Monica Basso

Baseline Cellular HIV DNA Load Predicts HIV DNA Decline and Residual HIV Plasma Levels during Effective Antiretroviral Therapy

ABSTRACT Cellular human immunodeficiency virus type 1 (HIV-1) DNA may be considered a marker of disease progression with significant predictive power, but published data on its correlation with plasma HIV RNA levels and CD4 counts in acute and chronic patients are not conclusive. We evaluated a cohort of 180 patients naïve for antiretroviral therapy before the beginning of treatment and after a virological response in order to define the indicators correlated with HIV DNA load decrease until undetectability. The following variables were evaluated as continuous variables: age, CD4 cell count and log10 HIV DNA level at baseline and follow-up, and baseline log10 HIV RNA level. Primary HIV infection at the start of therapy, an HIV RNA level at follow-up of <2.5 copies/ml, origin, gender, and transmission risk were evaluated as binary variables. The decline of HIV DNA values during effective therapy was directly related to baseline HIV DNA and HIV RNA values, to an increase in the number of CD4 cells, and to the achievement of an HIV RNA load of <2.5 copies/ml. An undetectable cellular HIV DNA load was achieved by 21.6% of patients at the follow-up time point and correlated significantly with lower baseline cellular HIV DNA values and with being in the primary stage of infection when therapy started. In conclusion, early treatment facilitated the achievement of undetectable levels of plasma viremia and cellular HIV DNA and a better recovery of CD4 lymphocytes. HIV DNA levels before and during highly active antiretroviral therapy may be used as a new tool for monitoring treatment efficacy.

Development and validation of a simple and robust HPLC method with UV detection for quantification of the hepatitis C virus inhibitor daclatasvir in human plasma

HIGHLIGHTSThe first HPLC‐UV method was developed for the determination of DCV in human plasma.This method was applied to quantification of daclatasvir in clinical samples.Daclatasvir is degraded by sunlight. ABSTRACT Daclatasvir is an inhibitor of hepatitis C virus NS5A protein that is used for the therapy of chronic hepatitis. So far, published methods for analysis of daclatasvir in plasma are exclusively based on mass spectrometry, which is not always available in standard clinical laboratories. Thus, we wished to develop and validate a simple, but still reliable and sensitive high‐performance liquid chromatography (HPLC) assay with UV detection for the quantification of daclatasvir, feasible for a wide‐spread clinical routine use. The method consisted of solid‐phase extraction of daclatasvir using Waters Oasis HLB 1cc cartridges, reversed‐phase liquid chromatography with a Waters XTerra RP18 (150 mm × 4.6 mm, 3.5 &mgr;m) column and a mobile phase of ammonium acetate buffer (pH 5.0, 10 mM) and acetonitrile (56:44, v/v), and UV detection at 318 nm. This assay proved to be sensitive (lower limit of quantification of 0.05 &mgr;g/mL), linear (correlation coefficients ≥0.997), specific (no interference with various potentially co‐administrated drugs), reproducible (both intra‐day and inter‐day coefficients of variation ≤8.9%), and accurate (deviations ranged from −2.2 to 8.0% and from −6.5 to 9.2% for intra‐day and inter‐day assays, respectively). The method was applied to therapeutic monitoring of patients undergoing daclatasvir therapy for hepatitis C and showed to be reliable and robust. Thus, this method provides a simple, sensitive, precise, and reproducible assay for dosing daclatasvir that can be readily adaptable to routine use by clinical laboratories with standard equipment. In addition, the stability of daclatasvir in plasma was evaluated under various conditions, including after the heating procedure required for inactivation of infectious viruses and in different light exposure conditions. These studies evidenced photo‐instability of the compound under sunlight exposure over time. Thus, blood sampling and the whole handling procedure have to be performed quickly and with minimal light exposure.

Present, old and future strategies for anti-HCV treatment in patients infected by genotype-1: estimation of the drug costs in the Calabria Region in the era of the directly acting antivirals.

BackgroundIn Italy, anti-HCV drugs are provided free of charge by the National Health System. Since 2011, three drug regimens including a directly acting antiviral (DAA) are considered the gold standard for HCV treatment. However, these drugs add a significant cost (roughly €26,000) to the combination of pegylated-interferon-α/ribavirin (PEG-IFN/RBV), which before DAA represented the unique treatment. To provide the National Health System potential useful information, we estimated costs to provide anti-HCV drugs to treat a population experienced for PEG-INF/RBV.MethodsGenotype 1 HCV mono-infected or HIV/HCV co-infected individuals who were treated with PEG-IFN/RBV between 2008 and 2013 were included. The cost to treat these patients with PEG-IFN/RBV was calculated (cost 1). We also estimated costs if we had to treat these patients with a lead-in period of PEG-INF/RBV followed by PEG-IFN/RBV and a DAA in naïves (cost 2), in addition to cost 1 plus the estimated cost to re-treat with PEG-IFN/RBV and a DAA patients who had a relapse or a non response (cost 3). Moreover, all costs were normalized by SVR. Rates of foreseen response with DAA were obtained from literature data.ResultsThe overall study population consisted of 104 patients. The rate of sustained virological response (SVR) was 55%, while it was estimated that SVR would be obtained in 75% of patients with a lead-in period with PEG-IFN/RBV followed by a DAA combination, and in 78% if this treatment is used to re-treat experienced patients with a DAA. Drug costs associated with these treatments were: €1,214,283 for cost 1, €3,474,977 for cost 2 and €3,002,095 for cost 3. Costs per SVR achieved were: €22,284 for cost 1, €44,643 for cost 2 and €38,322 for cost 3.ConclusionsTreatments including DAAs achieve a SVR in more patients than PEG-IFN/RBV but they cost around three times more than PEG-IFN/RBV alone regimens. Also, cost per SVR is almost twofold greater than PEG-IFN/RBV regimens. Therefore, it is mandatory to implement use of DAA in clinical practice, but the National Health System should allocate adequate resources to provide drugs, which challenges sustainability. Cost reduction for anti-HCV drugs should be pursued.

Soluble apoptosis molecules in primary biliary cirrhosis: analysis and commitment of the Fas and tumour necrosis factor-related apoptosis-inducing ligand systems in comparison with chronic hepatitis C

Apoptosis in the liver is generated mainly by the Fas system. Tumour necrosis factor‐related apoptosis‐inducing ligand (TRAIL) has been proposed recently as a new apoptotic inducer. In the liver environment hepatocytes and biliary epithelial cells express TRAIL receptors which are up‐regulated by increased levels of bile acids and during viral hepatitis. As for FasL, a soluble form of TRAIL has been described. To explore the commitment and level of activation of these two apoptotic systems in patients affected by primary biliary cirrhosis (PBC) or chronic hepatitis C (CH‐C), a comparative study was drawn. Thirty patients with PBC on ursodeoxycholic acid have been enrolled. This group was compared with 30 patients with CH‐C and with 20 healthy subjects. Soluble Fas ligand (sFasL) and soluble TRAIL (sTRAIL) levels were evaluated by double determinant immune assay and enzyme‐linked immunosorbent assay (ELISA), respectively. Soluble FasL molecules were higher in PBC compared to CH‐C (P = 0·009). Soluble FasL was not detected in controls. Soluble TRAIL was significantly higher in CH‐C patients compared to PBC (P = 0·0001). Soluble TRAIL levels were higher in PBC and in CH‐C than in controls (P = 0·015 and P < 0·001, respectively). No correlation between sFasL and sTRAIL, stage of disease, liver histology in each disease and cytolysis was present. Our data show different levels of commitment of TRAIL and Fas apoptosis‐inducing systems in CH‐C and PBC. Thus a different prominent role of TRAIL and Fas systems in the pathogenesis of these two conditions can be speculated: the former by inducing the death of infected hepatocytes, the latter by mediating the disappearance of bile duct.

Clinical and virological survey of patients with hepatitis B surface antigen in an Italian region: clinical considerations and disease burden.

The aim of this study was to determine the prevalence of hepatitis B virus (HBV) infection in an Italian region, Liguria (1,572,000 inhabitants), by means of a network of 12 referral centers for liver diseases. All patients with HBV surface antigen followed throughout 2006 were included. Personal data, infectious status with risk factors, other non‐infectious risk factors for liver disease, clinical status, and treatment were the questionnaire. Four hundred forty‐five patients (71% male) were evaluated. Their median age was 48 years (range 5–84), and 83.4% were of Italian origin. Community‐acquired infection was the principal mode of HBV transmission (82.5%), followed by previous intravenous drug use (9.4%), perinatal transmission (6.3%), and transfusion‐associated transmission (1.8%). Hepatitis B e‐antigen was present in 20.4% of the patients, while co‐infections with hepatitis D virus and/or hepatitis C virus and/or human immunodeficiency virus (HIV) were observed in 18.7% of the patients. Chronic active hepatitis was present in 62.5% of the patients, cirrhosis in 13.5%, hepatocellular carcinoma in 2.2%, and 21.8% of the patients were inactive carriers of HBV. In all, 42.5% of the patients were treated with interferon or lamivudine and/or adefovir‐dipivoxil. Forty‐nine patients were co‐infected with HIV (86% on highly active antiviral therapy). Nevertheless, this study identified only 2.2% of the expected patients with HBV. Hence, it has to be reasoned that few potential infectious or treatable patients are referred to liver disease centers. HBV infection is still an underestimated health problem, and few potential infectious or treatable patients are referred to tertiary centers. J. Med. Virol. 81:1882–1886, 2009.

Viral infections of the central nervous system in elderly patients: a retrospective study

OBJECTIVES Very few data exist on viral meningitis and encephalitis in elderly patients (>65 years old). METHODS This study investigated the detection of herpes simplex virus (HSV), varicella zoster virus (VZV), human herpes virus 6 (HHV-6), HHV-7, HHV-8, cytomegalovirus (CMV), Epstein-Barr virus (EBV), enterovirus (EV), human adenovirus (HAdV), human parechoviruses (HPeVs), and tick-borne encephalitis virus (TBEV) through real-time PCR (RT-PCR) in patients >65 years old who had cerebrospinal fluid (CSF) tested for a suspected central nervous system infection. RESULTS A total of 2868 RT-PCRs were performed on 502 CSF samples. Overall, 65 positive RT-PCRs were found: 23 for HSV (35.4% of positives), 15 for EV (23.1% of positives), 14 for EBV (21.5% of positives), 12 for VZV (18.5% of positives), and one for CMV (1.5% of positives). A positive RT-PCR in CSF was detected in 24 (17.4%) patients aged ≥ 80 years and in 35 (9.6%) patients aged 65-79 years (p=0.02). VZV was more frequently detected in the oldest subjects (5.9% vs. 1.6%, p=0.03). CONCLUSIONS HSV was the most common viral aetiology identified in the study, with VZV infection being recognized more frequently in those patients aged ≥ 80 years.

Strong and persistent correlation between baseline and follow-up HIV-DNA levels and residual viremia in a population of naïve patients with more than 4 years of effective antiretroviral therapy

In a longitudinal study on 181 naïve patients who responded to therapy (mean follow-up 4 years), high baseline human immunodeficiency virus (HIV)-RNA values correlated with high levels of cellular HIV-DNA at all time points (p < 0.0001, p 0.045, p 0.0055, and p 0.0025, respectively) and negatively correlated with undetectable residual viremia (URV; <2.5 copies/mL) at T1, T2, and T3 (p 0.026, p 0.0149, and p 0.0002, respectively). Baseline high HIV-DNA levels predicted the persistence of high values (p 0.0001) and negatively correlated with URV (p 0.0254, p 0.0481, and p 0.0085). These results suggest that baseline viral load, cellular HIV-DNA, and URV were strongly correlated over long-term follow-up of antiretroviral therapy responders.

Epstein-Barr and cytomegalovirus DNA salivary shedding correlate with long-term plasma HIV RNA detection in HIV-infected men who have sex with men.

The aim of the study was to evaluate cytomegalovirus (CMV) and Epstein–Barr virus (EBV) DNA salivary shedding in HIV‐positive men who have sex with men (MSM) and to determine whether viro‐immunological parameters and long‐term (24 months) plasma HIV RNA (pHIV) detection may predict herpesviruses replication. A total of 193 HIV‐positive MSM were consecutively recruited (mean CD4+ cell count 607 cells/mm3 and mean nadir value 333 cells/mm3); pHIV was analyzed for 24 months prior to saliva sampling: patients were categorized as successfully suppressed (SS) and not suppressed (NS). The EBV viral load was categorized as high viral load (HVL), intermediate (IVL), or low (LVL), CMV DNA as positive or negative. NS patients experienced both herpesviruses detectability more frequently respect to SS patients (P = 0.034); conversely, no salivary shedding was more frequent in SS patients (P = 0.014). HVL EBV was more frequent in NS patients than in SS subjects (P = 0.038 for isolated EBV detection and P = 0.001 when CMV shedding was associated). NS subjects with HVL EBV had a median pHIV of 43,820 copies/ml, significantly higher respect to IVL and LVL patients (P = 0.027 and P = 0.0005, respectively). CMV shedding was mostly associated to EBV shedding. NS patients showed a significantly higher frequency of saliva HVL EBV detection compared to SS patients; moreover, NS patients with HVL EBV had a higher pHIV respect to those with IVL and LVL shedding. Our results suggest that a successful pHIV suppression could reduce the burden of salivary EBV replication and likely the risk of herpesviruses‐related cancers. J. Med. Virol. 88:1211–1221, 2016.

Daclatasvir plasma level and resistance selection in HIV patients with hepatitis C virus cirrhosis treated with daclatasvir, sofosbuvir, and ribavirin.

OBJECTIVES Effective treatment with direct-acting antiviral drugs against hepatitis C virus (HCV) is a medical need in cirrhotic HIV-HCV co-infected patients. METHODS This study investigated the plasma levels of daclatasvir (DCV) and ribavirin (RBV) in HIV-HCV co-infected subjects treated with DCV, sofosbuvir, and RBV. Drug concentrations were quantified using validated high-performance liquid chromatography methods with ultraviolet detection. The HCV non-structural protein 5A and non-structural protein 5B coding regions were analyzed by population-based sequencing. RESULTS DCV was dosed at week 4 and at week 8 of treatment, and RBV at week 8. One patient had the lowest DCV level, corresponding to 32.7% of the overall median value of the other patients at week 4 and about 40% at week 8. The Y93H variant was detected in this subject at weeks 8, 16, and 20 of treatment, but not before treatment or at day 2, and the patient experienced virological failure. Another subject with the Y93H variant at baseline and appropriate DCV levels had HCV RNA <12 IU/ml at week 12 and undetectable at week 16. CONCLUSIONS Sub-optimal DCV drug levels allow the selection of resistance-associated variants and fail to contribute to antiviral activity. No definite reason for the low DCV level was found. Quantifying the drug is suggested in difficult-to-treat patients.

A stable CC-chemokine receptor (CCR)-5 tropic virus is correlated with the persistence of HIV RNA at less than 2.5 copies in successfully treated naïve subjects

BackgroundTo determine if tropism for CXCR4 or CCR5 correlates with cellular HIV DNA load, residual viraemia and CD4 count in 219 successfully treated naive subjects with HIV infection enrolled in five infectious diseases units in Northeastern Italy.MethodsA subset of subjects, achieving plasma HIV RNA level <50 copies/ml after initiation of first-line therapy and maintaining it until follow-up time points, was retrospectively selected from a prospective cohort. Blood samples were collected before the beginning of therapy (T0), at the first follow-up time (T1) and, when available, at a second (T2) follow-up time.ResultsHIV DNA, CD4 count and plasma viraemia were available from all 219 patients at T0 and T1, and in 86 subjects at T2, while tropism determinations were available from 109 subjects at T0, 219 at T1, and from 86 subjects at T2. Achieving residual viraemia <2.5 copies/ml at T1 correlated with having the same condition at T2 (p = 0.0007). X4 tropism at T1 was negatively correlated with the possibility of achieving viraemia<2.5 copies/ml at T2 (p = 0.0076). T1-T2 tropism stability was significant (p <0.0001). T0 tropism correlated with T1 and T2 tropism (p < 0.001); therefore the stability of the tropism over the two follow-up periods was significant (p = 0.0003). An effective viremic suppression (viraemia<2.5 copies/ml) correlated with R5 coreceptor affinity (p= 0.047).ConclusionsThe tropism of archived virus was stable during an effective treatment, with 15-18% of subjects switching over time, despite a viraemia<50 copies/ml. R5 tropism and its stability were related to achieving and maintaining viraemia<2.5 copies/ml.