RESUMEN
BACKGROUND: Postoperative pancreatic fistula is the leading cause of morbidity after distal pancreatectomy. Strategies investigated to reduce the incidence have been disappointing. Recent data showed a reduction in postoperative pancreatic fistula with the use of synthetic mesh reinforcement of the staple line. METHODS: An RCT was conducted between May 2014 and February 2016 at four tertiary referral centres in Sweden. Patients scheduled for distal pancreatectomy were eligible. Enrolled patients were randomized during surgery to stapler transection with biological reinforcement or standard stapler transection. Patients were blinded to the allocation. The primary endpoint was the development of any postoperative pancreatic fistula. Secondary endpoints included morbidity, mortality, and duration of hospital stay. RESULTS: Some 107 patients were randomized and 106 included in an intention-to-treat analysis (56 in reinforced stapling group, 50 in standard stapling group). No difference was demonstrated in terms of clinically relevant fistulas (grade B and C): 6 of 56 (11 per cent) with reinforced stapling versus 8 of 50 (16 per cent) with standard stapling (P = 0.332). There was no difference between groups in overall postoperative complications: 45 (80 per cent) and 39 (78 per cent) in reinforced and standard stapling groups respectively (P = 0.765). Duration of hospital stay was comparable: median 8 (range 2-35) and 9 (2-114) days respectively (P = 0.541). CONCLUSION: Biodegradable stapler reinforcement at the transection line of the pancreas did not reduce postoperative pancreatic fistula compared with regular stapler transection in distal pancreatectomy. Registration number: NCT02149446 (http://www.clinicaltrials.gov).
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Implantes Absorbibles , Pancreatectomía/efectos adversos , Fístula Pancreática/prevención & control , Grapado Quirúrgico/instrumentación , Grapado Quirúrgico/métodos , Adulto , Anciano , Anciano de 80 o más Años , Femenino , Humanos , Tiempo de Internación , Masculino , Persona de Mediana Edad , Fístula Pancreática/etiología , Complicaciones Posoperatorias/prevención & control , Estudios Prospectivos , Mallas QuirúrgicasRESUMEN
OBJECTIVES: Infectious gastroenteritis is one of the most common diseases among children and has a considerable impact on health and socio-economy. Day care centres are high-risk environments for infections. The aim of this study was to investigate if asymptomatic preschool children constitute a reservoir for potential enteropathogens. STUDY DESIGN: In total, 438 individual diapers were collected from day care centres in Uppsala, Sweden, during spring and autumn, and molecular techniques were used to estimate the prevalence of asymptomatic carriage of multiple enteropathogens. METHODS: Faecal samples were analysed with multiplex polymerase chain reaction (PCR) (xTAG® Gastrointestinal Pathogen Panel; Luminex Corporation, Toronto, Canada) targeting 21 different pathogens. Samples with a median fluorescence intensity above threshold were re-analysed with a second PCR assay. RESULTS: Sixteen of the 438 samples were positive for enteropathogens, 1.6% for enteric adenovirus, 0.7% for Campylobacter spp., and 0.7% for norovirus. CONCLUSIONS: Preschool children in Uppsala constitute a limited reservoir for potential enteropathogens.
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Portador Sano/epidemiología , Guarderías Infantiles , Heces/microbiología , Heces/virología , Adenoviridae/aislamiento & purificación , Campylobacter/aislamiento & purificación , Preescolar , Reservorios de Enfermedades , Gastroenteritis/epidemiología , Humanos , Lactante , Norovirus/aislamiento & purificación , Reacción en Cadena de la Polimerasa , Prevalencia , Medición de Riesgo , Suecia/epidemiologíaRESUMEN
BACKGROUND: Locoregional pancreatic ductal adenocarcinoma (PDAC) may progress rapidly and/or disseminate despite having an early stage at diagnostic imaging. A prolonged interval from imaging to resection might represent a risk factor for encountering tumour progression at laparotomy. The aim of this study was to determine the therapeutic window for timely surgical intervention. METHODS: This observational cohort study included patients with histologically confirmed PDAC scheduled for resection with curative intent from 2008 to 2014. The impact of imaging-to-resection/reassessment (IR) interval, vascular involvement and tumour size on local tumour progression or presence of metastases at reimaging or laparotomy was evaluated using univariable and multivariable regression. Risk estimates were approximated using hazard ratios (HRs). RESULTS: Median IR interval was 42 days. Of 349 patients scheduled for resection, 82 had unresectable disease (resectability rate 76.5 per cent). The unresectability rate was zero when the IR interval was 22 days or shorter, and was lower for an IR interval of 32 days or less compared with longer waiting times (13 versus 26.2 per cent; HR 0.42, P = 0.021). It was also lower for tumours smaller than 30 mm than for larger tumours (13.9 versus 32.5 per cent; HR 0.34, P < 0.001). Tumours with no or minor vascular involvement showed decreased rates of unresectable disease (20.6 per cent versus 38 per cent when there was major or combined vascular involvement; HR 0.43, P = 0.007). However, this failed to reach statistical significance on multivariable analysis (P = 0.411), in contrast to IR interval (P = 0.028) and tumour size (P < 0.001). CONCLUSION: Operation within 32 days of diagnostic imaging reduced the risk of tumour progression to unresectable disease by half compared with a longer waiting time. The results of this study highlight the importance of efficient clinical PDAC management.
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Carcinoma Ductal Pancreático/diagnóstico , Diagnóstico por Imagen/métodos , Pancreatectomía/métodos , Neoplasias Pancreáticas/diagnóstico , Adulto , Anciano , Anciano de 80 o más Años , Carcinoma Ductal Pancreático/cirugía , Progresión de la Enfermedad , Femenino , Estudios de Seguimiento , Humanos , Laparotomía , Masculino , Persona de Mediana Edad , Neoplasias Pancreáticas/cirugía , Pronóstico , Estudios Retrospectivos , Factores de TiempoRESUMEN
Exposure to Ljungan virus (LV) is implicated in the risk of autoimmune (type 1) diabetes but possible contribution by other parechoviruses is not ruled out. The aim was to compare children diagnosed with type 1 diabetes in 2005-2011 (n = 69) with healthy controls (n = 294), all from the Jämtland County in Sweden, using an exploratory suspension multiplex immunoassay for IgM and IgG against 26 peptides of LV, human parechoviruses (HPeV), Aichi virus and poliovirus in relation to a radiobinding assay (RBA) for antibodies against LV and InfluenzaA/H1N1pdm09. Islet autoantibodies and HLA-DQ genotypes were also determined. 1) All five LV-peptide antibodies correlated to each other (P < 0.001) in the suspension multiplex IgM- and IgG-antibody assay; 2) The LV-VP1_31-60-IgG correlated with insulin autoantibodies alone (P = 0.007) and in combination with HLA-DQ8 overall (P = 0.022) as well as with HLA-DQ 8/8 and 8/X subjects (P = 0.013); 3) RBA detected LV antibodies correlated with young age at diagnosis (P < 0.001) and with insulin autoantibodies (P < 0.001) especially in young HLA-DQ8 subjects (P = 0.004); 4) LV-peptide-VP1_31-60-IgG correlated to RBA LV antibodies (P = 0.009); 5) HPeV3-peptide-IgM and -IgG showed inter-peptide correlations (P < 0.001) but only HPeV3-VP1_1-30-IgG (P < 0.001) and VP1_95-124-IgG (P = 0.009) were related to RBA LV antibodies without relation to insulin autoantibody positivity (P = 0.072 and P = 0.486, respectively). Both exploratory suspension multiplex IgG to LV-peptide VP1_31-60 and RBA detected LV antibodies correlated with insulin autoantibodies and HLA-DQ8 suggesting possible role in type 1 diabetes. It remains to be determined if cross-reactivity or concomitant exposure to LV and HPeV3 contributes to the seroprevalence.
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Diabetes Mellitus Tipo 1/epidemiología , Diabetes Mellitus Tipo 1/etiología , Parechovirus/inmunología , Infecciones por Picornaviridae/complicaciones , Infecciones por Picornaviridae/epidemiología , Adolescente , Alelos , Anticuerpos Antivirales/sangre , Anticuerpos Antivirales/inmunología , Antígenos Virales/inmunología , Autoanticuerpos/sangre , Autoanticuerpos/inmunología , Estudios de Casos y Controles , Niño , Preescolar , Femenino , Genotipo , Antígenos HLA/genética , Antígenos HLA/inmunología , Humanos , Inmunoglobulina G/sangre , Inmunoglobulina G/inmunología , Inmunoglobulina M/sangre , Inmunoglobulina M/inmunología , Lactante , Masculino , Péptidos/química , Péptidos/inmunología , Infecciones por Picornaviridae/inmunología , Estudios Seroepidemiológicos , Suecia/epidemiologíaRESUMEN
BACKGROUND: The association between hospital teaching status and mortality after pancreatic resection is not well explored. Although hospital volume is related to short-term mortality, the effect on long-term survival needs investigation, taking into account hospital teaching status and selective referral patterns. METHODS: This was a nationwide retrospective register-based cohort study of patients undergoing pancreatic resection between 1990 and 2010. Follow-up for survival was carried out until 31 December 2011. The associations between hospital teaching status and annual hospital volume and short-, intermediate- and long-term mortality were determined by use of multivariable Cox regression models, which provided hazard ratios (HRs) with 95 per cent c.i. The analyses were mutually adjusted for hospital teaching status and volume, as well as for patients' sex, age, education, co-morbidity, type of resection, tumour site and histology, time interval, referral and hospital clustering. RESULTS: A total of 3298 patients were identified during the study interval. Hospital teaching status was associated with a decrease in overall mortality during the latest interval (years 2005-2010) (university versus non-university hospitals: HR 0·72, 95 per cent c.i. 0·56 to 0·91; P = 0·007). During all time periods, hospital teaching status was associated with decreased mortality more than 2 years after surgery (university versus non-university hospitals: HR 0·86, 0·75 to 0·98; P = 0·026). Lower annual hospital volume increased the risk of short-term mortality (HR for 3 or fewer compared with 4-6 pancreatic cancer resections annually: 1·60, 1·04 to 2·48; P = 0·034), but not long-term mortality. Sensitivity analyses with adjustment for tumour stage did not change the results. CONCLUSION: Hospital teaching status was strongly related to decreased mortality in both the short and long term. This may relate to processes of care rather than volume per se. Very low-volume hospitals had the highest short-term mortality risk.
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Hospitales de Alto Volumen/estadística & datos numéricos , Hospitales de Bajo Volumen/estadística & datos numéricos , Hospitales de Enseñanza/estadística & datos numéricos , Neoplasias Pancreáticas/mortalidad , Neoplasias Pancreáticas/cirugía , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Métodos Epidemiológicos , Femenino , Humanos , Masculino , Persona de Mediana Edad , Suecia/epidemiología , Adulto Joven , Neoplasias PancreáticasRESUMEN
INTRODUCTION: The use of outcomes to evaluate surgical quality implies the need for detailed risk adjustment. The physiological and operative severity score for the enumeration of mortality and morbidity (POSSUM) is a generally applicable risk adjustment model suitable for pancreatic surgery. A pancreaticoduodenectomy (PD)-specific intraoperative pancreatic risk assessment (IPRA) estimates the risk of postoperative pancreatic fistula (POPF) and associated morbidity based on factors that are not incorporated into POSSUM. OBJECTIVE: The aim of the study was to compare the risk estimations of POSSUM and IPRA in patients undergoing PD. METHODS: An observational single-center cohort study was conducted including 195 patients undergoing PD in 2008-2010. POSSUM and IPRA data were recorded prospectively. Incidence and severity of postoperative morbidity was recorded according to established definitions. The cohort was grouped by POSSUM and IPRA risk groups. The estimated and observed outcomes and morbidity profiles of POSSUM and IPRA were scrutinized. RESULTS: POSSUM-estimated risk (62 %) corresponded with observed total morbidity (65 %). Severe morbidity was 17 % and in-hospital-mortality 3.1 %. Individual and grouped POSSUM risk estimates did not reveal associations with incidence (p = 0.637) or severity (p = 0.321) of total morbidity or POPF. The IPRA model identified patients with high POPF risk (p < 0.001), but was even associated with incidence (p < 0.001) and severity (p < 0.001) of total morbidity. CONCLUSION: The risk factors defined by a PD-specific model were significantly stronger predictive indicators for the incidence and severity of postoperative morbidity than the factors incorporated in POSSUM. If available, reliable procedure-specific risk factors should be utilized in the risk adjustment of surgical outcomes. For pancreatic surgery, generally applicable tools such as POSSUM still have to prove their relevance.
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Fístula Pancreática/epidemiología , Fístula Pancreática/etiología , Pancreaticoduodenectomía/efectos adversos , Calidad de la Atención de Salud , Ajuste de Riesgo/métodos , Índice de Severidad de la Enfermedad , Adulto , Anciano , Anciano de 80 o más Años , Femenino , Mortalidad Hospitalaria , Humanos , Incidencia , Cuidados Intraoperatorios , Masculino , Persona de Mediana Edad , Estudios Prospectivos , Curva ROC , Factores de Riesgo , Síndrome de Respuesta Inflamatoria Sistémica/etiología , Adulto JovenRESUMEN
BACKGROUND: The use of prophylactic abdominal drainage following pancreaticoduodenectomy (PD) is controversial as its therapeutic value is uncertain. However, the diagnosis of postoperative pancreatic fistula (POPF), the main cause of PD-associated morbidity, is often based on drain pancreatic amylase (DPA) levels. The aim of this study was to assess the predictive value of DPA, plasma pancreatic amylase (PPA) and serum C-reactive protein (CRP) for diagnosing POPF after PD. METHODS: Patients undergoing PD with prophylactic drainage between 2008 and 2012 were studied prospectively. DPA, PPA and CRP levels were obtained daily. Differences between groups with clinically relevant POPF (International Study Group on Pancreatic Fistula (ISGPF) grade B/C) and without clinically relevant POPF (no POPF or ISGPF grade A) were evaluated. Receiver operating characteristic (ROC) analyses were performed to determine the value of DPA, PPA and CRP in prediction of POPF. Risk profiles for clinically relevant POPF were constructed and related to the intraoperative pancreatic risk assessment. RESULTS: Fifty-nine (18.7 per cent) of 315 patients developed clinically relevant POPF. DPA, PPA and CRP levels on postoperative day (POD) 1-3 differed significantly between the study groups. In predicting POPF, the DPA level on POD 1 (cut-off at 1322 units/l; odds ratio (OR) 24.61, 95 per cent confidence interval 11.55 to 52.42) and POD 2 (cut-off at 314 units/l; OR 35.45, 14.07 to 89.33) was superior to that of PPA on POD 1 (cut-off at 177 units/l; OR 13.67, 6.46 to 28.94) and POD 2 (cut-off at 98 units/l; OR 16.97, 8.33 to 34.59). When DPA was combined with CRP (cut-off on POD 3 at 202 mg/l; OR 16.98, 8.43 to 34.21), 90.3 per cent of postoperative courses could be predicted correctly (OR 44.14, 16.89 to 115.38). CONCLUSION: The combination of serum CRP and DPA adequately predicted the development of clinically relevant pancreatic fistula following PD.
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Amilasas/metabolismo , Proteína C-Reactiva/metabolismo , Drenaje/métodos , Fístula Pancreática/prevención & control , Pancreaticoduodenectomía/efectos adversos , Adulto , Anciano , Anciano de 80 o más Años , Carcinoma Ductal Pancreático/cirugía , Femenino , Humanos , Masculino , Persona de Mediana Edad , Páncreas/metabolismo , Neoplasias Pancreáticas/cirugía , Pancreatitis/cirugía , Cuidados Posoperatorios/métodos , Estudios Prospectivos , Curva ROC , Medición de Riesgo/métodos , Adulto JovenRESUMEN
In the present study, we evaluated rapid real-time PCR assays for ply, Spn9802, and lytA applied to plasma samples for the detection of Streptococcus pneumoniae in patients with community-acquired pneumonia (CAP). In a prospective study of CAP aetiology, an EDTA plasma sample was collected together with blood culture in 92 adult CAP patients and 91 adult controls. Among the 92 CAP patients, lytA PCR was positive in eight (9%), Spn9802 PCR was positive in 11 (12%) and ply PCR was positive in 19 (21%) cases. Of 91 controls, the ply PCR was positive in eight cases (9%), but no positive cases were noted by Spn9802 or lytA PCRs. Ten CAP patients had pneumococcal bacteraemia. Compared to blood culture, PCR for lytA, Spn9802 and ply had sensitivities of 70% (7/10), 60% (6/10) and 70% (7/10), and specificities of 96% (79/82), 94% (77/82) and 85% (70/82) respectively. With blood culture and/or culture of representative sputum, and/or urinary antigen detection, S. pneumoniae was identified in 31 CAP patients. Compared to these tests in combination, PCR for lytA, Spn9802 and ply showed sensitivities of 26% (8/31), 32% (10/31) and 42% (13/31), and specificities of 100% (61/61), 98% (60/61) and 90% (55/61) respectively. We conclude that Spn9802 and lytA PCRs may be useful for the rapid detection of bacteraemic pneumococcal pneumonia, whereas ply PCR is not specific enough for routine use and blood PCR with small plasma volumes is not useful for the detection of nonbacteraemic pneumococcal pneumonia.
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Proteínas Bacterianas/genética , Sangre/microbiología , Infecciones Comunitarias Adquiridas/microbiología , ADN Bacteriano/genética , Neumonía Neumocócica/diagnóstico , Reacción en Cadena de la Polimerasa/métodos , Streptococcus pneumoniae/genética , Adulto , Anciano , Anciano de 80 o más Años , Técnicas Bacteriológicas/métodos , ADN Bacteriano/aislamiento & purificación , Femenino , Humanos , Masculino , Persona de Mediana Edad , Estudios Prospectivos , Sensibilidad y Especificidad , Streptococcus pneumoniae/aislamiento & purificación , Estreptolisinas/genética , Factores de TiempoRESUMEN
The pneumolysin (ply) gene is widely used as a target in PCR assays for Streptococcus pneumoniae in respiratory secretions. However, false-positive results with conventional ply-based PCR have been reported. The aim here was to study the performance of a quantitative ply-based PCR for the identification of pneumococcal lower respiratory tract infection (LRTI). In a prospective study, fibreoptic bronchoscopy was performed in 156 hospitalized adult patients with LRTI and 31 controls who underwent bronchoscopy because of suspicion of malignancy. Among the LRTI patients and controls, the quantitative ply-based PCR applied to bronchoalveolar lavage (BAL) fluid was positive at >or=10(3) genome copies/mL in 61% and 71% of the subjects, at >or=10(5) genome copies/mL in 40% and 58% of the subjects, and at >or=10(7) genome copies/mL in 15% and 3.2% of the subjects, respectively. Using BAL fluid culture, blood culture, and/or a urinary antigen test, S. pneumoniae was identified in 19 LRTI patients. As compared with these diagnostic methods used in combination, quantitative ply-based PCR showed sensitivities and specificities of 89% and 43% at a cut-off of 10(3) genome copies/mL, of 84% and 66% at a cut-off of 10(5) genome copies/mL, and of 53% and 90% at a cut-off of 10(7) genome copies/mL, respectively. In conclusion, a high cut-off with the quantitative ply-based PCR was required to reach acceptable specificity. However, as a high cut-off resulted in low sensitivity, quantitative ply-based PCR does not appear to be clinically useful. Quantitative PCR methods for S. pneumoniae using alternative gene targets should be evaluated.
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Infecciones Neumocócicas/diagnóstico , Reacción en Cadena de la Polimerasa/métodos , Infecciones del Sistema Respiratorio/microbiología , Streptococcus pneumoniae/genética , Estreptolisinas/genética , Adulto , Anciano , Anciano de 80 o más Años , Proteínas Bacterianas/genética , Líquido del Lavado Bronquioalveolar/microbiología , Broncoscopía/métodos , Humanos , Persona de Mediana Edad , Estudios Prospectivos , Sensibilidad y EspecificidadRESUMEN
Highly conserved nucleotide stretches flanking the cleavage site of the haemagglutinin (HA) gene of influenza type A viruses were utilised for generating PCR amplicons from a broad range of avian influenza viruses (AIV) in a one-step real-time SYBR Green RT-PCR assay. The nucleotide sequencing of the amplified PCR products simultaneously reveals both the HA subtype and the pathotype of the AIV isolates, as we demonstrated in case of H5 subtype viruses. The specificity of the assay was confirmed by investigating 66 strains of AIV and nine heterologous pathogens, including influenza B, C and various avian pathogenic viruses. This assay enables a general HA subtype identification and pathotype determination of AIV isolates providing a useful alternative tool for avian influenza diagnosis.
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Aves/virología , Glicoproteínas Hemaglutininas del Virus de la Influenza/genética , Virus de la Influenza A/genética , Gripe Aviar/diagnóstico , Animales , Virus de la Influenza A/clasificación , Virus de la Influenza A/aislamiento & purificación , Gripe Aviar/virología , ARN Viral/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Sensibilidad y Especificidad , Análisis de Secuencia de ADNRESUMEN
Endogenous retroviruses (ERVs) most likely are remnants of ancient retroviral infections. ERVs preserve functions of exogenous retroviruses to a varying extent, and can be parasites, symbionts or more or less neutral genetic 'junk'.Their evolution has two facets, pre- and post-endogenization. Although the two are not clearly separated, the first pertains to retroviral evolution in general and the second to the fate of repetitive DNA and the evolution of the host organism and its genome. The study of ERVs provides much material for the understanding of retroviral evolution. This sequence archive reflects the history of successes and shortcomings of antiviral resistance, but also of strategic evolutionary decisions regarding genome organization and new gene acquisition. This review discusses retroviral evolution illustrated through HERVs, bioinformatic prerequisites for ERV studies, the endogenization process and HERV evolution post-endogenization, including relation to disease. (Part of a multi-author review).
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Retrovirus Endógenos/genética , Evolución Molecular , Animales , ADN Viral/genética , Bases de Datos Genéticas , Retrovirus Endógenos/clasificación , Transferencia de Gen Horizontal , Genoma , Interacciones Huésped-Patógeno/genética , Interacciones Huésped-Patógeno/fisiología , Humanos , ARN Viral/genética , Vertebrados/virología , Virulencia , Integración Viral , Replicación ViralRESUMEN
Enterovirus (EV) infection has been associated with Type 1 (T1D) diabetes and on a few occasions virus could be isolated at onset of the disease. Using two such isolates as antigens in a quantitative PCR enhanced immunoassay (T1D-EV-QPIA) we have measured IgM antibodies against such potentially diabetogenic viruses in serum from 33 newly diagnosed T1D children, 24 siblings, and 27 healthy children. Sera were also analysed with regard to autoantibodies against GAD65, the cytokine TNF-alpha and the chemokine IP-10. EV-RNA detection was performed on peripheral blood mononuclear cells (PBMC). IgM antibodies against this "new" EV antigen were more frequent in serum from T1D children than in serum from siblings and/or controls (P < 0.001). EV-RNA was detected more frequently in PBMC from T1D children than in healthy control children (P < 0.001) and also compared to the siblings (P < 0.003). The cytokine TNF-alpha was less frequently detected in serum from the T1D children compared with serum from siblings and/controls (P < 0.001). A positive correlation was found between the results obtained with the T1D-EV-QPIA and the EV-PCR (P < 0.001). These findings are in line with earlier findings of an increased frequency of enteroviral infections in newly diagnosed T1D patients. In addition, we found that T1D children at onset of the disease had lower frequencies of the chemokine TNF-alpha in their serum than age- and sex-matched controls had, suggesting an impaired immune response.
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Diabetes Mellitus Tipo 1/complicaciones , Infecciones por Enterovirus/complicaciones , Infecciones por Enterovirus/diagnóstico , Enterovirus/fisiología , Inmunoglobulina M/sangre , Adolescente , Antígenos Virales/inmunología , Niño , Diabetes Mellitus Tipo 1/inmunología , Enterovirus/inmunología , Infecciones por Enterovirus/inmunología , Ensayo de Inmunoadsorción Enzimática , Humanos , Leucocitos Mononucleares/virologíaRESUMEN
Earlier reports of a human exogenous retrovirus (HMTV) related closely to mouse mammary tumor virus (MMTV) led us to search for these viral sequences in breast cancer tissues and normal tissues. A real-time PCR was developed based on MMTV and published HMTV envelope sequences. The real-time PCR method can detect one to ten copies of MMTV target DNA. Tissue samples were collected prospectively from 18 breast cancer patients and 11 non-malignant control cases, as well as peripheral blood leukocytes from the same women. Despite the high sensitivity of the real-time PCR method used, none of the samples were positive for HMTV DNA or RNA. The absence of HMTV DNA in both breast cancer samples and controls indicates either that the concentration of putative HMTV DNA in the breast cancers was too low for detection or that it did not exist there.
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Betaretrovirus/aislamiento & purificación , Neoplasias de la Mama/virología , ADN Viral/análisis , Virus del Tumor Mamario del Ratón/genética , Adulto , Anciano , Anciano de 80 o más Años , Secuencia de Bases , Betaretrovirus/genética , ADN Viral/genética , Femenino , Humanos , Persona de Mediana Edad , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa/métodos , Estudios Prospectivos , Alineación de SecuenciaRESUMEN
Coronaviruses are etiologic agents of respiratory and enteric diseases in humans and in animals. In this study, a one-step real-time reverse transcription-polymerase chain reaction (RT-PCR) assay based on SYBR Green chemistry and degenerate primers was developed for the generic detection of coronaviruses. The primers, designed in the open reading frame 1b, enabled the detection of 32 animal coronaviruses including strains of canine coronavirus, feline coronavirus, transmissible gastroenteritis virus (TGEV), bovine coronavirus (BCoV), murine hepatitis virus (MHV) and infectious bronchitis virus (IBV). A specific amplification was also observed with the human coronaviruses (HCoV) HCoV-NL63, HCoV-OC43, HCoV-229E and severe acute respiratory syndrome coronavirus (SARS-CoV). The real-time RT-PCR detected down to 10 cRNA copies from TGEV, BCoV, SARS-CoV and IBV. In addition, the assay exhibited a high sensitivity and specificity on clinical samples from different animal species. The developed assay represents a potential tool for laboratory diagnostics and for detecting still uncharacterized coronaviruses.
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Coronaviridae/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Animales , Secuencia de Bases , Benzotiazoles , Coronaviridae/clasificación , Coronaviridae/aislamiento & purificación , Cartilla de ADN/genética , ADN Viral/genética , Diaminas , Colorantes Fluorescentes , Humanos , Datos de Secuencia Molecular , Compuestos Orgánicos , Filogenia , Quinolinas , ARN Complementario/genética , ARN Viral/genética , Reproducibilidad de los Resultados , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/estadística & datos numéricos , Sensibilidad y EspecificidadRESUMEN
Comprehensive two-dimensional gas chromatography (GCxGC) has proven to be an extremely powerful separation technique for the analysis of complex volatile mixtures. This separation power can be used to discriminate between highly similar samples. In this article we will describe the use of GCxGC for the discrimination of crude oils from different reservoirs within one oil field. These highly complex chromatograms contain about 6000 individual, quantified components. Unfortunately, small differences in most of these 6000 components characterize the difference between these reservoirs. For this reason, multivariate-analysis (MVA) techniques are required for finding chemical profiles describing the differences between the reservoirs. Unfortunately, such methods cannot discern between 'informative variables', or peaks describing differences between samples, and 'uninformative variables', or peaks not describing relevant differences. For this reason, variable selection techniques are required. A selection based on information between duplicate measurements was used. With this information, 292 peaks were used for building a discrimination model. Validation was performed using the ratio of the sum of distances between groups and the sum of distances within groups. This step resulted in the detection of an outlier, which could be traced to a production problem, which could be explained retrospectively.
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Cromatografía de Gases/métodos , Petróleo/clasificación , Análisis Multivariante , Análisis de Componente PrincipalRESUMEN
Characterization of complex petrochemical samples has been a classical subject of comprehensive two-dimensional (2D) gas chromatography (GC x GC). Macroscopic properties of these samples can be described accurately by separation of compounds in classes of identical molecular functionality. Ring structures in the carbon backbone of these compounds, which can be divided in saturated and unsaturated, are amongst the foremost functionalities affecting samples properties. Unfortunately, GC x GC tuned for separation of both saturated and unsaturated ring structures is likely to result in convoluted chromatograms when a distribution of both molecular properties is present in the sample. An independent liquid chromatographic (LC) separation preceding GC x GC could be used to resolve the mixture based on unsaturated rings, allowing saturated rings to be resolved separately in the GC x GC separation. This three-dimensional separation (abbreviated LC-GC x GC) was performed after rigorous evaluation of LC as part of a multidimensional separation using LC x GC. Group-type separation was achieved using this separation for components with either saturated or unsaturated rings. Results of this separation were used to compare information obtained by GC x GC with LC-GC x GC.
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Cromatografía de Gases/métodos , Cromatografía Liquida/métodos , Hidrocarburos/químicaRESUMEN
Few attempts have been made to study the transfer of DNA from ingested food across the intestinal barrier. A low uptake of ingested DNA has been observed in mice, cattle and poultry. There have been no reports on humans so far. Maintenance of species barriers, protection against retrotransposons, optimisation of oral DNA vaccines and the fate of genetically modified foodstuffs are issues where this topic is of importance. We therefore used the high-copy-number rabbit retrotransposon RERV-H, and rabbit mitochondrial DNA, to study the transfer of DNA from ingested rabbit meat into the bloodstream of two human volunteers. A quantitative PCR was used to measure RERV-H levels in food and in the blood. Amplification with the primers selected results in the generation of a 250-bp fragment of RERV-H. Transfer across the intestinal epithelium could be demonstrated in both subjects. Levels of the fragment in the bloodstream peaked at 1-3 h after ingestion of the experimental meal. One hour after a meal of rabbit meat containing 10(14) copies of RERV-H DNA, a maximum concentration of 200 copies of RERV-H DNA per ml of peripheral blood was observed, which corresponds to the uptake of approximately 10(6) RERV-H DNA copies in 1 h. RERV-H DNA was detected in both cellular and plasma compartments. Both rabbit retrotransposon and mitochondrial DNA was taken up from the human alimentary tract. The size of the fragments detected is similar to that of SINE retrotransposons (approximately 300 bp). The fate and functionality of alimentary DNA in humans will require further study.
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ADN/farmacocinética , Sistema Digestivo/metabolismo , Absorción Intestinal , Reacción en Cadena de la Polimerasa/métodos , Retroelementos , Administración Oral , Adulto , Animales , ADN/sangre , ADN Mitocondrial/sangre , ADN Mitocondrial/farmacocinética , Alimentos , Dosificación de Gen , Humanos , Cinética , Masculino , Persona de Mediana Edad , ConejosRESUMEN
In the past 50 years. gas chromatography (GC) has played a most important role in the analysis of oil. In this review, the early history is briefly reviewed; next developments in this highly relevant application area since about 1985 are highlighted. The main topic of interest are the introduction and decisive role of capillary GC, the use of selective detection techniques, the versatility of coupled-column techniques and, specifically, the additional power of comprehensive two-dimensional GC.
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Cromatografía de Gases/métodos , Aceites/análisis , PetróleoRESUMEN
DNA of a recently described fifth exogenous retrovirus (HRV-5) has been found in blood samples from patients with autoimmune diseases and lymphoma. We analyzed HRV-5 sequence in DNA extracted from whole blood of 17 patients with T cell non-Hodgkin's lymphoma (NHL) and 186 patients with hematological malignancies other than NHL, using a sensitive PCR technique. While all samples of patients with hematological malignancies other than NHL were negative, 2 of the 17 patients with T cell NHL were HRV-5 DNA positive. Both HRV-5-positive patients had T cell NHL of high-grade malignancy (stage IV) and diffuse distribution of the lymphoma, including infiltration of bone marrow or lung and pleura. The difference in HRV-5 DNA detection frequency between NHL and control groups is significant (p value of 0.0004 judged by the Fisher exact test). These data, together with our previous finding of HRV-5 DNA in three B cell NHL cases, are compatible with an association between HRV-5 and NHL, of both T cell and B cell origin.
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Linfoma no Hodgkin/virología , Retroviridae/aislamiento & purificación , Proteínas Virales/genética , Secuencia de Aminoácidos , Secuencia de Bases , Estudios de Cohortes , ADN Viral/química , Humanos , Linfoma no Hodgkin/sangre , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , Retroviridae/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Alineación de Secuencia , Linfocitos T/inmunologíaRESUMEN
The relationship between time of HIV-1 detection, appearance of symptoms and disease progression was studied in all 24 HIV-1-infected infants from a cohort of 117 children who were born to HIV-1-infected mothers and monitored from birth. HIV isolation from plasma and mononuclear cells, HIV-1 DNA PCR (polymerase chain reaction) and, retrospectively, a quantitative assay for HIV-1 RNA were used for virus detection. Two infants possibly exhibited a symptomatic primary HIV infection. More children with than without symptoms during the first year of life progressed to immunological class 3 (P=0.013) and to AIDS or death (P=0.003) during follow-up. HIV-1 was detected within 4 days of age in 4 of 16 infants: 3 of them became symptomatic within 1 year, as did 6 of the remaining 12 infants (not statistically significant). All four infants in whom virus was detected within 4 days of age progressed to severe immunosuppression, compared to 6 of 14 in whom the virus detection test was initially negative prior to the first positive result (n.s.). Two children with previous repeatedly negative HIV detection tests were diagnosed with HIV-1 infection at 8 and 9 months, respectively. Repeated blood sampling is needed for the diagnosis of HIV-1 infection in perinatally exposed infants, and virus detection tests for exclusion of HIV-1 infection must be used with caution.