RESUMEN
The presence of viral respiratory infections is associated closely with exacerbations in patients with cystic fibrosis. Viral and bacterial multiplex PCRs were developed and applied to nasal swab samples from children with cystic fibrosis. This showed a large number of individuals with cystic fibrosis were infected with rhinoviruses, and more were infected with viral than bacterial pathogens. All individuals with parainfluenza 3 virus had clinical exacerbations of their cystic fibrosis, and although 3/4 of these children were co-infected with HRV. The findings do not suggest a significant association for any other virus or bacteria with exacerbation. There is clear evidence some viral infections are associated with cystic fibrosis that dual infection is more likely to produce symptoms, and mechanisms of viral-induced exacerbation should be elucidated.
Asunto(s)
Infecciones Bacterianas/microbiología , Fibrosis Quística/complicaciones , Técnicas de Diagnóstico Molecular/métodos , Reacción en Cadena de la Polimerasa Multiplex/métodos , Infecciones del Sistema Respiratorio/microbiología , Infecciones del Sistema Respiratorio/virología , Virosis/virología , Adolescente , Bacterias/aislamiento & purificación , Niño , Preescolar , Femenino , Humanos , Masculino , Mucosa Nasal/microbiología , Mucosa Nasal/virología , Virus/aislamiento & purificación , Adulto JovenRESUMEN
Serology assays for standard screening are optimised for use with sera collected from living adults and children. Because of potential changes in the vascular compartments after death, methods used for screening sera from cadaveric organ donors need to be validated before testing these specimens. Serum was separated from blood collected from cadaveric donors within 24 h of death and biochemical parameters measured to detect dilution of protein and haemolysis. In order to demonstrate if any inhibitors that might interfere with the assays were present, pre and post-mortem specimens were spiked with aliquots of human immunodeficiency virus (HIV), hepatitis C virus (HCV), hepatitis B virus (HBV), human T-cell Lymphotropic Virus (HTLV) and T. pallidum-positive sera. Comparison of serum from living subjects with serum obtained post-mortem showed that while the concentration of total protein decreased, concentrations of albumin, immunoglobulin G (IgG) and immunoglobulin M (IgM) remained unchanged. The degree of haemolysis, as measured by free haemoglobin, was within the limits accepted for the Architect analyser. Spiking of pre- and post-mortem specimens with aliquots of HIV, HCV, HBV, HTLV and T. pallidum-positive sera showed no statistical difference in the signal between pre-mortem and post-mortem results when tested on the Abbott Architect analyser. Positive results were obtained in each of a further nine subjects who had tested positive for HIV (n=1), HCV (n=8), HBV (n=1) on pre-mortem serological testing. These findings suggest that the sensitivity of the Abbott Architect serological screening tests is not significantly affected in specimens collected within 24 h of the cessation of life.
Asunto(s)
Recolección de Muestras de Sangre/métodos , Enfermedades Transmisibles/sangre , Enfermedades Transmisibles/diagnóstico , Tamizaje Masivo , Cambios Post Mortem , Adulto , Anciano , Anciano de 80 o más Años , Cadáver , Anticuerpos Antideltaretrovirus/sangre , Demografía , Femenino , Anticuerpos Anti-VIH/sangre , Antígenos VIH/sangre , Anticuerpos contra la Hepatitis B/sangre , Antígenos de la Hepatitis B/sangre , Anticuerpos contra la Hepatitis C/sangre , Humanos , Masculino , Persona de Mediana Edad , Sífilis/sangre , Adulto JovenRESUMEN
The storage of biological samples may affect detection of viral nucleic acid, yet the stability of viral nucleic acid at standard laboratory storage temperatures (-70°C and -20°C) has not been comprehensively assessed. Deterioration of viral RNA and DNA during storage may affect the detection of viruses, thus leading to an increased likelihood of false-negative results on diagnostic testing. The viral loads of 99 hepatitis C virus (HCV), 41 HIV, and 101 hepatitis B virus (HBV) patient samples were measured before and after storage at -20°C and -70°C for up to 9.1 years using Versant branched DNA assays, Cobas Monitor assays, and/or AmpliPrep/AmpliScreen assays. Clinical samples stored at -20°C for up to 1.2 years and at -70°C for up to 9 years showed a statistically significant difference from baseline with respect to HCV RNA titer, although this difference was not greater than 0.5 log(10) unit. The concentration of HIV RNA in clinical samples stored at -20°C for 2.3 years and at -70°C for up to 9.1 years did not differ significantly from the baseline viral load. HBV DNA-positive clinical samples stored at -20°C for up to 5 years and at -70°C for up to 4 years differed significantly in viral load. In all studies, however, the loss of viral load of HCV, HIV, or HBV in clinical samples tested after storage at -20°C and -70°C for up to 9 years ranged from 0.01 to 0.35 log(10) IU/ml and did not exceed 0.5 log(10), which is the estimated intra-assay variation for molecular tests. Hence, the loss was considered of minimal clinical impact and adequate for the detection of HCV, HIV-1, and HBV nucleic acids using nucleic acid assays for the assessment of the infectious risk of cell, blood, and tissue donors.
Asunto(s)
ADN Viral/aislamiento & purificación , VIH-1/genética , Hepacivirus/genética , Virus de la Hepatitis B/genética , Plasma/virología , ARN Viral/aislamiento & purificación , Manejo de Especímenes/métodos , ADN Viral/genética , Congelación , VIH-1/aislamiento & purificación , Hepacivirus/aislamiento & purificación , Virus de la Hepatitis B/aislamiento & purificación , Humanos , ARN Viral/genética , Factores de Tiempo , Carga ViralRESUMEN
Mouse mammary tumor virus (MMTV) has been a long standing candidate as a potential cause of some human breast cancers. Forty years ago, electron microscopic images of MMTV-like particles were identified in milk from 5% of healthy lactating women. These observations, however, have not been confirmed by modern methods. The purpose of this study was to confirm the presence of MMTV-like DNA sequences in human milk from normal lactating women. Standard and in situ PCR analyses were conducted on DNA extracted from fresh breast milk samples collected from a group of 91 healthy lactating women volunteers. The MMTV-like viral positive PCR products were sequenced and a phylogenetic tree was constructed to compare these sequences. Immunohistochemistry analyses were performed on breast milk cells using polyclonal rabbit antibodies against affinity-purified MMTV envelope glycoproteins 52/36. MMTV-like envelope gene sequences were identified by PCR in 5% (4/91) of breast milk samples from healthy lactating women volunteers. These observations were confirmed by in situ PCR and immunohistochemistry using MMTV gp52/36 antibodies. These findings confirm the presence of MMTV-like gene sequences in human milk. As MMTV is transmitted via milk from mouse mothers to their newborn pups to cause mammary tumors when they become adults, this indicates a means of transmission of this virus in humans.
Asunto(s)
Virus del Tumor Mamario del Ratón/aislamiento & purificación , Leche Humana/virología , Animales , Secuencia de Bases , Retrovirus Endógenos/genética , Células Epiteliales/metabolismo , Femenino , Humanos , Lactancia , Virus del Tumor Mamario del Ratón/genética , Ratones , Datos de Secuencia Molecular , Filogenia , Prevalencia , Alineación de Secuencia , Proteínas del Envoltorio Viral/genética , Proteínas del Envoltorio Viral/metabolismoRESUMEN
Mouse mammary tumor virus (MMTV) sequences have been reported to be present in some human breast cancers, but it is unclear whether they have any causal role. In mice, MMTV promotes tumor formation indirectly by insertional mutagenesis of Wnt oncogenes that lead to their activation. In this study, we investigated the status of Wnt-1 in human breast cancers harboring MMTV-like sequences encoding viral envelope (env) genes. We confirmed the detection of env sequences in the nucleus of human breast cancer specimens that are similar in appearance to mouse mammary tumors expressing MMTV env sequences. MMTV env sequences in human breast cancers were also nearly indistinguishable from env sequences in mouse MMTV isolates. Further, Wnt-1 expression was higher in specimens of env-positive ductal carcinoma in situ and invasive ductal carcinoma, relative to env-negative specimens. Our findings extend the evidence that MMTV sequences found in naturally occurring mouse mammary tumors can be found in some human breast cancers, prompting further evaluation of causal roles in these settings.
Asunto(s)
Neoplasias de la Mama/genética , Neoplasias de la Mama/virología , Virus del Tumor Mamario del Ratón/genética , Animales , Antígenos Virales de Tumores/genética , Secuencia de Bases , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Carcinoma Ductal de Mama/genética , Carcinoma Ductal de Mama/metabolismo , Carcinoma Ductal de Mama/patología , Carcinoma Ductal de Mama/virología , Genes env , Humanos , Inmunohistoquímica , Ratones , Datos de Secuencia Molecular , Filogenia , Reacción en Cadena de la Polimerasa , Homología de Secuencia de Ácido Nucleico , Proteína Wnt1/biosíntesis , Proteína Wnt1/genéticaRESUMEN
Mouse mammary tumor virus (MMTV) is a hormonally regulated, oncogenic virus of mice. MMTV-like virus DNA has previously been detected in human breast cancers, liver disease, and liver cancers. It is hypothesized that local hormonal effects might be of primary importance in determining MMTV-like virus detection in human tumors. MMTV-like virus envelope (env) DNA was determined using nested PCR in 89 ovarian, 147 prostate, 50 endometrial, 141 skin, and 51 lung cancers. Viral-positive sequences were compared with published MMTV-like viral sequences from human breast cancer, liver cancer and MMTV. Immunohistochemistry for estrogen receptor (ER-alpha) and progesterone receptor (PgR) was performed on a subset of tumors. MMTV-like virus env DNA was detected in ovarian cancers (14/89; 16%), prostate cancers (53/147; 36%), endometrial cancers (5/50; 10%), skin cancers (13/141; 9%) but not in lung cancers (0/51). Phylogenetic analysis of the viral-positive sequences showed no clustering of the isolates according to tissue type. A significant association was observed between the presence of hormone receptors and detection of MMTV-like virus in the human cancers screened (P = 0.01). A significant association between MMTV-like virus and PgR was noted in skin cancers (P = 0.003). Therefore, unlike the mouse model, the detection of MMTV-like env sequences in human cancers in addition to breast indicates that MMTV-like viral expression is not breast cancer-specific and may relate to hormone-dependent viral expression.
Asunto(s)
Hormonas/farmacología , Neoplasias/virología , Retroviridae/aislamiento & purificación , ADN Viral/química , ADN Viral/genética , Neoplasias Endometriales/patología , Neoplasias Endometriales/virología , Receptor alfa de Estrógeno/análisis , Femenino , Humanos , Inmunohistoquímica , Neoplasias Pulmonares/patología , Neoplasias Pulmonares/virología , Masculino , Virus del Tumor Mamario del Ratón/genética , Datos de Secuencia Molecular , Neoplasias Ováricas/patología , Neoplasias Ováricas/virología , Reacción en Cadena de la Polimerasa , Neoplasias de la Próstata/patología , Neoplasias de la Próstata/virología , Receptores de Progesterona/análisis , Retroviridae/genética , Neoplasias Cutáneas/patología , Neoplasias Cutáneas/virología , Proteínas del Envoltorio Viral/genéticaRESUMEN
BACKGROUND/AIMS: The human equivalent of mouse mammary tumour virus (MMTV-LV) may have a role in the pathogenesis of primary biliary cirrhosis (PBC) and breast carcinogenesis. We investigated MMTV-LV prevalence in patients with liver disease of diverse aetiology and associations with hepatic expression of hormonal receptors, p53 protein and complicating hepatocarcinogenesis. METHODS: Nested PCR for MMTV-LV envelope was performed using tissue from 210 patients with liver disease and 20 patients with histologically normal liver. Hepatic expression of estrogen receptor-alpha (ER-alpha), progesterone receptor (PgR) and p53 protein was determined by immunohistochemistry. MMTV-LV expression was also determined in hepatocellular carcinoma (HCC) tissue from 21 patients, in 11 of whom paired non-tumourous liver tissue was available for comparison. RESULTS: MMTV-LV envelope sequences were present in 25% (53/210) of liver disease biopsies but not in histologically normal liver (0/20) (P=0.02). There was no association between MMTV-LV presence and hepatic expression of ER-alpha or p53 protein. No liver sections were PgR positive. MMTV-LV prevalence was not significantly different in HCC tissue, paired non-tumourous chronic liver disease tissue from the same patients and liver disease tissue from patients without complicating HCC. CONCLUSIONS: Hepatic expression of MMTV-LV is evident in a wide range of non-cirrhotic and cirrhotic liver diseases, irrespective of ER-alpha, PgR or p53 status, and unrelated to complicating HCC.