Vitor Duque

Characterization of JC human polyomavirus infection in a Portuguese population

JC virus (JCV) is ubiquitous in the human population, infecting children asymptomatically. After primary infection, JCV persists in the host throughout life and is often excreted in the urine. Two hundred thirty‐four urine samples and 78 serum samples, collected from 171 healthy individuals and 63 patients infected with HIV, were used to characterize JCV infection in a Portuguese population. Using PCR, JCV DNA was detected in 38% of the urine samples. A significant difference in the excretion rate was observed between patients infected with HIV (51%) and healthy individuals (33%). The frequency of JCV viruria increased with age in healthy individuals, but not in patients infected with HIV. JCV urinary load was determined by real‐time quantitative PCR and was independent of gender, age, HIV infection, and CD4+ cell count. Overall, the JCV genotype detected most commonly was 1B, followed by genotypes 2B and 4. The detection and quantitation of JCV‐specific antibodies were performed in serum samples by an established enzyme immunoassay (EIA). Antibodies to JCV were observed in 91% of the patients tested, irrespective of HIV infection. A positive correlation between JCV urinary load and antibody titers was demonstrated. The present study provides the first characterization of seroprevalence and urinary excretion of JCV in a Portuguese population and revealed similar results to those observed in other European countries. A comparison between healthy individuals and patients infected with HIV, despite identical values of seroprevalence, showed some differences in the pattern of urinary excretion. J. Med. Virol. 82:494–504, 2010.

An International Collaboration To Standardize HIV-2 Viral Load Assays: Results from the 2009 ACHIEV2E Quality Control Study

ABSTRACT Accurate HIV-2 plasma viral load quantification is crucial for adequate HIV-2 patient management and for the proper conduct of clinical trials and international cohort collaborations. This study compared the homogeneity of HIV-2 RNA quantification when using HIV-2 assays from ACHIEV2E study sites and either in-house PCR calibration standards or common viral load standards supplied to all collaborators. Each of the 12 participating laboratories quantified blinded HIV-2 samples, using its own HIV-2 viral load assay and standard as well as centrally validated and distributed common HIV-2 group A and B standards (http://www.hiv.lanl.gov/content/sequence/HelpDocs/subtypes-more.html). Aliquots of HIV-2 group A and B strains, each at 2 theoretical concentrations (2.7 and 3.7 log10 copies/ml), were tested. Intralaboratory, interlaboratory, and overall variances of quantification results obtained with both standards were compared using F tests. For HIV-2 group A quantifications, overall and interlaboratory and/or intralaboratory variances were significantly lower when using the common standard than when using in-house standards at the concentration levels of 2.7 log10 copies/ml and 3.7 log10 copies/ml, respectively. For HIV-2 group B, a high heterogeneity was observed and the variances did not differ according to the type of standard used. In this international collaboration, the use of a common standard improved the homogeneity of HIV-2 group A RNA quantification only. The diversity of HIV-2 group B, particularly in PCR primer-binding regions, may explain the heterogeneity in quantification of this strain. Development of a validated HIV-2 viral load assay that accurately quantifies distinct circulating strains is needed.

Detection of the protease codon 35 amino acid insertion in sequences from treatment-naïve HIV-1 subtype C infected individuals in the Central Region of Portugal

BACKGROUND Amino acids insertions in the protease (PR) coding region have been reported in protease inhibitors (PIs) treatment-naïve and experienced HIV-1 infected individuals ranging from 0.1% to 4.55% and have been rarely found in non-B HIV-1 subtype strains. OBJECTIVES To investigate the presence of amino acid insertions in the PR coding region in sequences from treatment-naïve HIV-1 infected individuals in the Central Region of Portugal. STUDY DESIGN Sequences of the pol gene from 260 treatment-naïve HIV-1 infected individuals between 2000 and 2008 were analyzed and phylogenetic analysis was performed. RESULTS A threonine insertion (E35E_T) was detected in 2.69% (n=7) of the sequences analyzed and all the sequences that possessed this insertion were identified as subtype C. All the seven inserted sequences clustered in the same lineage of the phylogenetic tree. Heterosexual and intravenous drug use were found to be the routes of infection. No major mutations in the PR coding region associated with resistance to PIs were detected. CONCLUSIONS It was found the highest prevalence of PR codon 35 insertion among treatment-naïve HIV-1 infected individuals ever reported in the western countries. Epidemiological data and Phylogenetic analysis indicated the possibility of transmission of this insertion. The results suggested that these inserted strains have normal susceptibility to PIs containing regimens. This study demonstrated the spreading epidemic of PR codon 35 inserted strains from subtype C in the Central Region of Portugal, during the past eight years.

Individuals infected with JC polyomavirus do not present detectable JC virus DNA in oropharyngeal fluids.

JC virus (JCV) is ubiquitous in the human population. Primary infection normally occurs during childhood and is followed by a lifelong persistent infection. The main mode of transmission remains unknown. Several authors have hypothesized that JCV transmission occurs through the respiratory route, and that respiratory secretions could represent a possible source of viral particles. The present study intended to evaluate oropharyngeal fluids from patients infected with JCV, in order to ascertain if respiratory secretions could indeed constitute a source of exposure to this polyomavirus. Oropharyngeal washing samples from 25 patients co-infected with JCV and human immunodeficiency virus type 1 were evaluated for the presence of JCV DNA. Regardless of the titre of antibodies or the presence of viral urinary excretion, JCV genome was not detected in oropharyngeal samples collected from any of the patients infected with JCV included in this study, which may suggest that oropharyngeal fluids are an unlikely source for JCV infection.

The early days of pandemic (H1N1) 2009 virus infection in the central region of Portugal

BACKGROUND The first case of pandemic (H1N1) 2009 virus infection was diagnosed in the central region of Portugal on June 16, 2009, in a woman infected in Canada. METHODS The aim of our study was, first to characterize the clinical and epidemiologic aspects of all the patients with clinical manifestations included in the definition of case for investigation with samples submitted to diagnosis of the pandemic (H1N1) 2009 virus infection, in the central region of Portugal; second to assess the precision of the case definition of case for investigation considered in the study according to the presence or the absence of fever at the moment of clinical observation. We reviewed the medical records of all the patients presenting with Influenza like-illness classified as case for investigation and the first cases of patients infected with the new pandemic (H1N1) 2009 virus, diagnosed in the central region of Portugal during the pandemic period between June and August, 2009, were analyzed. Real-time reverse-transcriptase polymerase-chain-reaction (RT-PCR) testing was used to confirm the pandemic (H1N1) 2009 virus infection. Data collection was performed on a standardized paper format in agreement with the General Health Directorate. RESULTS AND DISCUSSION Pandemic (H1N1) 2009 virus infection was confirmed in 255 patients. Overall, median age was 23 years and 42.7 % were included in the category of 20 to 29 years. Confirmed infection in patients with less than 2 years or greater than 50 years was a rare event. The first cases were imported from Europe, namely France, Spain and England. On a second phase, pandemic (H1N1) 2009 virus infection was acquired in the south of Portugal (Algarve), before de diagnosis of the first domestic case. The incidence rate for pandemic (H1N1) 2009 virus infection was 10.7 per 100,000 persons and was different according to the district. It was higher in the district of Coimbra and Guarda were the main roads connecting to Europe are. The median calculated incubation period for the for pandemic (H1N1) 2009 virus infection was 2 days. The length of the clinical manifestations until the patients look for medical observation had a median time of 2 days. All the cases were of mild to moderate severity. No deaths were observed. CONCLUSIONS The early days of pandemic (H1N1) 2009 virus infection was mild in our region. Most affected patients were young adults, with the extreme categories ages of life being spared. Early detection and diagnosis, combined with stringent isolation and treatment procedures could have slowed the spread of the infection in our region.

Prevalence of Etravirine Resistance Associated Mutations in HIV-1 Strains Isolated From Infected Individuals Failing Efavirenz: Comparison Between Subtype B and Non-B Genetic Variants

Etravirine (ETR) is a non‐nucleoside analogue reverse transcriptase inhibitor (NNRTI) with a high genetic barrier to the development of resistance and with potential activity against Human immunodeficiency virus type 1 (HIV‐1) strains resistant to first‐generation NNRTIs. The objective of this study was to investigate the prevalence of ETR resistance associated mutations (RAMs) in HIV‐1 strains isolated from infected individuals failing efavirenz (EFV), as well as to evaluate possible differences in the distribution of ETR RAMs between subtype B and non‐B genetic variants. Nucleotide sequences of the protease and partial reverse transcriptase (RT) coding regions of the pol gene of 55 HIV‐1 strains isolated from infected individuals failing EFV on regular follow‐up at a reference center in Portugal, were retrospectively analyzed. The most prevalent ETR RAMs observed were L100I, V90I, and K101E, with a prevalence of 16.4% (n = 9), 9.1% (n = 5), and 5.5% (n = 3), respectively. Overall, 47.3% (n = 26) of the nucleotide sequences had at least one ETR RAM: 38.2% (n = 21) had one ETR RAM, 7.3% (n = 4) had two ETR RAMs and 1.8% (n = 1) had three ETR RAMs. No statistically significant differences were found in the distribution of ETR RAMs between subtype B and non‐B genetic variants. The results demonstrate that ETR rescue therapy is a viable option in treatment‐experienced individuals failing EFV and suggests that ETR may be equally useful in HIV‐1 infections caused by different genetic variants. J. Med. Virol. 84:551–554, 2012.

Os dias iniciais da infecção pelo vírus da gripe pandémica (H1N1) 2009 na região centro de Portugal

Resumo Introducao O primeiro caso de infeccao pelo virus da gripe pandemica (H1N1) 2009 foi diagnosticado na regiao centro de Portugal no dia 16 de Junho de 2009, numa mulher infectada no Canada. Metodos O nosso estudo tem por objectivos, em primeiro lugar caracterizar os aspectos clinicos e epidemiologicos de todos os doentes que tiveram manifestacoes clinicas incluidas na definicao de caso para investigacao com amostras submetidas para diagnostico da infeccao pelo virus da gripe pandemica (H1N1) 2009; em segundo lugar, avaliar a precisao da definicao de caso para investigacao de acordo com a presenca ou ausencia de febre no momento da observacao clinica. Efectuamos a revisao dos registos medicos de todos os doentes classificados como caso para investigacao e analisaram-se os primeiros casos de doentes infectados com o novo virus da gripe pandemica (H1N1) 2009, diagnosticados na regiao centro de Portugal durante o periodo pandemico compreendido entre Junho e Agosto de 2009. Foi utilizado o metodo da reaccao em cadeia da polimerase de retrotranscripcao em tempo real para confirmacao da infeccao pelo virus da gripe pandemica (H1N1) 2009. A colheita de dados foi efectuada de forma padronizada em suporte de papel de acordo com as normas da Direccao Geral de Saude. Resultados e discussao A infeccao pelo virus da gripe pandemica (H1N1) 2009 foi confirmada laboratorialmente em 255 casos. A idade media foi de 23 anos e 42,7 % foram incluidos na categoria dos 20 aos 29 anos. A infeccao em doentes com menos de 2 anos ou mais de 50 anos foi um acontecimiento raro. Os primeiros casos foram importados da Europa: Franca, Espanha e Inglaterra. Numa segunda fase, a infeccao foi adquirida no sul de Portugal (Algarve). A taxa de incidencia de infeccao pelo virus da gripe pandemica (H1N1) 2009 foi de 10,7 por 100 000 pessoas e foi diferente consoante o distrito. Foi mais elevado no distrito de Coimbra e da Guarda onde estao as principais estradas de ligacao com a Europa. O periodo de incubacao calculado para a infeccao pelo virus da gripe pandemica (H1N1) 2009 foi de 2 dias. A duracao das manifestacoes clinicas ate os doentes procurarem observacao medica teve um valor mediano de 2 dias. Todos os casos foram de gravidade ligeira a moderada, sem casos de morte. Conclusoes Os dias iniciais da infeccao pelo virus da gripe pandemica (H1N1) 2009 foram caracterizados na nossa regiao por casos de doenca com gravidade ligeira a moderada. Os mais afectados foram os jovens adultos, com as idades extremas da vida poupadas. O diagnostico precoce, o isolamento estrito e o tratamento podem ter diminuido a disseminacao da infeccao.

Topoisomerase I Improve JC virus DNA detection

Introduction: Progressive Multifocal Leukoencephalopathy (PML) is a severe and often fatal CNS disease caused by JC human polyomavirus infection. Diagnosis of PML is based upon suggestive clinical symptoms and brain images, supported by the presence of JC virus genome in CSF samples. Objective: The main objective of our study was the search for an alternative method for JC virus DNA detection in CSF samples, with sensitivity and specificity characteristics close to that of standard techniques, but feasible at any clinical laboratory. Methods: In order to evaluate the effect of topoisomerase I treatment in the detection of JC virus genome, and its feasibility in laboratory diagnosis of PML, 129 CSF samples were examined for the presence of JC virus DNA by a nested-PCR protocol, with and without previous treatment with topoisomerase I. All CSF samples were also evaluated through a real-time PCR protocol. Results: Eleven CSF samples presented detectable JC virus DNA with all used protocols. On 9 CSF samples, JC virus DNA was only detectable with topoisomerase I modified nested-PCR and real-time PCR protocols. Real-time PCR was the only protocol able to detect JC virus genome in 4 CSF samples. One CSF sample revealed the presence of the expected amplified fragment only when tested with topoisomerase I modified nested-PCR protocol. Conclusion: The results of the present study point towards the benefit of using topoisomerase I DNA treatment before amplification reactions in JC virus DNA detection on CSF samples, and confirm that topoisomerase I modified nested-PCR protocol represents a good alternative method to detect JC virus DNA in CSF samples from patients with clinical signs and brain images suggestive of PML.

Treatment of HIV-2 infection: a retrospective study from a Portuguese center

7‐11 November 2010, Tenth International Congress on Drug Therapy in HIV Infection, Glasgow, UK

CD4 cell count response to first-line combination ART in HIV-2+patients compared with HIV-1+patients: a multinational, multicohort European study

Background CD4 cell recovery following first-line combination ART (cART) is poorer in HIV-2+ than in HIV-1+ patients. Only large comparisons may allow adjustments for demographic and pretreatment plasma viral load (pVL). Methods ART-naive HIV+ adults from two European multicohort collaborations, COHERE (HIV-1 alone) and ACHIeV2e (HIV-2 alone), were included, if they started first-line cART (without NNRTIs or fusion inhibitors) between 1997 and 2011. Patients without at least one CD4 cell count before start of cART, without a pretreatment pVL and with missing a priori-defined covariables were excluded. Evolution of CD4 cell count was studied using adjusted linear mixed models. Results We included 185 HIV-2+ and 30321 HIV-1+ patients with median age of 46 years (IQR 36-52) and 37 years (IQR 31-44), respectively. Median observed pretreatment CD4 cell counts/mm3 were 203 (95% CI 100-290) in HIV-2+ patients and 223 (95% CI 100-353) in HIV-1+ patients. Mean observed CD4 cell count changes from start of cART to 12 months were +105 (95% CI 77-134) in HIV-2+ patients and +202 (95% CI 199-205) in HIV-1+ patients, an observed difference of 97 cells/mm3 in 1 year. In adjusted analysis, the mean CD4 cell increase was overall 25 CD4 cells/mm3/year lower (95% CI 5-44; P = 0.0127) in HIV-2+ patients compared with HIV-1+ patients. Conclusions A poorer CD4 cell increase during first-line cART was observed in HIV-2+ patients, even after adjusting for pretreatment pVL and other potential confounders. Our results underline the need to identify more potent therapeutic regimens or strategies against HIV-2.