Stefano Novati

NATURALLY OCCURRING RESISTANCE MUTATIONS TO INHIBITORS OF HCV NS5A REGION AND NS5B POLYMERASE IN DAA TREATMENT-NAIVE PATIENTS

BackgroundDirect-acting antiviral (DAA) agents target HCV proteins; some of these have already been approved for the treatment of HCV infection, while others are in development. However, selection of DAA-resistant viral variants may hamper treatment. The aim of this study was to illustrate potential natural DAA-resistance mutations in the HCV NS5A and NS5B regions of HCV genotypes 1a and 1b from DAA-naïve patients.MethodsDirect sequencing of HCV NS5A and NS5B regions was performed in 32 patients infected with HCV genotype 1a and 30 patients infected with HCV genotype 1b; all subjects were naïve to DAAs.ResultsIn genotype 1a strains, resistance mutations in NS5A (M28V, L31M and H58P) were observed in 4/32 (12.5%) patients, and resistance mutations in NS5B (V321I, M426L, Y448H, Y452H) were observed in 4/32 (12.5%) patients. In genotype 1b, resistance mutations in NS5A (L28V, L31M, Q54H, Y93H and I280V) were observed in 16/30 (53.3%) patients, while resistance mutations in NS5B (L159F, V321I, C316N, M426L, Y452H, R465G and V499A) were observed in 27/30 (90%) patients.ConclusionsMutations conferring DAA resistance were detected in NS5A and NS5B of HCV genotypes 1a and 1b from DAA-naïve patients. Although some mutations confer only a low level of resistance, the presence at baseline of mutated HCV variants should be taken into consideration in the context of DAA therapy.

Naturally occurring mutations to HCV protease inhibitors in treatment-naïve patients

BackgroundProtease inhibitors (PIs) to treat hepatitis C (HCV) virus infection have been approved and others are under development.ResultsThe aims of this study were to illustrate natural polymorphisms in the HCV protease and measure the frequency of PI resistance mutations in different HCV genotypes from PI-naïve patients.Direct sequencing of HCV NS3/4A protease was performed in 156 HCV patients naïve to PIs who were infected with genotype 1a (n = 31), 1b (n = 39), 2 (n = 30), 3 (n = 33) and 4 (n = 23).Amino acid (aa) substitutions associated with HCV PI resistance were found in 17/156 (10.8%) sequences. Mutations V36L, T54S, V55A/I, and Q80K/L were observed in 29% of patients with genotype 1a, and V55F, Q80L/N and M175L in 10% of patients with genotype 1b. The mutation V158M was found in 3% of patients with genotype 2, D168Q was present in 100% of patients with genotype 3 and D168E was observed in 13% of patients with genotype 4. In addition, multiple aa polymorphisms not associated with PI resistance were detected in patients with genotypes 1a, 1b and 4.ConclusionsAlthough major PI resistance mutations were not detected, other resistance mutations conferring low level resistance to PIs together with a number of natural polymorphisms were observed in proteases of PI naïve HCV patients. A more extensive analysis is needed to better evaluate the impact of baseline resistance and compensatory mutations in the efficacy of HCV PI treatment.

Performance of liver stiffness measurements by transient elastography in chronic hepatitis

AIM To compare results of liver stiffness measurements by transient elastography (TE) obtained in our patients population with that used in a recently published meta-analysis. METHODS This was a single center cross-sectional study. Consecutive patients with chronic viral hepatitis scheduled for liver biopsy at the outpatient ward of our Infectious Diseases Department were enrolled. TE was carried out by using FibroScan™ (Echosens, Paris, France). Liver biopsy was performed on the same day as TE, as day case procedure. Fibrosis was staged according to the Metavir scoring system. The diagnostic performance of TE was assessed by using receiver operating characteristic (ROC) curves and the area under the ROC curve analysis. RESULTS Two hundred and fifty-two patients met the inclusion criteria. Six (2%) patients were excluded due to unreliable TE measurements. Thus, 246 (171 men and 75 women) patients were analyzed. One hundred and ninety-five (79.3%) patients had chronic hepatitis C, 41 (16.7%) had chronic hepatitis B, and 10 (4.0%) were coinfected with human immunodeficiency virus. ROC curve analysis identified optimal cut-off value of TE as high as 6.9 kPa for F ≥ 2; 7.9 kPa for F ≥ 3; 9.6 kPa for F = 4 in all patients (n = 246), and as high as 6.9 kPa for F ≥ 2; 7.3 kPa for F ≥ 3; 9.3 kPa for F = 4 in patients with hepatitis C (n = 195). Cut-off values of TE obtained by maximizing only the specificity were as high as 6.9 kPa for F ≥ 2; 9.6 kPa for F ≥ 3; 12.2 kPa for F = 4 in all patients (n = 246), and as high as 7.0 kPa for F ≥ 2; 9.3 kPa for F ≥ 3; 12.3 kPa for F = 4 in patients with hepatitis C (n = 195). CONCLUSION The cut-off values of TE obtained in this single center study are comparable to that obtained in a recently published meta-analysis that included up to 40 studies.

JC virus load in cerebrospinal fluid and transcriptional control region rearrangements may predict the clinical course of progressive multifocal leukoencephalopathy

Progressive multifocal leukoencephalopathy (PML) is a severe disease of the central nervous system (CNS), caused by infection with the Polyomavirus JC virus (JCV). Because there are no known treatments or prognostic factors, we performed a long‐term study focusing mainly on cerebrospinal fluid (CSF) samples from PML patients to describe the virological features akin to the different forms of the disease. Twenty‐eight PML patients were enrolled: 10 HIV‐1+ patients with classical PML (CPML), 9 HIV‐1+ patients with slowly progressing or stable neurological symptoms (benign PML), 3 HIV‐1+ asymptomatic patients, and 6 HIV‐1‐negative patients. CSF, urine, and blood samples were collected at the enrollment (baseline) and every 6 months afterwards when possible. The JCV DNA and HIV‐1 RNA loads were determined, and the JCV strains were characterized. At baseline, the mean CSF JCV load was log 6.0 ± 1.2 copies/ml for CPML patients, log 4.0 ± 1.0 copies/ml for benign PML patients, log 4.2 ± 0.5 copies/ml for asymptomatic PML patients, and log 5.8 ± 1.3 copies/ml for HIV‐1‐negative PML patients (CPML vs. benign: P < 0.01; CPML vs. asymptomatic: P < 0.05; HIV‐1 negative vs. benign: P < 0.01). Organization of the JCV transcriptional control region (TCR) showed unusual archetype structures in two long‐term survival patients; the NF1 sequence was found most commonly, whereas the Sp1 binding site was the most common for both CPML patients and HIV‐1 negative patients. Our results suggest that the JCV load in the CSF and the organization of the TCR should be considered as indicators of PML clinical outcome. J. Cell. Physiol. 227: 3511–3517, 2012.

Lymphotropic Polyomavirus is detected in peripheral blood from immunocompromised and healthy subjects

BACKGROUND Lymphotropic Polyomavirus (LPV) was isolated from a B-lymphoblastoid cell line of an African green monkey. This virus shares some characteristics with human polyomaviruses, but it is antigenically distinct from BK Virus (BKV) and JC Virus (JCV). Seroepidemiological studies revealed that human sera react in the presence of LPV antigens, and, recently, the viral genome was amplified in the peripheral blood from patients affected with HIV-related leukoencephalopathies. OBJECTIVES The aims of the study were to investigate and compare the presence of LPV DNA with that of JCV and BKV in different biological samples and patient groups. STUDY DESIGN LPV, JCV and BKV DNA were searched and quantified in peripheral blood and CSF from HIV+ patients and in peripheral blood from healthy subjects. RESULTS The LPV genome was detected in peripheral blood of both HIV+ patients and healthy subjects, with a prevalence of 7.2% and 4.7% respectively, but not in CSF. However, its presence was less frequent than that of JCV and BKV. CONCLUSIONS The amplification of LPV genome from human peripheral blood confirms the fact that LPV can infect the human population. LPV DNA was amplified from patients affected with HIV-related leukoencephalopathies but also from HIV patients without neurological disorders and from healthy subjects. Therefore, the results do not support the hypothesis of an association between LPV infection and any neurological disease. However, given their high similarity, it is possible that LPV, as well as BKV and JCV, could establish latency in humans and cause disease only in rare circumstances.

Plasma Levels of Bacterial DNA in HIV Infection: The Limits of Quantitative Polymerase Chain Reaction

To the Editor—In a recent article, Jiang et al [1] showed that blood levels of bacterial DNA, as determined by quantitative polymerase chain reaction (PCR) on 16S ribosomal DNA fragments, were significantly higher in human immunodefi-ciency virus (HIV)–infected patients than were those in uninfected control patients. Moreover, the level of bacterial DNA in the blood was shown to decrease after an-tiretroviral treatment. Blood levels of 16S ribosomal DNA could be regarded as an indicator of the translocation of microbial molecules from the gut lumen to other body compartments. This could be responsible for chronic activation of the immune system and could have a role in AIDS progression.

Viremic Dengue virus infections in travellers: Potential for local outbreak in Northern Italy

Dengue virus infection is a growing global problem and reports of outbreaks in Asia and Latin America as well as sporadic infections in international travellers are increasing. Two imported cases of serotype-1 Dengue virus infection in a Northern Italy region with high density of Aedes albopictus vector are described: a 27-year-old man returning from Bali and a 25-years-old woman returning from Brazil. In both patients, viremia lasted for several days before disappearance. These cases stress the importance of investigating Dengue virus infections in febrile travellers, point to the potential for local outbreak of Dengue virus infection and emphasize the necessity of maintaining surveillance in non-endemic countries.

Liver fibrosis, microbial translocation and immune activation markers in HIV and HCV infections and in HIV/HCV co-infection

BACKGROUND Liver fibrosis is accelerated in patients co-infected with human immunodeficiency virus and hepatitis C viruses. AIMS We investigated the correlation between liver fibrosis, immune activation and microbial translocation. METHODS This cross-sectional study included patients with hepatitis C virus (HCV) and human immunodeficiency virus (HIV) mono-infections, HIV/HCV co-infection, and healthy controls (20 subjects/group). Peripheral blood was analysed to determine the levels of Forkhead box 3 (Foxp3) T cells, TGF-β1, CD14 (soluble and surface isoforms), IL-17 and bacterial translocation products. These measurements were correlated to the severity of liver fibrosis, measured with the FIB-4 score and transient elastography. RESULTS Foxp3T cell levels were significantly elevated in HIV mono-infected and co-infected groups (p<0.0005). FIB-4 and liver stiffness values inversely correlated with TGF-β1 (p=0.0155 and p=0.0498). Bacterial DNA differed significantly in the HIV-positive compared to the other groups: HIV/HCV co-infected subjects had significantly higher serum levels of bacterial translocation products, CD14, and IL-17 levels (p<0.001). CONCLUSIONS Fibrosis stage in HIV/HCV co-infection may be influenced by immune activation due either by viral infections or to bacterial translocation.

Development and persistence of DAA resistance associated mutations in patients failing HCV treatment

BACKGROUND Direct-acting antiviral agents (DAAs) combined with pegylated-interferon (PegIFN) and ribavirin (RBV) are still a standard treatment in patients with genotype 1HCV infection. However, virologic response could be impaired by baseline or early selection of resistant HCV strains. OBJECTIVES The aim of this study was to determine the onset and persistence of resistance-associated mutations (RAMs) in the NS3 and NS5B genes of DAA-naïve patients failing treatment. STUDY DESIGN Direct sequencing of HCV NS3 was performed in 49 DAA-naïve patients with HCV genotype 1 infection. RESULTS Eight out of 23 patients (34.7%) failed PegIFN/RBV/telaprevir during the 12-weeks of therapy. Treatment failure was associated with the development of RAMs at amino-acids 36,54,80 and 155 of the HCV protease in 6/8 patients (75%). Among patients treated with PegIFN/RBV/boceprevir treatment, 4/18 (22.2%) failed therapy. Of these, 2 (50%) carried virus strains which developed a RAM at amino-acids 54 and 155. Among HCV strains with RAMs, 7 belonged to genotype 1a and 1 to 1b. Finally, in 6/10 (60%) patients, drug-resistant variants could still be detected for up to 3-7 months after stopping therapy. CONCLUSIONS A higher rate (p=0.49) of treatment failure was observed in patients receiving telaprevir- compared to the boceprevir-based combination. In addition, compared with genotype 1b, genotype 1a was associated with higher rates (p=0.01) of treatment failure due to virus resistant strains. Resistance testing at baseline and during DAA treatment should be taken into consideration when treating patients with new HCV combination therapies.

Development of a broad-range 23S rDNA real-time PCR assay for the detection and quantification of pathogenic bacteria in human whole blood and plasma specimens

Molecular methods are important tools in the diagnosis of bloodstream bacterial infections, in particular in patients treated with antimicrobial therapy, due to their quick turn-around time. Here we describe a new broad-range real-time PCR targeting the 23S rDNA gene and capable to detect as low as 10 plasmid copies per reaction of targeted bacterial 23S rDNA gene. Two commercially available DNA extraction kits were evaluated to assess their efficiency for the extraction of plasma and whole blood samples spiked with different amount of either Staphylococcus aureus or Escherichia coli, in order to find the optimal extraction method to be used. Manual QIAmp extraction method with enzyme pre-treatment resulted the most sensitive for detection of bacterial load. Sensitivity of this novel assay ranged between 10 and 10(3) CFU per PCR reaction for E. coli and S. aureus in human whole blood samples depending on the extraction methods used. Analysis of plasma samples showed a 10- to 100-fold reduction of bacterial 23S rDNA in comparison to the corresponding whole blood specimens, thus indicating that whole blood is the preferential sample type to be used in this real-time PCR protocol. Our results thus show that the 23S rDNA gene represents an optimal target for bacteria quantification in human whole blood.