Characterization of NS5A and NS5B Resistance-Associated Substitutions from Genotype 1 Hepatitis C Virus Infected Patients in a Portuguese Cohort.

This study is focused on the prevalent NS5 coding region resistance-associated substitutions (RASs) in DAA-naive genotype (GT)1 HCV-infected patients and their potential impact on success rates. Plasma RNA from 81 GT1 HCV-infected patients was extracted prior to an in-house nested RT-PCR of the NS5 coding region, which is followed by Sanger population sequencing. NS5A RASs were present in 28.4% (23/81) of all GT1-infected patients with 9.9% (8/81) having the Y93C/H mutation. NS5B RASs showed a prevalence of 14.8% (12/81) and were only detected in GT1b. Overall 38.3% (31/81) of all GT1 HCV-infected patients presented baseline RASs. The obtained data supports the usefulness of resistance testing prior to treatment since a statistically significant association was found between treatment failure and the baseline presence of specific NS5 RASs known as Y93C/H (p = 0.04).

Improve screening of HCV infection by targeting high prevalence aged groups: analysis of a cohort of HCV and HIV co-infected patients.

INTRODUCTION: Hepatitis C constitutes a major public health burden. In Portugal, the prevalence is estimated at 1-1.5% (1). Of these, only 30% are presumed to be diagnosed, which reveals that most infections go unknown. The objective of this study is to identify the age-range distribution at HCV diagnosis and to identify the high-prevalence birth groups that could be targeted for screening, as a strategy to increase diagnosis and identify patients who would benefit most from treatment. METHODS: Retrospective observational study of a cohort of chronic HCV-infected and HIV co-infected patients followed at an Infectious Diseases Center, diagnosed between 1979 and 2014 (Figure 1). Hepatic fibrosis evaluation was performed by real time elastography using METAVIR score. Epidemiological, demographic, clinical, virological and therapeutic data was retrieved from clinical registries. Statistical analysis was performed using Microsoft Excel 2010®. Chi2, Student T were used for a significant p value of <0.05. RESULTS: Our study assessed a cohort of 665 patients: 442 (66.5%) HCV/HIV co-infected and 223 (33.5%) HCV monoinfected. There was a male predominance in both groups (74.9% vs 70.9%). The mean age was 47 HCV/HIV vs 49 years; Portuguese origin in 80% vs 83% and African in 14% vs 12%. The most frequently assumed transmission route was by intravenous drug use (IVDU) (81% vs 72%), followed by sexual contact (18% vs 20%). Mean age at diagnosis was 32 vs 40 years. Mean time since HCV diagnosis was 14, 6 vs 9, 6 years. Fibrosis stage evaluation by real time elastography was available for 133 (30%) and 99 (44.4%) patients (HCV/HIV vs HCV): 16% vs 13% F1; 32% vs 33% F2; 31% vs 35% F3; 21% vs 18% F4. The peak prevalence occurred between the birth intervals of 1960-1969 and 1970-1979 for both groups, corresponding to 81% vs 66,8% (p=0.003) (Figure 1). About three quarters of all patients (76%) were born between the year of 1960 and 1979, with a prevalence of 70% of IVDU. CONCLUSIONS: In our cohort we identify a high risk population for chronically HCV infection, which comprises people born between 1960 and 1979, findings common to those with mono or HIV co-infection. This finding is concordant with the epidemic of IVDU in Portugal around 1980-1990. These patients should be screened for diagnosis in order to be treated and to prevent further disease progression.

Risk of liver decompensation assessed in HIV/HCV co-infected individuals with advanced liver fibrosis: a faster countdown experience.

INTRODUCTION: Cirrhosis secondary to HCV infection is expected to peak in the next decade, particularly in the HIV co-infected subgroup and has become a leading cause of morbidity among these individuals. Efforts must be done to estimate the risk of liver decompensation (LD) in the short term, in order to define the appropriate time for HCV treatment. MATERIALS AND METHODS: Retrospective observational cohort study aimed to assess the risk of LD among a group of HIV/HCV co-infected patients diagnosed in the past 23 years in a central hospital of Lisbon. INCLUSION CRITERIA: (1) advanced liver fibrosis ≥F3; (2) HCV treatment naïve or without sustained virologic response (SVR). Patients had a one to five years period of follow-up. Multiple linear regression, Mann-Whitney and Kendall were the statistical tests performed. RESULTS: From 444 HIV/HCV co-infections, 66 met the inclusion criteria, with preponderance of male gender (82%), 35-45 years of age (55%), genotype 1a (52%), a mean of 13 years of co-infection and an AIDS stage documented in 65%, though the majority is under antiretroviral therapy (86%) and have TCD4+>500 µ/L (59%). Half (52%) showed evidence of steatosis, many of these (41%) presenting a history of alcoholism or overweight (BMI ≥25 Kg/m(2)). Pre-cirrhotic (F3 or F3/4) or cirrhotic (F4) stage was documented in 36 and 30 patients respectively. After staging, 28 (42%) initiated HCV treatment and SVR was achieved in 8 (29%) of those. Five (14%) pre-cirrhotic and twelve (40%) cirrhotic patients experienced at least one LD episode: 8 vs 28 cumulative events at five years and 2.8 vs 1.8 average years up to the first LD episode for pre-cirrhotic vs cirrhotic. The probability of remaining free of LD for pre-cirrhotic vs cirrhotic patients was 97% vs 78% (p≤0.01) at one year; 88% vs 65% (p≤0.001) at three years and 71% vs 44% (p≤0.001) at five years. Positive correlation was found between LD and the cirrhotic stage (vs pre-cirrhosis, p≤0.001), baseline AST ≥100 µ/L (vs <100 µ/L, p≤0.01) and platelet count <120 x 109/L (vs >120 x 109/L, p≤0.05). CONCLUSIONS: Cirrhosis accounts for a significant superior risk of LD. The time up to the first LD event differed in only one year between pre-cirrhotic and cirrhotic, standing for the importance of a rapid treatment referral in both subgroups. Modifiable risk factors that accelerate fibrosis are prevalent in HIV/HCV co-infected patients. Low platelet count, elevated AST and F4 stage predict the rapid progression to LD and the need for early HCV treatment. Large studies are required for further support of these results.

Analysis of hepatitis non-treatment causes in a cohort of HCV and HCV/HIV infected patients.

INTRODUCTION: The decision to start hepatitis C virus (HCV) treatment and its timing remains controversial. As new treatment regimens are approved, it is essential to identify patients eligible for each regimen in a timed and tailored approach. This study aims to identify the reasons to defer treatment of chronic hepatitis C infection in both HCV and HCV/HIV infected patients. MATERIALS AND METHODS: Retrospective observational study of a cohort of HCV chronically infected patients with or without HIV infection, followed in an infectious disease clinic in Lisbon. Demographic, epidemiological, clinical, immunologic and virologic data were collected. Statistical analysis was performed with Microsoft Office(®)- Excel 2012. Kolmogorov-Smirnov, t-test, Chi-square and correlation analysis were performed for a significant p value<0.05. RESULTS: The study included 669 patients, 225 patients infected with HCV (group A) and 444 patients co-infected with HCV/HIV (group B). The comparative analysis of those groups (A vs. B) showed: mean age was 49.4 years versus 46.9 (p<0.01), mean time since HCV diagnosis was 9.5 versus 14.6 years (p=0.558) both groups shared a male predominance and HCV acquisition due to intravenous drug use. Regarding genotype characterization, the predominant was 1a in both groups (p<0.01). Evaluation of IL28B polymorphism revealed CC 15.5% (A) versus 9.45% (B) (p<0.01). Group B mean TCD4 count was 585 cells/µL (mean percentage 27.1%). There was spontaneous viral clearance in 10.7% (A) versus 4.1% (B) (p<0.01). There were treated 52.0% (A) versus 32.2% (B) patients (p<0.01). For the untreated ones (107 - group A vs 270 - group B), no reason was identified for treatment deferral in 32.5% (A) versus 48.0% (B) patients. The most frequent reasons for deferring treatment were: withdrawal to follow-up (33.7%), active staging of disease (7.2%), alcohol abuse (6.0%) and advanced age (6.0%) in group A versus low TCD4 cell count (17.1%), loss to follow-up (7.5%), poor adherence (7.5%) and alcohol abuse (3.2%) in group B. CONCLUSIONS: One of the highlighted cause for treatment deferral in both mono and co-infected patients was withdrawal to follow-up. In co-infected patients, low TCD4 cell count and poor adherence, also gain prominence, suggesting that strategies to improve retention in care may be needed. Additionally, emergence of direct-acting antiviral agents is expected to reduce these determinants in starting treatment, namely reduce the impact of low TCD4 count in co-infected patients.

Evolution trends over three decades of HIV infection late diagnosis: the experience of a Portuguese cohort of 705 HIV-infected patients.

INTRODUCTION: Late HIV diagnosis is common and associated with an increased risk of clinical progression, blunted immune response on antiretroviral (ARV) therapy and higher risk of drug toxicity. Across Europe, more than a third of patients are diagnosed late and consequently delay medical care. European Consensus definition group identify as late presentation (LP) persons, presenting for care, with a CD4 count below 350 cell/mm(3) or presenting with AIDS-defining event, regardless of CD4 cell count. Additionally, advanced HIV disease (AD) is defined by a CD4 count below 200 cell/mm(3) or an AIDS defining condition in persons presenting to care. MATERIALS AND METHODS: Retrospective observational study of a cohort of 705 HIV-infected patients diagnosed between 1986 and 2014 and medically followed at an Infectious Diseases Service in Lisbon. OBJECTIVES: Evaluate LP rate evolution in the last three decades (10-year time intervals considered: 1986-1995; 1996-2005; 2006-2014); compare clinic, immunologic, virologic and therapeutic response over time. Identify main reasons responsible for late HIV diagnosis in order to promote optimized intervention strategies. SPSS version 20.0 was used for statistical analysis. RESULTS: Study included 705 patients HIV diagnosed during 3 time intervals: group A n=82 [1986-1995]; group B n=332 [1996-2005]; group C n=291 [2006-2014]. Demographic and epidemiological characterization revealed (A vs B vs C): male predominance of 79% vs 66% vs 66%; mean age at diagnosis 30 vs 36 vs 42 years; Portugal (82% vs 70% vs 58%) and Africa (13% vs 23% vs 29%) as the main places of birth; transmission by heterosexual contact in 21% vs 47% vs 62%, MSM in 21% vs 15% vs 23% and IVDU in 57% vs 35% vs 13%. Mean CD4 at diagnosis was 362 vs 344 vs 377 cell/mm(3). Considering the time intervals, LP was found in 52% vs 56% vs 52% of patients and AD in 31% vs 38% vs 35%, respectively. At first health care encounter, 46% vs 43% vs 39% of individuals presented with AIDS. Over follow up, the vast majority initiated ARV (95% vs 98% vs 84%) and mean CD4 at that time was 254 vs 282 vs 250 cell/mm(3). The last immunologic and virologic determination available registered mean CD4 of 657 vs 644 vs 584 cell/mm(3) and undetectable HIV plasma RNA in 92% vs 84% vs 82% of treated patients. CONCLUSIONS: This study evidenced a maintained LP rate, slightly above 50% in each of the three analyzed last decades, and one-third of patients presented AD at HIV diagnosis. At initial health care contact, nearly 40% of individuals met AIDS clinical or immunological criteria.

Durability of first antiretroviral treatment in HIV chronically infected patients: why change and what are the outcomes?

INTRODUCTION: First antiretroviral therapy (ART) is often switched to simpler, more potent or better tolerated regimens (1, 2). Although discontinuation rates are frequently studied, the durability of regimens is rarely approached. MATERIALS AND METHODS: Retrospective study with the following objectives: analyze first ART schemes and their durability in naive patients with chronic HIV-1 and 2 infections, evaluate factors influencing ART change, second-line ART and consequent virologic and immunologic responses. Patients had follow-ups in a Central University Hospital, started ART between January 2007 and December 2012 and changed first regimens. Clinical data was obtained from medical records and analyzed using the Statistical Package for the Social Sciences (version 20). RESULTS: Of the 652 naive patients who started ART, 164 changed regimens. The majority had HIV-1 infection (n=158). The mean age was 43.9 years (standard deviation±14.3), with a male predominance of 57.9%. Regimens with efavirenz were the most common amongst HIV-1 patients (50%) followed by lopinavir/r (22%). In HIV-2 patients, lopinavir/r (n=3) regimens were most prevalent. First ART regimens had a mean duration of 12.1 months. There was no difference between NNRTI (59.8%) and protease inhibitor (40.2%) schemes regarding durability. Adverse reactions were the major cause of ART switching (55.5%) followed by therapy resistance (12.1%). Age was inversely related to durability (p=0.007 Mann-Whitney, Phi coefficient -0.161) and associated with the appearance of adverse reactions (p=0.04, Chi-square). Younger patients had a reduced risk of adverse reactions by 27%. Adverse reactions increased the risk of inferior durability by 40%. Psychiatric symptoms (28.4%) were the most prevalent, all attributed to efavirenz. The year of ART initiation was associated with different durability rates (p=0.005, Mann-Whitney). Patients started on ART before the year 2010 reduced the probability of inferior ART duration by 25.8%. After second-line ART regimens, TCD4+ counts>500 cell/µL were increased by 38% and favourable virologic outcome achieved in 84%. CONCLUSIONS: Adverse reactions were the main cause for ART switching, supporting a cautious approach when initiating regimens, particularly in older patients. All ART naive patients who changed initial therapy had favourable immunological and virologic responses.

Atypical presentation of entomophthoromycosis caused by Conidiobolus coronatus.

A male patient from Guinea-Bissau was admitted to Egas Moniz Hospital, Lisbon, complaining of fever and exhibiting a productive cough with mucopurulent discharge and weight loss. He had been using empirical medication with dexamethasone to treat his generalized facial swelling. At admission, he was cachectic and presented with soft facial edema, oropharyngeal thrush, and two fistulas of the palate. Acid-fast bacilli were detected in the sputum and were later identified as Mycobacterium tuberculosis. Cultures of the palatine exudate and biopsy resulted in the growth of Candida albicans. The patient was administered antituberculosis drugs and fluconazole, but his clinical situation deteriorated progressively. Extensive investigation of his clinical condition did not result in a conclusive diagnosis until he began to experience respiratory distress and subcutaneous nodules appeared on his face. Biopsies of the hypopharynx and nodules revealed the presence of Conidiobolus coronatus. After initiating combined antifungal and antibiotic therapy, the patient's clinical condition improved significantly. We report an unusual presentation of entomophthoromycosis and describe the clinical difficulties that delayed this diagnosis.

[Increased IL-4 production in response to virulent Mycobacterium tuberculosis in tuberculosis patients with advanced disease]. / Aumento da produção de IL-4: resposta ao Mycobacterium tuberculosis virulento em doentes com tuberculose em estádio avançado.

The study was designed to compare immune responses to Mycobacterium tuberculosis bacilli and antigens in healthy Portuguese subjects and pulmonary tuberculosis patients (TB), and to correlate immune status with clinical severity of tuberculosis disease. PBMC were cultured and stimulated with live and killed M. tuberculosis H37Rv and purified protein derivative (PPD) and lymphoproliferation and production of IFN-gamma and IL-5/IL-4 by these cultures were evaluated by the use of ELISA and multi-parameter flow cytometry. PBMC from 30 tuberculosis patients demonstrated significantly reduced amounts of proliferation and IFN-gamma when stimulated with live M. tuberculosis compared the control group. Of 15 tuberculosis patients tested for intracellular IL-4 following stimulation with M. tuberculosis, 7 showed greatly increased IL-4 production in CD8+ and gammadelta+ T cells. Tuberculosis patients demonstrated an increase of intracellular IL-4 after PBMC were stimulated with live M. tuberculosis in the CD4+ phenotype, but more notably in CD8+ and gammadelta TCR+ subsets. Increased production of IL-4 in tuberculosis patients was primarily in individuals with advanced involvement of lung parenchymal with high bacterial loads in sputum. These results suggest that an alteration in type 1 and type 2 cytokine balance can occur in patients with tuberculosis at an advanced clinical stage of disease.

Gamma delta T cell responses associated with the development of tuberculosis in health care workers.

This study evaluated T cell immune responses to purified protein derivative (PPD) and Mycobacterium tuberculosis (Mtb) in health care workers who remained free of active tuberculosis (HCWs w/o TB), health care workers who went on to develop active TB (HCWs w/TB), non-health care workers who were TB free (Non-HCWs) and tuberculosis patients presenting with minimal (Min TB) or advanced (Adv TB) disease. Peripheral blood mononuclear cells (PBMC) were stimulated with Mtb and PPD and the expression of T cell activation markers CD25+ and HLA-DR+, intracellular IL-4 and IFN-gamma production and cytotoxic responses were evaluated. PBMC from HCWs who developed TB showed decreased percentages of cells expressing CD8+CD25+ in comparison to HCWs who remained healthy. HCWs who developed TB showed increased gammadelta TCR+ cell cytotoxicity and decreased CD3+gammadelta TCR- cell cytotoxicity in comparison to HCWs who remained healthy. PBMC from TB patients with advanced disease showed decreased percentages of CD25+CD4+ and CD25+CD8+ T cells that were associated with increased IL-4 production in CD8+ and gammadelta TCR+ phenotypes, in comparison with TB patients presenting minimal disease. TB patients with advanced disease showed increased gammadelta TCR+ cytotoxicity and reduced CD3+gammadelta TCR- cell cytotoxicity. Our results suggest that HCWs who developed TB show an early compensatory mechanism involving an increase in lytic responses of gammadelta TCR+ cells which did not prevent TB.

Increased Interleukin-4 production by CD8 and gammadelta T cells in health-care workers is associated with the subsequent development of active tuberculosis.

We evaluated immune responses to Mycobacterium tuberculosis in 10 health-care workers (HCWs) and 10 non-HCWs and correlated their immune status with the development of active tuberculosis (TB). Twenty individuals were randomly recruited, tested, and monitored longitudinally for TB presentation. Peripheral blood mononuclear cells (PBMCs) from donors were stimulated with M. tuberculosis and tested for cell proliferation and the production of interferon (IFN)- gamma, interleukin (IL)-5, and IL-4, by use of enzyme-linked immunosorbent or flow-cytometric assays. HCWs had higher levels of cell proliferation (24,258 cpm) and IFN- gamma (6373 pg/mL) to M. tuberculosis than did non-HCWs (cell proliferation, 11,462 cpm; IFN- gamma, 3228 pg/mL). Six of 10 HCWs showed increased median percentages of CD8+IL-4+ (4.7%) and gammadelta +IL-4+ (2.3%) T cells and progressed to active TB. HCWs who remained healthy showed increased median percentages of CD8+IFN- gamma+ (25.0%) and gammadelta +IFN- gamma+ (8.0%) and lower percentages of CD8+IL-4+ (0.05%) and gammadelta +IL-4+ (0.03%) T cells.