RESUMO
Early diagnosis and genotyping of high-risk human papillomavirus (HR-HPV) in cervical tissue specimens is significant for cervical cancer prevention. A sensitive microplate fluorometric hybridization assay (MFHA) was designed for the detection of HPV DNA 16 and 18 in cervical tissue. Following optimization and validation of the method, 60 formalin-fixed and paraffin-embedded cervical samples representing different cervical intraepithelial neoplasia grades of HPV-associated lesions were tested to determine the sensitivity and specificity of the assay. Using consensus GP5+/6+ biotin-labeled primers to amplify a conserved region within the L1 gene, the amplicons were added to the microplate wells coated with specific probes for the hybridization of HPV 16 and 18 individually. Final detection was performed with streptavidin-AlexaFluor488 conjugated. The results were then compared with type-specific nested polymerase chain reaction (PCR) and colorimetric microplate assay. While the agreement between the results obtained by the type-specific nested PCR and fluorometric assay for the detection of both HR-HPV types was 100%, this agreement for the detection of HPV type 16 and 18 using microplate colorimetric assay was 94.2% and 85% respectively. Overall, the results of the fluorometric and colorimetric assays are promising for detecting both HR-HPV types in a large number of cervical tissue samples with the higher MFHA assay sensitivity.
Assuntos
Colo do Útero/virologia , DNA Viral/genética , Fluorometria/métodos , Papillomavirus Humano 16/genética , Papillomavirus Humano 18/genética , Hibridização de Ácido Nucleico/métodos , Adulto , Bancos de Espécimes Biológicos , Primers do DNA , Feminino , Corantes Fluorescentes , Genótipo , Técnicas de Genotipagem , Humanos , Pessoa de Meia-Idade , Inclusão em Parafina , Sensibilidade e Especificidade , Neoplasias do Colo do Útero/virologia , Adulto JovemRESUMO
OBJECTIVES: To investigate the prevalence of human polyomavirus (BK and JC viruses) infection in peripheral blood mononuclear cells of healthy blood donors. METHODS: The study included 250 healthy blood donors. Five-milliliter blood was drawn into sterile EDTA tubes and PBMCs were isolated from whole blood. The isolated PBMCs were counted and stored at -70°C for future investigation. DNA was extracted and subjected to simple, sensitive and specific semi-nested PCR as well as QPCR using both general and specific primers for different assays. RESULTS: Of 250 blood samples, 66 (26.4%) were positive for BKV DNA (146-34,514 copies/106 cells). JC DNA was found in 45 (18%) blood samples (65-21,250 copies/106 cells). Co-infection with these viruses were found in 11 (4.4%) out of 250 blood samples. DISCUSSION: Our study provides important data on polyomavirus infection in peripheral blood mononuclear leukocytes in immunocompetent individuals. These data indicate significant differences between the prevalence of BKV and JCV infection in healthy blood donors. The prevalence of BK and JC virus infection is higher in the age range 30-39 years compared to other age ranges.
Assuntos
Vírus BK/isolamento & purificação , Doadores de Sangue , Vírus JC/isolamento & purificação , Leucócitos Mononucleares/virologia , Infecções por Polyomavirus/virologia , Infecções Tumorais por Vírus/virologia , Adulto , Distribuição por Idade , Vírus BK/genética , DNA Viral/isolamento & purificação , Feminino , Humanos , Irã (Geográfico)/epidemiologia , Vírus JC/genética , Masculino , Pessoa de Meia-Idade , Infecções por Polyomavirus/sangue , Infecções por Polyomavirus/epidemiologia , Prevalência , Reação em Cadeia da Polimerase em Tempo Real , Infecções Tumorais por Vírus/sangue , Infecções Tumorais por Vírus/epidemiologia , Carga Viral , Adulto JovemRESUMO
ABSTRACT Objectives: To investigate the prevalence of human polyomavirus (BK and JC viruses) infection in peripheral blood mononuclear cells of healthy blood donors. Methods: The study included 250 healthy blood donors. Five-milliliter blood was drawn into sterile EDTA tubes and PBMCs were isolated from whole blood. The isolated PBMCs were counted and stored at −70 °C for future investigation. DNA was extracted and subjected to simple, sensitive and specific semi-nested PCR as well as QPCR using both general and specific primers for different assays. Results: Of 250 blood samples, 66 (26.4%) were positive for BKV DNA (146-34,514 copies/106 cells). JC DNA was found in 45 (18%) blood samples (65-21,250 copies/106 cells). Co-infection with these viruses were found in 11 (4.4%) out of 250 blood samples. Discussion: Our study provides important data on polyomavirus infection in peripheral blood mononuclear leukocytes in immunocompetent individuals. These data indicate significant differences between the prevalence of BKV and JCV infection in healthy blood donors. The prevalence of BK and JC virus infection is higher in the age range 30-39 years compared to other age ranges.