DNMT1 modulation in chronic hepatitis B patients and hypothetic influence on mitochondrial DNA methylation status during long-term nucleo(t)side analogs therapy.

Inhibition of viral replication is the most important goal in patients with Hepatitis B virus chronic infection (CHB). Currently, five oral nucleo(t)side analogs (NAs), including Lamivudine, Adefovir, Telbivudine, Entecavir, and Tenofovir, have been approved for treatment. The widespread use of NAs has also been linked with a progressive growth of unlikely anomaly attributable to mitochondrial dysfunctions, not previously recognized. Here, we explore the hypothesis that NAs may cause persistent epigenetic changes during prolonged NAs therapy in CHB patients. We obtained peripheral blood mononuclear cells (PBMC) from whole blood samples of consecutive patients with chronic HBV infection, 18 receiving NAs and 20 untreated patients. All patients were Caucasian and Italians. Epigenetic analysis was performed by Bisulphite sequencing PCR to search the existence of methylated cytosine residues in the Light (L)-strands of mitochondrial DNA control region (D-loop). Gene expression analysis of DNA methyltransferases 1 was performed by a quantitative relative Real-Time Polymerase Chain Reaction (PCR). DNMT1 expression was significantly (P < 000001) higher in NA treated patients (4.09, IQR 3.52-5.15) when compared with HBV naives (0.61, IQR 0.34-0.82). Besides, DNMT1 expression was significantly correlated with NA therapy duration (Spearman Rho = 0.67; P < 0.05). Furthermore, NA therapy duration was the only significant predictor of DNMT1 expression at multivariate analysis (Beta = 0.95, P < 0.0000001). Bisulphite PCR sequencing showed that methylation of cytosine residues occurred in a higher percentage in patients treated with NAs in comparison with untreated patients and healthy controls. Our data showed a DNMT1 overexpression significantly correlated to NA therapy duration and an higher regional mtDNA hypermethylation. This might suggest an epigenetic alteration that could be involved in one of the possible mechanisms of mitochondrial gene regulation during NAs therapy.

Mediterranean spotted fever-like illness in Sardinia, Italy: a clinical and microbiological study.

INTRODUCTION: Rickettsioses represent a group of emerging infectious diseases in Europe. Climate changes and the anthropization of rural environment have favored vectors' biological cycle and geographic spread. In Sardinia, Mediterranean spotted fever (MSF) is endemic and represents an important public health problem. PURPOSE: We investigated the etiology and the clinical presentation of MSF-like illness in northern Sardinia by enrolling patients admitted to the Infectious Disease Unit of the University of Sassari. RESULTS: Diagnostic tests included ELISA, Indirect immunofluorescence (IFI), DNA isolation from blood and from eschar samples with real-time PCR and genotyping. Eighty-seven patients with a mean age of 53 ± 14 years, of whom 65 (75 %) males, were included in the study. The most common diagnosis was MSF (79 %), followed by Q fever (8 %), and anaplasmosis (2 %). A tache noire was found in 58 % of rickettioses and 28 % of Coxiella burnetii infections. MSF was confirmed in 47 % of the cases by IFI and 43 % by ELISA antibody tests. The isolation of rickettsial DNA from the eschar was positive in 10/13 (77 %) of the cases due to Rickettsia conorii. Using this method, we identified the first case of R. monacensis infection in Italy. CONCLUSIONS: In conclusion, antibody-based tests confirmed the diagnosis in less than 50 % of the cases, whereas DNA isolation confirmed the diagnosis in 77 % of tested cases and allowed the identification of a new pathogenic species in Italy. Therefore, DNA isolation should be implemented to better identify the etiology of MSF-like illnesses and help the clinician in the management of patients.

Ebola virus disease: Case management in the Institute of Infectious Diseases, University Hospital of Sassari, Sardinia, Italy.

Since the onset of the worst epidemic of Ebola virus disease in December 2013, 28,637 cases were reported as confirmed, probable, or suspected. Since the week of 3 January 2016, no more cases have been reported. The total number of deaths have amounted to 11,315 (39.5%). In developed countries, seven cases have been diagnosed: four in the United States, one in Spain, one in the United Kingdom, and one in Italy. On 20 July 2015, Italy was declared Ebola-free. On 9 May 2015, an Italian health worker came back to Italy after a long stay in Sierra Leone working for a non-governmental organization. Forty-eight hours after his arrival, he noticed headache, weakness, muscle pains, and slight fever. The following day, he was safely transported to the Infectious Diseases Unit of University Hospital of Sassari. The patient was hospitalized for 19 hours until an Italian Air Force medical division transferred him to Rome, to the Lazzaro Spallanzani Institute. Nineteen people who had contacts with the patient were monitored daily for 21 days by the Public Health Office of Sassari and none presented any symptoms. So far, neither vaccine nor treatment is available to be proposed on an international scale. Ebola is considered a re-emerging infectious disease which, unlike in the past, has been a worldwide emergency. This case study aimed to establish a discussion about the operative and logistic difficulties to be faced and about the discrepancy arising when protocols clash with the reality of facts.

Could inducible protein-10 and heparin-binding hemagglutinin improve the detection of Mycobacterium tuberculosis-infected subjects in a country with low incidence of tuberculosis?

BACKGROUND: This study aimed to evaluate inducible protein-10 (IP-10) as a biomarker besides interferon-gamma (IFN-γ) to improve the identification of active tuberculosis (TB) and latent tubercular infection (LTBI) in a country with a low incidence of TB. METHODS: Whole blood from Mycobacterium tuberculosis-infected subjects was stimulated with region-of-difference-1 (RD1)-specific peptides and with heparin-binding hemagglutinin (HBHA) to determine the release of IP-10 and IFN-γ. RESULTS: No statistically significant difference was observed between positive rates of IP-10 and IFN-γ after RD1-specific peptide stimulation in the TB and LTBI groups; a different response was detected in QuantiFERON TB-gold test-negative (QFT-) subjects. A significantly different proportion of positive responses was observed between IP-10 and IFN-γ following HBHA stimulation in the TB group and in the QFT- group but not in the LTBI group. CONCLUSIONS: The IP-10 test seemed to identify false-negative QFT results in some subjects with a positive IFN-γ/IP-10/HBHA pattern.

Hepatitis C virus RNA levels at week-2 of telaprevir/boceprevir administration are predictive of virological outcome.

BACKGROUND: Triple therapy with telaprevir/boceprevir + pegylated-interferon+ribavirin can achieve excellent antiviral efficacy, but it can be burdened with resistance development at failure. AIMS: To evaluate kinetics of hepatitis C virus (HCV) RNA decay and early resistance development, in order to promptly identify patients at highest risk of failure to first generation protease inhibitors. METHODS: HCV-RNA was prospectively quantified in 158 patients receiving pegylated-interferon+ribavirin+telaprevir (N = 114) or+boceprevir (N = 44), at early time-points and during per protocol follow-up. Drug resistance was contextually evaluated by population sequencing. RESULTS: HCV-RNA at week-2 was significantly higher in patients experiencing virological failure to triple-therapy than in patients with sustained viral response (2.3 [1.9-2.8] versus 1.2 [0.3-1.7]log IU/mL, p < 0.001). A 100 IU/mL cut-off value for week-2 HCV-RNA had the highest sensitivity (86%) in predicting virological success. Indeed, 23/23 (100%) patients with undetectable HCV-RNA reached success, versus 26/34 (76.5%) patients with HCV-RNA<100 IU/mL, and only 11/31 (35.5%) with HCV-RNA > 100 IU/mL (p < 0.001). Furthermore, differently from failing patients, none of the patient with undetectable HCV-RNA at week-2 had baseline/early resistance. CONCLUSIONS: With triple therapy based on first generation protease inhibitors, suboptimal HCV-RNA decay at week-2 combined with early detection of resistance can help identifying patients with higher risk of virological failure, thus requiring a closer monitoring during therapy.

Timing of antiretroviral therapy initiation after a first AIDS-defining event: temporal changes in clinical attitudes in the ICONA cohort.

BACKGROUND: Time of starting antiretroviral therapy (ART) after diagnosis of specific AIDS-defining event (ADE) is a crucial aspect. Objectives of this study were to evaluate if in patients diagnosed with ADE the time to ART initiation may vary according to year of diagnosis and type of ADE. METHODS: All HIV+ persons diagnosed with an ADE over the 6 months prior to or after enrolment in the Icona Foundation study cohort and while ART-naive were grouped according to type of diagnosis: Those with ADE requiring medications interacting with ART [group A], those with ADE treatable only with ART [B] and other ADE [C]. Survival analysis by Kaplan-Meier was used to estimate the percentage of people starting ART, overall and after stratification for calendar period and ADE group. Multivariable Cox regression model was used to investigate association between calendar year of specific ADE and time to ART initiation. RESULTS: 720 persons with first ADE were observed over 1996-2013 (group A, n=171; B, n=115; C, n=434). By 30 days from diagnosis, 27% (95% CI: 22-32) of those diagnosed in 1996-2000 had started ART vs. 32% (95% CI: 24-40) in 2001-2008 and 43% (95% CI: 33-47) after 2008 (log-rank p=0.001). The proportion of patients starting ART by 30 days was 13% (95% CI 7-19), 40% (95% CI: 30-50) and 38% (95% CI 33-43) in ADE groups A, B and C (log-rank p=0.0001). After adjustment for potential confounders, people diagnosed after 2008 remained at increased probability of starting ART more promptly than those diagnosed in 1996-1999 (AHR 1.72 (95% CI 1.16-2.56). CONCLUSIONS: In our "real-life" setting, the time from ADE to ART initiation was significantly shorter in people diagnosed in more recent years, although perhaps less prompt than expected.

Mucocutaneous leishmaniasis as presentation of HIV infection in Sardinia, insular Italy.

Leishmaniasis is endemic in Sardinia but only cutaneous and visceral cases have been reported to date. We report a case of mucocutaneous leishmaniasis as presentation of HIV infection in a Sardinian patient who had never visited endemic areas. Serological and clinical diagnosis was cytologically and histopathologically confirmed. The patient had a good response to treatment with liposomal amphotericin combined with highly active antiretroviral therapy without recurrences after four years. Our case report highlights the need to better assess the circulation of species, risk factors and clinical spectrum of Leishmania infection in the Italian Mediterranean islands.

Response to raltegravir-based salvage therapy in HIV-infected patients with hepatitis C virus or hepatitis B virus coinfection.

OBJECTIVES: To define the impact of coinfection with hepatitis B virus (HBV) or hepatitis C virus (HCV) on viroimmunological response to raltegravir-based salvage regimens that also include new HIV inhibitors such as maraviroc, darunavir and etravirine. METHODS: We used data from a national observational study of patients starting raltegravir-based regimens to compare virological suppression and CD4 cell change from baseline in patients with and without concomitant HBV or HCV infection. RESULTS: Overall, 275 patients (107 coinfected and 168 non-coinfected) were evaluated. Coinfected patients were more commonly former intravenous drug users and had a longer history of HIV infection and higher baseline aminotransferase levels. Both HIV-RNA and CD4 response were similar in the two groups. Mean time to first HIV-RNA copy number <50 copies/mL was 4.1 months (95% CI 3.5-4.6) in non-coinfected patients and 3.9 months (95% CI 3.3-4.5) in coinfected patients (hazard ratio 1.039, 95% CI 0.761-1.418, P = 0.766, log-rank test). The risk of developing new grade 3-4 hepatic adverse events was significantly higher in coinfected patients (hazard ratio 1.779, 95% CI 1.123-2.817, P = 0.009). The two groups of coinfected and non-coinfected patients had similar rates of interruption of any baseline drug (hazard ratio 1.075, 95% CI 0.649-1.781, P = 0.776) and of raltegravir (hazard ratio 1.520, 95% CI 0.671-3.447, P = 0.311). Few AIDS-defining events and deaths occurred. CONCLUSIONS: Viroimmunological response to regimens based on raltegravir and other recent anti-HIV inhibitors is not negatively affected by coinfection with HBV or HCV. Liver toxicity, either pre-existing or new, is more common in coinfected patients, but with no increased risk of treatment interruption.

HIV type 1 origin and transmission dynamics among different risk groups in sardinia: molecular epidemiology within the close boundaries of an Italian island.

In Italy, the HIV-1 epidemic is still mainly sustained by the subtype B genetic form, although other and novel subtypes and circulating recombinant forms (CRFs) have been reported. A total of 215 HIV-1 pol gene sequences were collected between 1992 and 2010. Multiple alignments spanning subtype-specific HIV-1 B pol sequences were analyzed by Bayesian phylogenetic methods. Subtype B represented 90.7% (n=195) of the sequences. Three main clusters were detected. The root of the tree dated to 1987. Most of the observed viral gene flow events occurred from heterosexual to intravenous drug users (IDUs). Phylogenetic and molecular clock analysis showed an early HIV-1 subtype B introduction in the mid-1980s and dissemination within local risk-specific clusters. This is the first study to describe in detail the HIV-1 molecular epidemiology in one of the largest islands in the European basin. The future potential of the Sardinian epidemic as a hub between Southern and Northern Europe has to been considered.

Novel sensitive, specific and rapid pharmacogenomic test for the prediction of abacavir hypersensitivity reaction: HLA-B*57:01 detection by real-time PCR.

AIM: International HIV treatment guidelines recommend HLA-B*57:01 typing before abacavir administration, in order to reduce the incidence of abacavir hypersensitivity reactions, the major cause of early therapy discontinuation. A fast, sensitive and specific test for HLA-B*57:01 detection has been developed in the present study. MATERIALS & METHODS: Two sets of sequence-specific primers were designed, and amplification rapidly detected by real-time PCR. RESULTS: A total of 108 samples were analyzed in a single-blind fashion, and 41 samples were identified as positive. Complete agreement, with κ = 1 (standard error = 0.0962, p < 0.0001), was found, with a validated methodology used in the EPI109367 clinical trial funded by GlaxoSmithKline, and consisting of low-resolution sequence-specific oligonucleotide PCR, followed by high-resolution sequence-specific oligonucleotide PCR carried out on the HLA-B*57-positive samples. CONCLUSION: We provided a detailed characterization of a novel HLA-B*57:01 screening test, which can be easily implemented by those laboratories already involved in the detection of viral load and virus genotyping. Original submitted 26 October 2010; Revision submitted 13 December 2010.

First HAART in HIV-infected patients with high viral load: value of HIV RNA levels at 12 weeks to predict virologic outcome.

The aim of this study is to identify the role of incomplete suppression during the first months of highly active antiretroviral treatment (HAART) to predict virologic failure in patients with high levels of HIV replication. In a retrospective, longitudinal, and multicenter study, response to HAART was assessed in treatment-naive adults with HIV RNA >100 000 copies/mL, and factors predicting failure were analyzed through regression analyses. A total of 118 patients were included. Virologic failure occurred more often in patients with >500 copies/mL at week 12 (Cox regression: Exp (B) 3.22; P = .02). HIV RNA >500 copies/mL at week 12 predicted incomplete virologic response (odds ratio [OR] = 9.33; P = .002] but not viral rebound. Major antiretroviral resistant mutations were present in 11 of 14 patients. HIV RNA >500 copies/mL at week 12 of first HAART predicts incomplete virologic response in patients with high levels of replication at baseline. Most patients carried resistance mutations at the time of failure.

Trends in the European HIV/AIDS epidemic: a perspective from Italy.

The HIV/AIDS epidemic represents one of the major public-health challenges in present days. Despite the relevant improvement in the prognosis of HIV disease following the introduction of highly active antiretroviral therapy (HAART), numerous new challenges are progressively emerging as patient characteristics evolve. In this article, we give an insight into more actual topics in the HIV/AIDS epidemic in Italy and Europe, including recent epidemiological trends, emerging drug resistance and non-B-subtype circulation, the lights and shadows of HAART and the potential of HARRT to reduce sexual transmission of HIV.

Pneumococcal pneumonia: clinical features, diagnosis and management in HIV-infected and HIV noninfected patients.

PURPOSE OF REVIEW: In this review, we focus on the clinical features, diagnosis and management of pneumococcal pneumonia in HIV-infected and noninfected patients, with particular attention to the most recent advances in this area. RECENT FINDINGS: Classical clinical features are found in young adults, whereas atypical forms occur in immunocompromised patients including HIV-infected individuals. Bacteremic pneumococcal pneumonia is more frequently observed in HIV-infected and also in low-risk patients, according to the Pneumonia Severity Index (PSI). Pneumococcal pneumonia diagnostic process includes physical examination, radiologic findings and microbiologic diagnosis. However, etiologic diagnosis using traditional culture methods is difficult to obtain. In this setting, urinary antigen test, which recognizes Streptococcus pneumoniae cell wall C-polysaccharide, increases the probability of etiologic diagnosis. A correct management approach is crucial in reducing pneumococcal pneumonia mortality. The use of the PSI helps clinicians in deciding between inpatient and outpatient management in immunocompetent individuals, according to Infectious Diseases Society of America (IDSA)-American Thoracic Society (ATS) guidelines. Recent findings support PSI utility also in HIV-infected patients. Recently, efficacy of pneumococcal vaccine in reducing pneumococcal disease incidence has been evidenced in both HIV-infected and noninfected individuals. SUMMARY: Rapid diagnosis and correct management together with implementation of preventive measures are crucial in order to reduce pneumococcal pneumonia related incidence and mortality in HIV-infected and noninfected patients.

Successful prevention of multidrug resistant HIV mother-to-child transmission with enfuvirtide use in late pregnancy.

The availability of antiretroviral therapy has dramatically reduced the risk of HIV mother-to-child transmission (MTCT). However, mothers infected with multidrug resistant HIV (MDR-HIV) are at increased risk of MTCT. We report the case of a pregnant patient infected with MDR-HIV in whom MTCT was avoided with enfuvirtide use in late pregnancy and elective caesarean section.

Antiretroviral-associated portal hypertension: a new clinical condition? Prevalence, predictors and outcome.

BACKGROUND: Cases of severe unexplained liver disease in HIV-infected individuals have recently been reported and are often associated with exposure to didanosine (ddl) and nodular regenerative hyperplasia. Herein, we examine the clinical outcome following ddl removal. METHODS: From 3,300 HIV-infected patients attending three clinics since 2004, all who exhibited persistently elevated aminotransferases and/or significant liver fibrosis in the absence of any known cause of liver damage were identified. RESULTS: Thirty-two individuals (prevalence approximately 1%) met the inclusion criteria - all were on antiretroviral therapy. Of these, 84% were male and 68% had acquired HIV through homosexual contact. Liver biopsy was performed in 12, of whom three showed nonspecific advanced liver fibrosis, two nodular regenerative hyperplasia and three showed only periportal fibrosis. On follow up, nine patients developed episodes of hepatic decompensation, mainly as a consequence of portal hypertension; in eight cases (25%) portal thrombosis was diagnosed. No association was found with plasma HIV RNA or CD4+ T-cell count. All patients but three had been exposed to ddl for a median of 44 months; removal of ddl in 27 was followed 12 months later by improvement in clinical and laboratory parameters in 13 (48%) patients. Finally, a trend towards liver fibrosis improvement was recognised using FibroScan. CONCLUSIONS: Idiopathic persistent liver enzyme elevations in HIV-infected individuals are often associated with cirrhotic and non-cirrhotic portal hypertension. Although this is a relatively rare condition, prolonged exposure to ddl seems to play a pathogenic role and removal of the drug is associated with clinical and laboratory improvements.

Tenofovir renal safety in HIV-infected patients: results from the SCOLTA Project.

OBJECTIVE: To evaluate the prevalence and incidence of nephrotoxicity in HIV-infected patients enrolled in the SCOLTA Project tenofovir cohort and to identify possible risk factors. DESIGN: The SCOLTA Project is a prospective, observational, multicenter study involving 25 infectious disease departments in Italy created to assess the incidence of severe adverse events in patients receiving new antiretroviral drugs. PATIENTS: The SCOLTA Project tenofovir cohort includes a total of 754 HIV infected patients. RESULTS: Data including grade II-IV creatinine elevations according to ACTG scale were available in 354 patients, 237 (67%) males with a mean age of 40.1+/-7.6 years enrolled in the SCOLTA Project tenofovir cohort. During a mean follow up of 19.5+/-11.5 months creatinine elevations were reported in 9/354 (2.5%) patients, all males. Mean duration of tenofovir therapy at the event was 9.5+/-5 months. The overall incidence was 1.6 (95% CI 1.5-1.7) per 100 person-years (p-y) and 0.5 (95% CI 0.4-0.6) p-y for grade III. No grade IV creatinine elevations were reported. Patients with nephrotoxicity were older and more frequently male, HCV infected, in CDC stage C and their CD4 cell count was significantly lower than those without nephrotoxicity. No significant difference was found between tenofovir co-administered antiretroviral drugs. CONCLUSIONS: Both prevalence and incidence of nephrotoxicity were low in patients receiving tenofovir in a non-selected clinical setting. Renal injury in patients receiving tenofovir seems associated with the presence of co-morbidities and with advanced HIV infection.