RESUMEN
OBJECTIVE: To assess the prevalence of herpesvirus DNA in ocular fluids obtained from healthy patients undergoing vitreoretinal surgery. BACKGROUND: Polymerase chain reaction (PCR) has been used to detect herpesvirus DNA in patients with acute retinal necrosis and cytomegalovirus retinitis. Little is known regarding the prevalence of detectable herpesvirus DNA in ocular fluids collected from healthy seropositive patients with no clinical evidence of viral retinitis. METHODS: Seventy-five intraocular specimens (35 aqueous and 40 vitreous samples) were collected from 75 patients undergoing scleral buckling or vitrectomy. Using a PCR-based assay, the authors tested each specimen for the presence of herpesvirus genome DNA with primers specific for cytomegalovirus, Epstein-Barr virus, herpes simplex virus types 1 and 2, and varicella zoster virus. Serologic testing for immunoglobulin G (IgG) and IgM levels corresponding to each of the herpesviruses was also performed. RESULTS: Of the 75 samples tested, none was found to harbor herpesvirus DNA. The assay did not give false-positive results in patients with active intraocular inflammation. The sensitivity of the assay was 0.08 infection-forming units for cytomegalovirus, 0.6 tissue culture infectious doses for herpes simplex virus, 0.5 infected-cell equivalents for Epstein-Barr virus, and 0.03 focus-forming units for varicella zoster virus. The percentage of patients with positive herpesvirus serology ranged from 86% to 100% and was consistent with rates observed in the general population. CONCLUSIONS: The prevalence of herpesvirus DNA detectable by PCR techniques in ocular fluids appears to be quite low despite the high proportion of patients who tested positive for herpesvirus antibodies. Therefore, a positive result obtained in a patient presenting with vitreoretinal inflammation should be regarded as significant.
Asunto(s)
Humor Acuoso/virología , Retinitis por Citomegalovirus/inmunología , ADN Viral/análisis , Herpesviridae/genética , Huésped Inmunocomprometido/inmunología , Reacción en Cadena de la Polimerasa , Síndrome de Necrosis Retiniana Aguda/inmunología , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Anticuerpos Antivirales/análisis , Humor Acuoso/metabolismo , Niño , Preescolar , Retinitis por Citomegalovirus/virología , Ensayo de Inmunoadsorción Enzimática , Femenino , Herpesviridae/inmunología , Humanos , Inmunoglobulina G/inmunología , Inmunoglobulina G/metabolismo , Inmunoglobulina M/inmunología , Inmunoglobulina M/metabolismo , Lactante , Masculino , Persona de Mediana Edad , Síndrome de Necrosis Retiniana Aguda/virología , Cuerpo Vítreo/metabolismo , Cuerpo Vítreo/virologíaRESUMEN
BACKGROUND: Cytokeratins are predominantly expressed in epithelial cells and their malignant counterparts. Ultrasensitive methods for cytokeratin messenger RNAs (mRNAs) can detect rare circulating tumor cells consistent with hematogenous dissemination in epithelial-derived malignancies, including breast carcinomas. Intraoperative tumor-cell shedding may contribute to this process; this hypothesis is based on the assumption that only tumor cells can be mobilized during surgical manipulation. METHODS AND RESULTS: The present study addresses this issue by using cytokeratin 19 mRNA detection by reverse transcription-polymerase chain reaction (RT-PCR) in preoperative and postoperative blood samples from 54 patients undergoing excisional biopsy for benign breast disease; 22 healthy volunteers represented the control group. No cytokeratin RT-PCR positivity was found in the control or preoperative samples. Cytokeratin RT-PCR positivity was found in 21 postoperative samples (39%). CONCLUSIONS: This finding shows that benign epithelial cells can be mobilized during breast surgery; this effect of surgical manipulation warrants caution in the interpretation of RT-PCR positivity for cytokeratin mRNA in the peripheral blood of patients undergoing surgery for breast cancer.
Asunto(s)
Enfermedades de la Mama/sangre , Queratinas/sangre , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Adulto , Anciano , Biomarcadores , Enfermedades de la Mama/cirugía , Separación Celular/métodos , Células Epiteliales/metabolismo , Femenino , Humanos , Persona de Mediana Edad , ARN Mensajero/sangreRESUMEN
The use of molecular diagnostic testing is increasing in the clinical setting; therefore, data regarding DNA stability in clinical specimens are essential for correct test performance and interpretation. This study was designed to determine DNA stability in peripheral blood and solid tissue under different storage conditions. DNA quality and yield were assayed by spectrophotometric absorbance, gel electrophoresis, and suitability for Southern hybridization and polymerase chain reaction (PCR), the most widely employed clinical DNA analyses. A second goal of the study was to evaluate DNA stability during storage at 4 degrees C for 1 month to 3 years. The data show that freezing or refrigeration of separated leukocytes is preferable for short- to intermediate-term storage and freezing is preferable for solid tissue. DNA degradation varying from slight to severe is seen inconsistently with such specimens, probably due to sampling of unevenly frozen-tissue areas. Depending on the degree of DNA degradation, analysis may still be possible by PCR and in some cases even by Southern hybridization. Once isolated, DNA was stable at 4 degrees C for at least 3 years. These results suggest a more flexible approach to specimen requirements for molecular pathology, as some samples that would routinely be rejected gave interpretable results.
Asunto(s)
Citogenética , ADN/análisis , Técnicas Genéticas , Manejo de Especímenes , Células Sanguíneas/ultraestructura , Southern Blotting , ADN/aislamiento & purificación , Enzimas de Restricción del ADN , Electroforesis , Humanos , Placenta/ultraestructura , Reacción en Cadena de la Polimerasa , Temperatura , Conservación de TejidoRESUMEN
The detection of viral nucleic acids in intraocular fluids and tissues by PCR has become increasingly important in clinical ophthalmology. While much attention has been directed toward minimizing false-positive reactions resulting from specimen contamination or amplicor carryover, relatively little attention has been given to the causes of false-negative PCRs. This report describes a PCR inhibitor in normal aqueous and vitreous fluids that can produce false-negative PCR results. As little as 0.5 microliter of vitreous fluid and 20 microliters of aqueous fluid can completely inhibit DNA amplification in a 100-microliters PCR mixture. This inhibition was not primer specific, nor was it due to chelation of Mg2+ ions or DNase activity in the ocular fluid. The inhibitor was completely resistant to boiling for 15 min. However, the inhibitory effects were completely removed by a single chloroform-isoamyl alcohol (24:1) extraction. The extent of PCR inhibition depended upon the type of thermostable DNA polymerase used in the reaction. Taq DNA polymerase was very sensitive to the inhibitor, while thermostable DNA polymerases from Thermus thermophilus HB-8 (Tth) and Thermus flavus (Tfl) were completely resistant. Thus, the inhibitory effects of intraocular fluids on PCRs can be removed by diluting the specimen, by chloroform extraction, or by using Tth or Tfl DNA polymerases.
Asunto(s)
Alphaherpesvirinae/aislamiento & purificación , Humor Acuoso/virología , Infecciones Virales del Ojo/diagnóstico , Infecciones por Herpesviridae/diagnóstico , Reacción en Cadena de la Polimerasa/métodos , Cuerpo Vítreo/virología , Alphaherpesvirinae/genética , Secuencia de Bases , Desoxirribonucleasas/análisis , Inhibidores Enzimáticos , Estabilidad de Enzimas , Reacciones Falso Negativas , Herpesvirus Humano 3/aislamiento & purificación , Calor , Humanos , Magnesio/farmacología , Datos de Secuencia Molecular , Inhibidores de la Síntesis del Ácido Nucleico , Reacción en Cadena de la Polimerasa/efectos de los fármacos , Simplexvirus/aislamiento & purificaciónRESUMEN
T-cell activation and cytokine production play an important role in several chronic inflammatory diseases. Because n-3 fatty acids exert beneficial effects on the clinical state of some of these diseases, we examined the effect of dietary supplementation of n-3 fatty acids on T-cell proliferation, expression of CD25 (interleukin-2 receptor alpha-chain), secretion of interleukin-2, interleukin-6 and tumour necrosis factor from T-cells from patients with psoriasis and atopic dermatitis. During 4 months, 21 patients supplied 6 g of highly concentrated ethyl esters of EPA and DHA in gelatin capsules daily to their diet. In the control group 20 patients supplied 6 g per day of corn oil in gelatin capsules to their diet. Eicosapentaenoic acid (20:5, n-3) of serum phospholipids increased from 14 (min 4-max 42) to 81 (min 59-max 144) mg l-1 (P < 0.01) in patients with atopic dermatitis receiving n-3 fatty acids, and from 25 (min 7-max 66) to 74 (min 46-max 142) mg l-1 (P < 0.01) in patients with psoriasis, whereas docosahexaenoic acid (22:6, n-3) increased from 65 (min 46-max 120) to 92 (min 54-max 121) mg l-1 (P < 0.05) and from 81 (min 38-max 122) to 92 (min 63-max 169) mg l-1 (NS) in atopic and psoriatic patients, respectively. The changes in the serum phospholipid fatty acid profile in the groups receiving n-3 fatty acids, correlate to the dietary intake of corresponding fatty acids.(ABSTRACT TRUNCATED AT 250 WORDS)