RESUMO
BACKGROUND: Hepatitis C virus (HCV) genotyping is crucial in clinical practise for determining the type and duration of antiviral therapy. Between 2009 and 2014, 24 (7.95%) of all HCV genotype 3 (HCV-3) cases obtained indeterminate results via the RealTime HCV Genotype II assay (Abbott) at a tertiary care center in Spain. HCV-3 is the second most common genotype worldwide. Moreover, it has been associated with a higher risk of liver disease progression and a lower response to the latest antivirals. OBJECTIVE: Given the clinical significance of accurately identifying HCV-3, we aimed to characterize the genetic diversity of the HCV 5' untranslated region (5' UTR), the target of genotyping assays, by ultradeep pyrosequencing (UDPS). STUDY DESIGN: For the 24 indeterminate samples, the 5' UTR-core was amplified and subjected to UDPS with the 454/GS-Junior platform (Roche). The genotype/subtype of each identified haplotype was assigned by phylogenetic analysis. For comparison, three additional samples correctly identified as HCV-3 by the real-time assay were also analyzed. RESULTS: HCV genotyping based on 5' UTR-core UDPS was in agreement with NS5B Sanger sequencing in all cases, confirming the absence of mixed infections and recombination events. The generated 5' UTR sequences proved the presence of one to three polymorphisms at the probe-binding site of the Abbott assay, thereby differentiating indeterminate from correctly genotyped HCV-3 samples. CONCLUSIONS: The observed naturally occurring polymorphisms provide insight into regional differences observed with genotype 3, their impact on genotyping assay performance, and potential improvement and designing options.
Assuntos
Variação Genética , Genótipo , Hepacivirus/genética , Hepacivirus/isolamento & purificação , Hepatite C/diagnóstico , Hepatite C/virologia , Regiões 5' não Traduzidas , Adulto , Idoso , Análise por Conglomerados , Feminino , Hepacivirus/classificação , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Masculino , Pessoa de Meia-Idade , Filogenia , Espanha , Adulto JovemRESUMO
INTRODUCTION: Sexually transmitted infections (STI) are currently on the increase worldwide. New molecular tools have been developed in the past few years in order to improve their diagnosis. An evaluation was carried out using a new commercially available real-time PCR assay, Anyplex™ II STI-7 (Seegene, Seoul, Korea), which detects seven major pathogens in a single reaction - Chlamydia trachomatis, Neisseria gonorrhoeae, Trichomonas vaginalis, Mycoplasma hominis, Mycoplasma genitalium, Ureaplasma urealyticum, and Ureaplasma parvum - and compared with conventional methods performed in our laboratory. MATERIALS AND METHODS: Two different populations were included, and 267 specimens from different sites of infection (urines, endocervical swabs, rectal swabs, vaginal swabs, urethral swabs and one inguinal adenopathy) were processed for both methods. RESULTS: The parameters of clinical performance were calculated for C. trachomatis, N. gonorrhoeae, and T. vaginalis, and the assay achieved sensitivities (SE) from 93.94% to 100%, and specificities (SP) from 96.55% to 100%, with negative predictive values (NPV) from 93.33% to 98.85%, and positive predictive values (PPV) from 96.88% to 100%, with a very good agreement (kappa index from 0.88 to 1). CONCLUSIONS: Anyplex™ II STI-7 is a good tool for the reliable diagnosis of STI. Its ease of use and processing allows it to be incorporated into the day to day laboratory work.
Assuntos
Reação em Cadeia da Polimerase Multiplex/métodos , Infecções Sexualmente Transmissíveis/diagnóstico , Chlamydia trachomatis/isolamento & purificação , Estudos Transversais , Feminino , Humanos , Mycoplasma genitalium/isolamento & purificação , Mycoplasma hominis/isolamento & purificação , Neisseria gonorrhoeae/isolamento & purificação , Estudos Retrospectivos , Sensibilidade e Especificidade , Infecções Sexualmente Transmissíveis/microbiologia , Trichomonas vaginalis/isolamento & purificação , Ureaplasma/isolamento & purificação , Ureaplasma urealyticum/isolamento & purificaçãoRESUMO
BACKGROUND: Only about 50% of patients chronically infected with HCV genotype 1 (HCV-1) respond to treatment with pegylated interferon-alfa and ribavirin (dual therapy), and protease inhibitors have to be administered together with these drugs increasing costs and side-effects. We aimed to develop a predictive model of treatment response based on a combination of baseline clinical and viral parameters. METHODOLOGY: Seventy-four patients chronically infected with HCV-1b and treated with dual therapy were studied (53 retrospectively -training group-, and 21 prospectively -validation group-). Host and viral-related factors (viral load, and genetic variability in the E1-E2, core and Interferon Sensitivity Determining Region) were assessed. Multivariate discriminant analysis and decision tree analysis were used to develop predictive models on the training group, which were then validated in the validation group. PRINCIPAL FINDINGS: A multivariate discriminant predictive model was generated including the following variables in decreasing order of significance: the number of viral variants in the E1-E2 region, an amino acid substitution pattern in the viral core region, the IL28B polymorphism, serum GGT and ALT levels, and viral load. Using this model treatment outcome was accurately predicted in the training group (AUROC = 0.9444; 96.3% specificity, 94.7% PPV, 75% sensitivity, 81% NPV), and the accuracy remained high in the validation group (AUROC = 0.8148, 88.9% specificity, 90.0% PPV, 75.0% sensitivity, 72.7% NPV). A second model was obtained by a decision tree analysis and showed a similarly high accuracy in the training group but a worse reproducibility in the validation group (AUROC = 0.9072 vs. 0.7361, respectively). CONCLUSIONS AND SIGNIFICANCE: The baseline predictive models obtained including both host and viral variables had a high positive predictive value in our population of Spanish HCV-1b treatment naïve patients. Accurately identifying those patients that would respond to the dual therapy could help reducing implementation costs and additional side effects of new treatment regimens.
Assuntos
Genótipo , Hepacivirus/genética , Hepatite C Crônica , Interleucinas/genética , Modelos Biológicos , Polimorfismo Genético , Adulto , Feminino , Hepatite C Crônica/sangue , Hepatite C Crônica/genética , Hepatite C Crônica/terapia , Humanos , Interferons , Interleucinas/sangue , Masculino , Pessoa de Meia-Idade , Estudos Prospectivos , Estudos Retrospectivos , Carga ViralRESUMO
BACKGROUND: Hepatitis C virus (HCV) genotyping is mandatory for tailoring dose and duration of pegylated interferon-α plus ribavirin treatment and for deciding on triple therapy eligibility. Additionally, subtyping may play a role in helping to select future treatment regimens that include directly-acting antivirals. However, commercial assays for HCV genotyping fail to identify the genotype/subtype in some cases. OBJECTIVE: Our aims were (i) to determine the success rate of the commercial genotyping assay Abbott RealTime HCV Genotype II at identifying the genotype and the HCV-1 subtype; and (ii) to phylogenetically characterise the obtained indeterminate results. STUDY DESIGN: HCV genotyping results obtained between 2009 and 2012 in a Spanish reference hospital were reviewed. A total of 896 people were genotyped with the Abbott RealTime HCV Genotype II assay. Specimens with an indeterminate result were retrospectively genotyped using the reference method based on the phylogenetic analysis of HCV NS5B sequences. RESULTS: Using the commercially available assay, an indeterminate HCV genotype result was obtained in 20 of 896 patients (2.2%); these corresponded to genotypes 3a, 3k and 4d. Importantly, 8.6% of all cases where genotype 3 was detected were indeterminate. In addition, the HCV-1 subtype was not assigned in 29 of 533 cases (5.4%). CONCLUSIONS: The implementation in the clinical microbiology laboratory of the reference method for HCV genotyping allows indeterminate genotype/subtype results to be interpreted and may lead to the identification of previously uncharacterised subtypes.
Assuntos
Hepacivirus/classificação , Hepacivirus/genética , Hepatite C/diagnóstico , Hepatite C/virologia , Técnicas de Diagnóstico Molecular/métodos , Kit de Reagentes para Diagnóstico , Adulto , Feminino , Genótipo , Hepacivirus/isolamento & purificação , Hospitais , Humanos , Masculino , Pessoa de Meia-Idade , Dados de Sequência Molecular , Análise de Sequência de DNA , Espanha , Proteínas não Estruturais Virais/genéticaRESUMO
OBJECTIVE: To determine the prevalence of Chlamydia trachomatis (CT) and high risk factors for acquisition in preventive prisoners in Catalonia. METHODS: Cross-sectional study of a convenience sample of 478 prisoners aged between 18 and 35 years was analysed using real-time polymerase chain reaction. A standardized questionnaire was used to collect behavioural data. Significant differences were analysed in the descriptive study using Pearson's χ(2). The association between CT and its determinants was analysed using the Mantel-Haenszel test and a multivariate logistic regression model. RESULTS: The overall prevalence of CT was 5.4%. The independent risk factors for infection by CT were as follows: foreign origin, having had concurrent sexual partners, and alcohol consumption. CONCLUSIONS: This is the first study performed in prisons of Catalonia that shows the prevalence of CT in young prisoners. The high mobility of young detainees could explain the similarity in prevalence obtained about young people in Catalonia. Systematic monitoring of CT infection in young preventive prisoners is important in order to prevent further problems in themselves and in the general population, since they become a 'bridge population' in sexually transmissible infection spreading.
Assuntos
Infecções por Chlamydia/diagnóstico , Infecções por Chlamydia/epidemiologia , Chlamydia trachomatis/isolamento & purificação , Prisioneiros/estatística & dados numéricos , Comportamento Sexual/estatística & dados numéricos , Parceiros Sexuais , Adulto , Distribuição por Idade , Técnicas Bacteriológicas/métodos , Estudos Transversais , Feminino , Humanos , Masculino , Reação em Cadeia da Polimerase/métodos , Prevalência , Prisões , Fatores de Risco , Assunção de Riscos , Espanha , Inquéritos e Questionários , Adulto JovemRESUMO
Cervical cancer is the second-most prevalent cancer in young women around the world. Infection with human papillomavirus (HPV), especially high-risk HPV types (HR-HPV), is necessary for the development of this cancer. HPV-DNA detection is increasingly being used in cervical cancer screening programs, together with the Papanicolau smear test. We evaluated the usefulness of introducing this new array-based HPV genotyping method (i.e., Clinical Arrays Papillomavirus Humano) in the cervical cancer screening algorithm in our center. The results obtained using this method were compared to those obtained by the hybrid capture II high-risk HPV DNA test (HC-II) and Papanicolau in a selected group of 408 women. The array-based assay was performed in women that were HC-II positive or presented cytological alterations. Among 246 array-positive patients, 123 (50%) presented infection with >or=2 types, and HR-HPV types were detected in 206 (83.7%), mainly HPV-16 (24.0%). Up to 132 (33.2%) specimens were classified as ASCUS (for atypical squamous cells of undetermined significance), and only 48 (36.4%) of them were HPV-DNA positive by either assay; however, 78.7% of these cases were caused by HR-HPV types. The agreement between both HPV-DNA detection techniques was fairly good (n = 367). Screening with Papanicolau smear and HC-II tests, followed by HPV detection and genotyping, provided an optimal identification of women at risk for the development of cervical cancer. Furthermore, with the identification of specific genotypes, either in single or multiple infections, a better prediction of disease progression was achieved. The array method also made allowed us to determine the possible contribution of the available vaccines in our setting.