur Institute of New Caledonia were processed in a retrospective analysis (9723 samples tested for the detection of IgM to hepatitis A). Over the 2004-2006 period, a phylogenetic study of representative strains from New Caledonia and other Pacific islands was carried out by the French National Reference Laboratory for hepatitis A (Paul-Brousse hospital, Villejuif, France). RESULTS: The continuous improvement of hygiene that occurred in New Caledonia during the last two decades led to a dramatic drop in the frequency of hepatitis A among patients tested, ranging from an average value of 79 cases (14%) for the 1986-1999 period to 0 case from 2002. However, in 2005, a strong increasing number of confirmed cases was notified, mainly among young people (78% were under the age of 20). In 2006, this epidemic reached the island of Futuna where it involved more than 1% of the total population (56 cases). The phylogenetic study has confirmed the clonality of the virus circulating during this epidemic, not related to other regional strains (Fiji, Vanuatu, New Zealand) nor with a New Caledonian strain from the previous endemic period. This transition situation, with persistence of a high epidemic risk, should encourage the health authorities to implement adapted response strategies, based in particular on systematic case declaration and targeted immunisation programmes.
Asunto(s)
Brotes de Enfermedades/estadística & datos numéricos , Enfermedades Endémicas/estadística & datos numéricos , Hepatitis A/epidemiología , Secuencia de Bases , Virus de la Hepatitis A/clasificación , Virus de la Hepatitis A/genética , Virus de la Hepatitis A/aislamiento & purificación , Humanos , Melanesia/epidemiología , Nueva Caledonia/epidemiología , Filogenia , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Improvements in hygienic and sanitary conditions, especially in developing countries, have lead to a progressive shift in the epidemiologic pattern of hepatitis A. This infection, most commonly transmitted via feco-oral route, is now observed in adult patients reflecting the reduced circulation of the hepatitis A virus in many countries. Paradoxically, the risk of more severe disease forms and outbreaks is increased. Vaccination against hepatitis A appears as an effective way to reduce hepatitis A incidence, HAV could be eradicated if routine vaccination of children is implemented in all countries. However, this vaccination is not presently recommended for areas with high overall and age-specific incidence.
RESUMEN
OBJECTIVE: To describe the spontaneous course, before the introduction of highly active antiretroviral therapy (HAART), of HIV-1 RNA during the AIDS-free period of the disease. To assess the predictive value of changes in HIV-1 RNA levels. DESIGN: A total of 330 patients with a known date of infection followed in the SEROCO cohort. METHODS: HIV-1 RNA levels (threshold, 200 copies/ml) were evaluated from 2243 frozen sera obtained from enrolment until the onset of AIDS or until February 1996. Lowess curves were used to describe the variations of viraemia during follow-up. A Cox regression model was used to assess the predictive value of early and updated CD4 cell count and viral load. RESULTS: In addition to a lower early viral load, patients who remained AIDS-free had, on average, a longer period of viral load decrease after infection (36 versus 18 months), followed by a slower viral load increase compared with those who progressed to AIDS. A true plateau-phase after the seroconversion period, lasting approximately 4 years, was identified only in patients who remained AIDS-free for at least 90 months. In multivariate analysis, both early viral load and later changes were significant predictors of progression to AIDS. A decrease in the CD4 cell count to less than 200 cells/microl and the onset of a group B condition remained significant predictors of progression. CONCLUSION: Our study extends to the early post-seroconversion phase the prognostic value of extracellular HIV-1 RNA levels. Moreover, our data suggest that, in most HIV-infected individuals, a progressive loss of control of viral replication arises during the early years of HIV-1 infection.
Asunto(s)
Infecciones por VIH/virología , VIH-1/aislamiento & purificación , Síndrome de Inmunodeficiencia Adquirida/inmunología , Síndrome de Inmunodeficiencia Adquirida/virología , Recuento de Linfocito CD4 , Femenino , Estudios de Seguimiento , Infecciones por VIH/inmunología , Infecciones por VIH/patología , Humanos , Masculino , Valor Predictivo de las Pruebas , ARN Viral/análisis , Juego de Reactivos para Diagnóstico , Abuso de Sustancias por Vía Intravenosa/virología , Factores de Tiempo , Reacción a la Transfusión , Carga ViralRESUMEN
From November 1985 to August 1987, 22 children underwent orthotopic liver transplantation (OLT). Primary cytomegalovirus (CMV) infection was observed in 3 of 10 seronegative recipients (30%). It was severe in 2 cases, which were treated successfully by a combination of DHPG and CMV hyperimmune globulin. From September 1987 to August 1988, a virologic survey was undertaken. It enabled us to take prophylactic measures and to make an early diagnosis of CMV infection, permitting early antiviral treatment. During this period, 26 children received liver grafts. Nine of 13 seronegative recipients (69, 2%) developed primary CMV infection. Seven had received immunoprophylaxis and 8 were treated by DHPG. None had severe disease and all improved rapidly.
Asunto(s)
Infecciones por Citomegalovirus/epidemiología , Trasplante de Hígado , Aciclovir/administración & dosificación , Anticuerpos Antivirales/administración & dosificación , Niño , Preescolar , Infecciones por Citomegalovirus/prevención & control , Infecciones por Citomegalovirus/terapia , Humanos , Inmunización Pasiva , Estudios Prospectivos , Virosis/epidemiologíaRESUMEN
To determine the prevalence of antibodies to hepatitis C virus (HCV) and the short-term evolution of HCV infection in children undergoing orthotopic liver transplantation, we retrospectively studied the sera and medical records of 149 children surviving from 9 months to 5 years after OLT. Fourteen children (9.4%) were found to be positive for anti-HCV with second-generation ELISA and RIBA tests. They were individualized in 2 distinctive groups. In 5 children, anti-HCV was present before OLT, and in 1 patient only HCV RNA was detected at that time. All 6 patients were positive for anti-HCV after OLT. In the other 8 children, anti-HCV and HCV RNA were only detected after OLT and likely reflect infection during or shortly after OLT. The antibody reactivities against the 3 antigens included in the second-generation RIBA test varied in a given patient throughout follow-up and between these 2 groups of children. In all patients, serum transaminase (ALT) activities returned to normal levels when prednisone therapy was lowered and given every other day. These results indicate that the search for HCV infection in these children is necessary in the differential diagnosis of other liver complications in order to avoid excessive immunosuppressive treatment.
Asunto(s)
Hepatitis C/epidemiología , Hepatitis C/etiología , Trasplante de Hígado/efectos adversos , Adolescente , Secuencia de Bases , Niño , Ensayo de Inmunoadsorción Enzimática , Estudios de Seguimiento , Anticuerpos Antihepatitis/sangre , Hepatitis C/sangre , Anticuerpos contra la Hepatitis C , Virus de Hepatitis/inmunología , Humanos , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , Estudios RetrospectivosRESUMEN
BACKGROUND: Conventional approaches to virus detection failed to provide convincing evidence of a viral etiology in sudden unexplained deaths in infants (SUDI). Many viruses may not have been detected by the routinely used methods; among them enteroviruses (EV) have seldom been found in SUDI. METHODS: In this study EV were sought directly in stools, in pharyngeal and tracheal samples and in myocardial and lung tissues, by using a nested PCR; they were also sought indirectly by detecting IgM antibodies with a new capture immunoassay. Twenty-four SUDI cases were divided into two groups: Group I, certainly associated with; or Group II, not associated with clinical, biologic or histologic signs of viral infection. RESULTS: EV were found in stools but their prevalence was not significantly different between Group I and Group II (20 and 22.2%, respectively). On the contrary EV were detected in respiratory tract and/or lung samples in 53.8% of infants of Group I and in none of Group II. Anti-EV IgM antibodies were detected in 55.5% of infants of Group I and in none of Group II. CONCLUSIONS: These results indicate that EV infection may be specifically associated with the subgroup of SUDI with viral signs, raising the question of its role in this condition.
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Infecciones por Enterovirus/complicaciones , Muerte Súbita del Lactante/etiología , Secuencia de Bases , Preescolar , Infecciones por Enterovirus/diagnóstico , Infecciones por Enterovirus/epidemiología , Femenino , Humanos , Incidencia , Lactante , Recién Nacido , Masculino , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , Pronóstico , Factores de RiesgoRESUMEN
An enzyme-linked immunosorbent assay (ELISA) for the detection of IgG and IgM antibodies against Mycoplasma pneumoniae, performed with commercial antigen and reagents, is compared with the complement fixation test (CF) in a serological study of 209 human sera. Concordant results were usually obtained by CF test and by IgG ELISA in sera from patients with recent M pneumoniae infection. In contrast, when used for an immunological survey of a general population, approximately 27% of the sera negative in the CF test were positive for IgG by the ELISA, and sera with low CF titres were found to have a broad range of IgG titre by the ELISA. This may be due to the greater sensitivity of the ELISA technique and/or to different types of antibody measured by both tests. IgM was detected by ELISA in sera from all patients with recent M pneumoniae infection diagnosed on the basis of clinical findings and by CF assay. Occasionally false-positive IgM antibodies were due to rheumatoid factor (RF); this potential interference necessitates routine testing of IgM antibody positive sera for RF.
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Anticuerpos Antibacterianos/análisis , Neumonía por Mycoplasma/inmunología , Adulto , Niño , Preescolar , Pruebas de Fijación del Complemento , Ensayo de Inmunoadsorción Enzimática , Humanos , Inmunoglobulina G/análisis , Inmunoglobulina M/análisis , Lactante , Persona de Mediana Edad , Factores de TiempoRESUMEN
A non-radioactive hybridization microtiter plate assay was developed and evaluated for detection of the HHV-6 genome and to identify HHV-6 variants A and B. The viral DNA is amplified by the polymerase chain reaction using a 5'-end-biotinylated primer. The biotinylated amplimers are captured on avidin-coated microtiter plates, denaturated with sodium hydroxide and hybridized to a 3'-end-digoxigenin-labelled probe. Subsequently, anti-digoxigenin Fab fragments conjugated with alkaline phosphatase are used for the revelation of the hybridized probe. The result is obtained by measuring the intensity of light emitted with a spectrophotometer. This new assay was compared to the standard analysis of amplified products by Southern hybridization consisting of gel electrophoresis of the amplimers, transfer onto a nylon membrane, and hybridization with a 32P-labelled oligomeric probe. Both methods exhibited the same sensitivity and specificity. Thus, a non-radioactive hybridization microtiter plate assay may be a suitable alternative to isotopic techniques.
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Biotina , Cartilla de ADN , Variación Genética , Infecciones por Herpesviridae/virología , Herpesvirus Humano 6/aislamiento & purificación , Hibridación de Ácido Nucleico/métodos , Southern Blotting , Células Cultivadas , ADN Viral/análisis , Infecciones por Herpesviridae/patología , Herpesvirus Humano 6/genética , HumanosRESUMEN
The Amplicor HCV Monitor test for quantitative determination of serum or plasma hepatitis C virus (HCV) RNA was modified recently and introduced onto the Cobas Amplicor instrument to automate fully amplification, detection and calculation of results. This new version (v2.0) was evaluated in a routine diagnostic laboratory. The sensitivity and reproducibility were assessed on well-characterized panels (Eurohep) and clinical samples. HCV RNA levels measured by the v2.0 Monitor test were about 1log(10) higher than those detected by the previous version test, and genotypes 1 and 3 were quantified with equal sensitivity. Within the linear dynamic range of 10(3) to 10(6) copies/ml, the coefficients of variation for both intra- and inter-assay reproducibility ranged from 1.9 to 2.95%. This test system was found to be a reliable and labor saving assay for the quantification of HCV RNA.
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Hepacivirus/aislamiento & purificación , Hepatitis C/diagnóstico , Reacción en Cadena de la Polimerasa/métodos , ARN Viral/sangre , Automatización , Estudios de Evaluación como Asunto , Hepacivirus/genética , Hepatitis C/virología , Humanos , Juego de Reactivos para Diagnóstico , Reproducibilidad de los Resultados , Sensibilidad y EspecificidadRESUMEN
The analytical variability of the new commercially available Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR) assay, Amplicor HIV-1 Monitor, has been assessed to establish criteria for assessing the significance of HIV-1 RNA level measurements. Estimations of the standard deviations (SD) of log-copies in inter-assay (mean 0.09 log) and in inter-laboratory (mean 0.14 log) reproducibility experiments demonstrated that the assay can discriminate with 95% confidence between 3-fold (inter-assay) and 5-fold differences (inter-laboratory). The inter-lot reproducibility (mean 0.10 log) was similar to the inter-assay reproducibility. The HIV-1 RNA concentrations measured in plasma collected in potassium EDTA anticoagulant were slightly higher than those measured in plasma collected in sodium citrate. The HIV-1 RNA concentrations measured in sera were about 50% of the HIV-1 RNA concentrations measured in paired plasma samples. However, there was a strong correlation between these two measurements (P < 0.0001). The assay was used to measure viral RNA in the plasma of 50 HIV-1 positive individuals at different stages of infection. All the individuals had detectable HIV-1 RNA (300-957000 copies/ml). There was no correlation between HIV-1 RNA and Immune Complex Dissociated (ICD) p24 antigen, but HIV-1 RNA was correlated with CD4+ cell counts (P < 0.0001) and the clinical stage (P = 0.0042), with higher HIV-1 RNA concentrations in patients with a more advanced stage of the disease. The significant association of HIV-1 RNA with major markers of HIV infection and the reliability of this sensitive, easy-to-use RT-PCR assay indicate its suitability for use in clinical trials and suggest that this assay is appropriate for routine clinical applications.
Asunto(s)
Infecciones por VIH/virología , VIH-1/aislamiento & purificación , Reacción en Cadena de la Polimerasa/métodos , ARN Viral/sangre , Adulto , Recolección de Muestras de Sangre , Recuento de Linfocito CD4 , Femenino , Proteína p24 del Núcleo del VIH/sangre , VIH-1/genética , Humanos , Masculino , Persona de Mediana Edad , Plasma/virología , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , Viremia/virologíaRESUMEN
The aim of this study was to determine whether multicentre quality controls for the detectability of viral genomes could contribute to the improvement of diagnostic performance in the participating laboratories. The study was carried out during two successive rounds, during which 18 laboratories specialized in nucleic acid testing analyzed, through a polymerase chain reaction (PCR) assay, a common panel of GB virus C (GBV-C)/hepatitis G virus (HGV) RNA-positive and -negative samples. During the first round, the laboratories used either an 'in-house' PCR procedure or a partly standardized commercial test. After decoding the results of the first round, the procedures of the participating laboratories were compared in order to establish a consensus procedure deduced from those of the laboratories which provided the best results. During the second round, each participating laboratory could use the resulting consensus procedure, or its own procedure, or both. The results of this quality control study indicated that, whatever method used, even specialized and trained laboratories may give false-negative or false-positive results. The commercial assay did not guarantee a systematic high quality level of results. The striking heterogeneity of results observed among laboratories using the same commercial assay confirm that molecular biology methods need skilled technicians. The results of this quality control study suggest that full standardization of viral genome detection, including all steps of the procedure, is necessary and that the laboratories performing PCR should participate in repeated quality control studies, whatever technique is being used.