Prevalence of resistance-associated substitutions to NS3, NS5A and NS5B inhibitors at DAA-failure in hepatitis C virus in Italy from 2015 to 2019.

Despite the high efficacy of direct-acting antivirals (DAAs), the selection of resistance-associated substitutions (RASs) after virological failure of hepatitis C virus (HCV) DAAs can impair the cure of chronic HCV. The aim of the study was to characterize RASs after virological failure of DAAs in Italy over the years. Within the Italian network VIRONET-C, the change in prevalence of NS3/4A-NS5A-NS5B RASs was retrospectively evaluated in patients who failed a DAA regimen over the years 2015-2019. NS3, NS5A and NS5B Sanger sequencing was performed using homemade protocols and the geno2pheno system was used to define HCV-genotype/subtype and predict drug resistance. The changes in the prevalence of RASs over time were evaluated using the chi-square test for trend. Predictors of RASs at failure were analysed by logistic regression. Among 468 HCV-infected patients, HCV genotype 1 was the most prevalent (1b in 154, 33% and 1a in 109, 23%). DAA regimens were: ledipasvir (LDV)/sofosbuvir (SOF) in 131 patients (28%), daclatasvir (DCV)/SOF in 109 (23%), ombitasvir/paritaprevir/ritonavir+dasabuvir (3D) in 89 (19%), elbasvir (EBR)/grazoprevir (GRZ) in 52 (10.5%), velpatasvir (VEL)/SOF in 53 (11%), glecaprevir (GLE)/pibrentasvir (PIB) in 27 (6%) and ombitasvir/paritaprevir/ritonavir (2D) in 7 (1.5%); ribavirin was administered in 133 (28%). The NS5A fasta sequence was available for all patients, NS5B and NS3/4A both for 93%. The prevalence of NS5A and NS3/4A RASs significantly declined from 2015 to 2019; NS5B RAS remained stable. Independent predictors of any RASs included older age and genotype 1a (vs G2 and vs G4). Notably, at least partial susceptibility to all the agents included in the GLE/PIB and VEL/SOF/Voxilaprevir (VOX) combinations was predicted in >95% of cases. As RASs remain common at the failure of DAAs, their identification could play a crucial role in optimizing re-treatment strategies. In Italy RAS prevalence has been decreasing over the years and susceptibility to the latest developed drug combinations is maintained in most cases.

Analysis of genetic and viral determinants of HBsAg levels in patients with chronic HBV infection.

Single nucleotide polymorphisms (SNPs) in the interleukin 28B (IL28B) gene can influence the course of treated and untreated HBV infection. However, the correlation between different IL28B-SNPs and HBVDNA and quantitative HBsAg (qHBsAg) in chronic HBV infection remains to be fully elucidated. Patients with chronic HBV infection were analysed for qHBsAg, HBVDNA, HBV genotype and six IL28B-SNPs (rs12980275, rs8105790, rs8099917, rs7248668, rs12979860, rs10853728). Seventy patients were recruited: 80% Caucasian, 56% genotype D, 44% treated with nucleos(t)ide analogues, 11% cirrhotic, 37% inactive carriers (IC). Median (IQR) qHBsAg and HBVDNA were 3.2 log10 IU/ml (2.2-3.9) and 2.2 log10 IU/ml (0.3-3.3), respectively. Lower levels of qHBsAg were associated in the whole study population with rs12979860 CC vs. CT (p=0.05), rs12980275 AA vs. AG (p=0.04), rs8105790 TT vs. CT (p=0.05) and genotype D vs. A+E (p=0.01). rs8105790 TT was present in 81% of IC vs. 46% non-IC (p=0.005). These data were also confirmed in the untreated patients' subgroup. In multivariate analysis, IL28B-SNP haplogroups were associated with lower qHBsAg: CC/AA at rs12979860/rs12980275 (-0.70 log IU/mL, 95% CI -1.26;-0.14; p=0.01), CC/TT at rs12979860/rs8105790 (-0.78 log IU/mL, 95% CI -1.33;-0.23; p=0.006) and AA/TT at rs12980275/rs8105790 (-0.71 log IU/mL, 95% CI -1.27;-0.17; p=0.01) both in the whole population and in the untreated subgroup. Specific IL28-SNP haplogroups might be associated with lower qHBsAg.

Hepatitis C Genotype 4 Virus Nonstructural 3 and Nonstructural 5A Resistance-associated Substitutions in a 16-year-old Adolescent Failing Ombitasvir/Paritaprevir/Ritonavir Plus Ribavirin.

Preexistence and appearance of resistance-associated substitutions limit the efficacy of direct-acting antivirals in treatment of hepatitis C. This is the first case report of an adolescent with chronic hepatitis C virus genotype 4 infection and cirrhosis who failed treatment with ombitasvir/paritaprevir/ritonavir and ribavirin. Resistance analysis showed baseline resistance-associated substitutions M28V and Y93C and emergent D168H.

Two Distinct Hepatitis C Virus Genotype 1a Clades Have Different Geographical Distribution and Association With Natural Resistance to NS3 Protease Inhibitors.

Background. Hepatitis C virus (HCV) genotype 1 is the most prevalent worldwide. Subtype 1a, compared with 1b, shows lower response rates and higher propensity to select for drug resistance to NS3 and selected NS5A and nonnucleoside NS5B inhibitors. Two distinct clades of subtype 1a have been described. Methods. Using Bayesian methodology, we performed a time-scaled phylogeny reconstruction of clade separation and characterized the geographic distribution, phylodynamics, and association with natural resistance variants of NS3 sequences from 362 patients carrying subtype 1a HCV. Results. All sequences segregated in 2 clearly distinct clades. Clade I showed an earlier origin from the common ancestor compared with clade II. Clade I virus was more prevalent in non-European countries, represented mostly by United States, compared with European (75.7% vs 49.3%; P < .001). The prevalence of the natural NS3 variant Q80K, associated with resistance to the macrocyclic protease inhibitor simeprevir, was detected in 51.6% of clade I and 0% of clade II (P < .001); clade I showed a lower genetic barrier for Q80K, whereas no sign of selective pressure at any protease inhibitor resistance-associated codon was detected. Conclusions. Hepatitis C virus subtype 1a clades have a clearly different distribution in Europe and the United States, and the natural resistance mutation Q80K is exclusively associated with clade I.

Stability of unfrozen whole blood DNA for remote genotypic analysis of HIV-1 coreceptor tropism.

BACKGROUND: Maraviroc is an HIV-1 coreceptor antagonist that has shown good efficacy and tolerability in treatment-naive and treatment-experienced patients harboring CCR5-tropic virus. The use of Maraviroc in treatment simplification in patients with suppressed plasma HIV-1 RNA requires analysis of HIV-1 DNA. Coreceptor tropism testing is often performed remotely at reference laboratories. In this study paired whole blood stored at + 4 °C and at-20°C were compared as a source for genotypic coreceptor tropism testing. METHODS: Two hundred paired whole blood samples from different patients were analysed. Each sample was stored in two different conditions: one aliquot was stored at-20 °C until spin column DNA extraction (WB20) and one aliquot was stored at +4°C for two weeks and then placed at room temperature (22-24 °C) for two days before DNA extraction (WB4). Subsequently, a fragment encompassing the HIV-1 gp120 V3 domain was amplified by a singlicate nested PCR followed by triplicate nested PCR in the negative samples. A randomly selected panel of 20 paired WB4 and WB20 duplicate amplification products were sequenced and coreceptor tropism was inferred by geno2pheno [coreceptor]. RESULTS: WB20 yielded a higher amount of DNA than WB4 (median [IQR] values 332.5 ng/µl [117.5-401] and 107 ng/µl [56.6-318], respectively; P < 0.001). However, the DNA purity was higher for WB4 than for WB20 (median distance from the optimal OD260/280 ratio, 0.14 [0.07-0.79] and 0.96 [0.36-1.10], respectively; P < 0.0001). The number of samples successfully amplified was 152 (76.0%) for WB20 and 155 (77.5%) for WB4 with the first PCR and 179 (89.5%) for WB20 and 181 (90.5%) for WB4 (P = ns) following subsequent triplicate analysis. The inferred coreceptor tropism was concordant in 18 out of 20 paired WB4 and WB20 samples. Two samples yielded discordant results, consistent with the discordance rate within duplicates from the same sample source (2/20 with WB4 and 1/20 with WB20) due to the inherent gp120 V3 variability. CONCLUSIONS: Storing whole blood at +4°C for up to two weeks and shipping at room temperature is a convenient method for obtaining HIV-1 gp120 V3 sequence information via testing at a remote laboratory in patients with suppressed viremia.

Naturally occurring hepatitis C virus (HCV) NS3/4A protease inhibitor resistance-related mutations in HCV genotype 1-infected subjects in Italy.

OBJECTIVES: To assess the prevalence of hepatitis C virus (HCV) NS3/4A protease inhibitor (PI) resistance mutations in HCV genotype 1-infected PI-naive individuals in Italy. PATIENTS AND METHODS: One hundred and twelve patients infected with HCV genotype 1a or 1b (based on Versant HCV Genotype 2.0 or 5'UTR/core sequencing) and never treated with any HCV PI were evaluated. The whole NS3 region was analysed by population sequencing and mutations related to resistance to linear and macrocyclic PIs were recorded. RESULTS: Forty-six HCV-monoinfected and 66 HCV/HIV-coinfected subjects were studied. Complete NS3 sequence information was obtained for 109 (97.3%) samples: 67 subtype 1a and 42 subtype 1b. Subtype assignment by NS3 sequencing was concordant in 100.0% and 83.9% of cases with the original 5'UTR sequencing and Versant result, respectively. At least one mutation related to PI resistance was detected in 21 (19.3%) isolates. However, 11 of these had only Q80K, expected to confer resistance to one investigational macrocyclic compound, and were detected only in subtype 1a. Boceprevir and telaprevir resistance-related mutations were detected in 10 (9.2%) isolates and included V36L, T54S and V55A. Only one isolate harboured two mutations (V36L and T54S). There was no association between HCV PI resistance and HIV coinfection or exposure to HIV PIs. CONCLUSIONS: A minority of untreated HCV genotype 1 patients in Italy harbour a virus population carrying HCV PI resistance-related mutations. The clinical implications of this finding warrant further analysis.

Prevalence of skin allograft discards as a result of serological and molecular microbiological screening in a regional skin bank in Italy.

BACKGROUND: Postmortem skin is widely used in the treatment of patients with severe burns. Skin specimens must be screened for transmissible agents including human immunodeficiency virus (HIV), hepatitis B (HBV) and C (HCV) virus, human T-cell lymphotropic virus (HTLV), cytomegalovirus (CMV) and Treponema pallidum. METHODS: Four hundred and sixty-one cadaveric donors underwent serological and molecular microbiological (polymerase chain reaction, PCR) screening at Siena Skin Bank between 2000 and 2004. RESULTS: 74/461 donors (16.1%) were found ineligible under current regulations. CONCLUSIONS: These results are interesting in a local context and underline the importance of screening involving both routine serological procedures and molecular microbiological investigation. The latter has not been uniformly introduced in many countries and very limited data is available to assess its cost-benefit ratio in the field of skin donor screening.

Structurally driven selection of human hepatitis C virus mimotopes.

A structural genomics approach is proposed for the development of new diagnostic kits. It combines molecular modelling, peptide synthesis and immunological tests. The preliminary step is the development of a reliable three-dimensional structure of an immunodominant protein of the target pathogenic organism using the various bioinformatic strategies that are now available to structural biologists. Once the protein structure is obtained, the most surface-exposed fragments with minimal sequence variability among the different strains reported in the genomic data bank are reproduced synthetically as linear peptides. These peptides are then tested for immunoreactivity with the plasma of infected patients to determine whether the synthetic molecules have antigenic activity and can therefore be used to detect infecting agents. This structurally driven selection of mimotopes was successfully performed for the human hepatitis C virus, as five peptides that specifically interact with the plasma of HCV-infected patients were identified solely on the basis of the three-dimensional structure predicted for the E2 homodimer of the la viral subtype. A similar approach could easily be extended to a large variety of immunogenic proteins from other pathogenic organisms.