Fifteen years of a nationwide culture collection of Neisseria gonorrhoeae antimicrobial resistance in Portugal.

Neisseria gonorrhoeae antimicrobial resistance (AMR) and gonorrhea disease burden remain major public health concerns worldwide. To contribute to the supranational demands to monitor and manage the spread of antimicrobial-resistant N. gonorrhoeae, the Portuguese NIH promoted the creation of the National Laboratory Network for Neisseria gonorrhoeae Collection (PTGonoNet). The present study reports the N. gonorrhoeae major AMR trends observed from 2003 up to 2018. All isolates described in the present study constitute the opportunistic ongoing N. gonorrhoeae isolate collection supported by the National Reference Laboratory for Sexually Transmitted Infections of the Portuguese NIH, enrolling strains isolated in 35 different public and private laboratories. Minimum inhibitory concentrations were determined using E-tests for azithromycin, benzylpenicillin, cefixime, ceftriaxone, ciprofloxacin, gentamicin, spectinomycin and tetracycline. Molecular typing was determined using NG-MAST. AMR data of 2596 country-spread isolates show that 87.67% of all N. gonorrhoeae isolates presented decreased susceptibility to at least one antimicrobial. A continuous decreased susceptibility and resistance to penicillin, tetracycline and ciprofloxacin can be observed along the years. However, no decreased susceptibility to cephalosporins was observed until 2018, while for azithromycin, this was always low. The most common observed NG-MAST genogroups were G1407, G7445, G225, G2, and G1034. This study evidences the advantages of a nationwide collection of isolates and of centralized AMR testing to respond to supranational (EURO-GASP) requirements while providing unprecedented data on AMR in the context of 15 years of surveillance.

Hepatitis C in a Mobile Low-Threshold Methadone Program.

INTRODUCTION: Data on the epidemiology of hepatitis C among individuals who use drugs in low-threshold settings are lacking, although crucial to assess the burden of disease and aid in the design of treatment strategies. OBJECTIVE: The aim of this study was to characterize the epidemiology and disease related to hepatitis C in a population attending a low-threshold methadone program. MATERIALS AND METHODS: A cross-sectional study in the population attending the Mobile Low-Threshold Methadone Program in Lisbon, Portugal, was carried out. The survey included assessment of risk factors for infection with hepatitis C virus (HCV) and liver disease, HCV serology and RNA detection, HCV genotyping, and liver disease staging. RESULTS: A total of 825 participants were enrolled, 81.3% men, mean age 44.5 years. Injecting drug use (IDU) was reported by 58.4% - among these, 28.2% were people who inject drugs. Excessive drinking and HIV coinfection were reported by 33.4 and 15.9%, respectively. Among participants with active infection, 16.9% were followed up in hospital consultation. The overall seroprevalence for HCV was 67.6% (94.2% in IDU, 30.0% in non-IDU, 97.1% in people who inject drugs, and 75.6% in excessive drinkers). Among seropositives for HCV, active infection was present in 68.4%. Among individuals with active infection, the most common genotypes were 1a (45.3%) and 3a (28.7%), whereas 30% had severe liver fibrosis or cirrhosis. Age 45 years or older, HCV genotype 3, and coinfection with HIV were significant predictors of cirrhosis. CONCLUSION: This population has a high burden of hepatitis C and several characteristics that favor dissemination of infection. Healthcare strategies are urgently needed to address hepatitis C in this setting.

[Assessment of Hepatitis C Virus Diversity in Addition to the Frequency of Genotypes in Samples Analyzed Between 2009 and 2014 at the Reference Laboratory of National Health Institute Dr. Ricardo Jorge]. / Conhecer a Diversidade do Vírus da Hepatite C para Além da Frequência dos Genótipos em Amostras Analisadas entre 2009 e 2014 no Laboratório de Referência do Instituto Nacional de Saúde Dr. Ricardo Jorge.

INTRODUCTION: The identification of genotypes was essential for the prognosis and treatment of hepatitis C virus chronic patients in recent years. The aims of the study were to know the frequency of genotypes diagnosed in the last six years at the laboratory, and reveal the contribution of an in-house assay for molecular characterization of viruses. MATERIAL AND METHODS: The genotyping of hepatitis C virus by LiPA was performed in 923 samples, mostly from male individuals. The subtyping of hepatitis C virus by an in-house assay to target regions in the Core/E1 and/or NS5B was performed in 112 samples. RESULTS: We observed a high prevalence of genotype 1 (56.6%), with a frequency of subtype 1a four times higher compared to 1b. All cases of genotype 3 (27.5%) were subtype 3a. For the cases of genotype 4 (12.9%), it were identified subtypes 4a (65.5%), 4d (31%), 4b (1.7%) and 4c (1.7%). Recombinants intragenotype 2, the RF1_2k/1b, and mixed infections, were also identified. DISCUSSION: The most prevalent subtypes (1a and 3a) obtained are usually described in injecting drug users. Although most of the samples analysed match to inmates (78.4%), we cannot exclude any possible risk behaviors associated with illicit drug use. CONCLUSIONS: The high prevalence of subtype 1a, the frequency and diversity of genotype 4, and the identification of recombined virus suggest modification of the molecular pattern of hepatitis C virus infection described in the past. The in-house assay proved to be useful for the correct classification of hepatitis C virus and improving knowledge about the diversity of virus circulating in the country.

Introdução: A identificação dos genótipos do vírus da hepatite C foi essencial para o prognóstico e tratamento dos doentes crónicos durante os últimos anos. Foram objetivos deste estudo conhecer a frequência de genótipos do vírus da hepatite C nos últimos seis anos, e revelar o contributo de um ensaio in-house para caracterização molecular do vírus. Material e Métodos: A genotipagem do vírus da hepatite C por LiPA foi realizada em 923 amostras, maioritariamente provenientes de indivíduos do sexo masculino. A subtipagem do vírus da hepatite C pelo ensaio in-house com alvo nas regiões Core/E1 e/ou NS5B foi efetuada em 112 amostras. Resultados: Observámos elevada prevalência do genótipo 1 (56,6%), sendo a frequência do subtipo 1a quatro vezes superior ao subtipo 1b. Todos os casos de genótipo 3 (27,5%) foram classificados em subtipo 3a. Nas infeções pelo genótipo 4 (12,9%), identificaram-se os subtipos 4a (65,5%), 4d (31%), 4b (1,7%) e 4c (1,7%). Foram identificadas a RF1_2k/1b, recombinantes intragenótipo 2 e potenciais infeções mistas na população analisada. Discussão: Os subtipos mais prevalentes, 1a e 3a, estão descritos como comuns em utilizadores de drogas injetáveis. Apesar da maioria das amostras analisadas corresponder a reclusos (78,4%), não podemos excluir eventuais comportamentos de risco associados ao consumo de drogas ilícitas. Conclusões: A prevalência elevada do subtipo 1a, a frequência e diversidade do genótipo 4 e a identificação de vírus geneticamente recombinados, sugerem alteração do padrão molecular vírus da hepatite C descrito no passado. O ensaio in-house implementado revelou ser útil para a correta classificação do vírus da hepatite C e melhoria do conhecimento sobre a diversidade do vírus em circulação no país.

Hepatitis C virus subtyping based on sequencing of the C/E1 and NS5B genomic regions in comparison to a commercially available line probe assay.

Hepatitis C virus (HCV) genotype determination is required in clinical practice to establish the dose and duration of antiviral treatment. Although subtype identification does not impact on current therapy this is changing with new specific inhibitors of HCV enzymes and functions which are becoming available worldwide. These new drugs may yield different antiviral responses and resistance profiles. Accurate classification of HCV genotype and subtype is therefore crucial. An "in-house" method was developed for improving HCV subtyping and the results were compared with a second-generation line probe assay (LiPA) used extensively in Portugal. Phylogenetic analysis was undertaken of the C/E1 and NS5B genomic regions of HCV isolated from 72 prisoners with chronic HCV infection and from reference samples. Although LiPA is considered to be a good method for genotyping, HCV was subtyped in only 47.2% of cases compared with 95.8% of cases by the "in-house" method. Molecular data for both C/E1 and NS5B regions were obtained in 88.9% of the samples. Two out of 23 cases of subtype 1a were misclassified as subtype 1b by LiPA. A putative recombinant like RF1_2k/1b, two potential inter-genotypic recombinants 1b/4a and 3a/4a, and also a potential intra-genotypic recombinant 2q/2k in C/E1 and 2k/2a in NS5B were also identified. The "in-house" method enabled HCV to be subtyped accurately with the detection, in some cases, of recombinant viruses or dual HCV infections. Near full-length genomic analysis to characterize these potential recombinant viruses is planned.