RESUMO
BACKGROUND: Diagnosis of human papillomavirus (HPV) disease remains a challenge due to several factors related to the cost, the workload of available commercial assays to detect and genotype HPV, and to the low prevalence of infected patients. OBJECTIVE: Our study aimed to develop a real-time PCR, based on SPF10 primers, in order to combine HPV-DNA detection and genotype identification avoiding the negative samples. STUDY DESIGN: Validation of SYBR-green based SPF10 real-time PCR on HPV-DNA plasmids followed by the investigation of the viral status in 92 samples from oropharyngeal (94%) cutaneous biopsies (3%) and anal smears (3%) which had previously been HPV-genotyped by LiPA hybridization. In-house HPV viral loads were performed to evaluate the SPF10 real-time PCR sensitivity. RESULTS: Data showed that 100% of HPV plasmids, assessable by LiPA hybridization, were detected and genotyped appropriately after SPF10 real-time PCR assays. These results defined a range of melting temperature peaks for HPV positivity by real-time PCR. The efficient determination of the presence of HPV-DNA by SPF10 real-time PCR was validated for 98% of clinical samples compared to commercial method. Discordant results were due to a low HPV-DNA amount and to a supplementary HPV genotype identified. The SPF10 real-time PCR sensitivity was evaluated between 1 and 10 copies/10(3)cells using in-house HPV (6, 11 and 16) viral load assays. CONCLUSION: The real-time PCR method was efficient in combining screening and genotyping of HPV-DNA. Cost and workload reduction by SPF10 real-time PCR approach may facilitate earlier diagnosis and clinical management of HPV infected patients.
Assuntos
Técnicas de Diagnóstico Molecular/métodos , Papillomaviridae/classificação , Papillomaviridae/isolamento & purificação , Infecções por Papillomavirus/diagnóstico , Infecções por Papillomavirus/virologia , Reação em Cadeia da Polimerase em Tempo Real/métodos , Virologia/métodos , Canal Anal/virologia , Benzotiazóis , Custos e Análise de Custo , Primers do DNA/genética , Diaminas , Genótipo , Humanos , Técnicas de Diagnóstico Molecular/economia , Sondas de Oligonucleotídeos/genética , Compostos Orgânicos/metabolismo , Orofaringe/virologia , Quinolinas , Reação em Cadeia da Polimerase em Tempo Real/economia , Sensibilidade e Especificidade , Pele/virologia , Coloração e Rotulagem/métodos , Virologia/economiaRESUMO
Defective hepatitis B virus (dHBV) generated from spliced RNA is detected in the sera of HBV-chronic carriers. Our study was designed to determine whether the proportion of dHBV changed during the course of infection, and to investigate whether dHBV might interfere with HBV replication. To achieve this, HBV wild-type and dHBV levels were determined by Q-PCR in sera from 56 untreated chronic patients and 23 acute patients, in sequential samples from 4 treated-patients and from liver-humanized mice after HBV infection. The proportion of dHBV was higher in patients with severe compared to null/moderate liver disease or with acute infection. Follow-up showed that the proportion of dHBV increased during disease progression. By contrast, a low and stable proportion of dHBV was observed in the humanized-mouse model of HBV infection. Our results highlight a regulation of the proportion of dHBV during liver disease progression that is independent of interference with viral replication.