[Show abstract][Hide abstract] ABSTRACT: Drug metabolism via the cytochrome P450 (CYP450) system has emerged as an important determinant in the occurrence of several drug interactions (adverse drug reactions, reduced pharmacological effect, drug toxicities). In particular, CYP3A4 and CYP3A5 (interacting with more than 60% of licensed drugs) exhibit the most individual variations of gene expression, mostly caused by single nucleotide polymorphisms (SNPs) within the regulatory region of the CYP3A4 and CYP3A5 genes which might affect the level of enzyme production.In this study, we sought to improve the performance of sensitive screening for CYP3A polymorphism detection in twenty HIV-1 infected patients undergoing lopinavir/ritonavir (LPV/r) monotherapy.
BMC Medical Genetics 07/2014; 15(1):76. · 2.54 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: To evaluate the replication capacity and phenotypic susceptibility to dolutegravir and raltegravir of wild-type and raltegravir-resistant HIV-1 strains in several cellular systems.
Journal of Antimicrobial Chemotherapy 05/2014; · 5.34 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The spectrum of HIV-1 cellular reservoirs is highly diversified, and their role varies according to the milieu of the anatomical sites in which the virus replicates. In this light, mechanisms underlying HIV-1 persistence in anatomical compartments may be profoundly different from what is observed in peripheral blood. This scenario is further complicated by sub-optimal drug penetration in tissues allowing persistent and cryptic HIV-1 replication in body districts despite undetectable viremia. On this basis, this review aims at providing recent insights regarding the critical role of HIV-1 cellular reservoirs in different anatomical compartments, and their relationship with the pathogenesis of HIV-1 infection. A comprehensive definition of the complex interplay between the virus and its reservoir is critical in order to set up prophylactic and therapeutic strategies aimed at achieving the maximal virological suppression and hopefully in the near future the cure of HIV-1 infection (either functional or biological).
[Show abstract][Hide abstract] ABSTRACT: Aims: Because of the extreme genetic variability of HCV, we analyzed the NS5B polymerase genetic variability in circulating HCV genotypes/subtypes and its impact on the genetic barrier for the development of resistance to clinically relevant NIs/NNIs.Methods: The study includes 1145 NS5B-polymerase sequences retrieved from Los-Alamos HCV database and Genbank. Genetic barrier was calculated for drug-resistance emergence. Prevalence and genetic barrier was calculated for 1 major-NI- and 32 NNI-resistance variants (13 major and 19 minor) at 21 total NS5B positions. Docking calculations were used to analyze sofosbuvir affinity towards the diverse HCV-genotypes.Results: Overall, NS5B polymerase was moderately conserved among all HCV-genotypes, with 313/591 amino-acid residues (53.0%) showing ≤1% variability and 83/591 residues (14.0%) with high variability (≥25.1%). Nine NNIs-resistance variants (2-major: 414L-423I; 7-minor: 316N-421V-445F-482L-494A-499A-556G) were found as natural polymorphisms in selected genotypes. In particular, 414L and 423I were found in HCV-4 (N=14/38, 36.8%) and in all HCV-5 sequences (N=17, 100%), respectively.Regardless of HCV-genotype, 282T major-NI-resistance variant and 10 major-NNIs-resistance variants (316Y-414L-423I/T/V-448H-486V-495L-554D-559G) always required a single nucleotide substitution to be generated. Conversely, the other 3 major-NNI-resistance variants (414T-419S-422K) were associated with a different genetic barrier score development among the six HCV-genotypes. Sofosbuvir docking analysis highlighted a better ligand affinity towards HCV-2 in comparison to HCV-3, in agreement with the experimental observations.Conclusion: The genetic variability among HCV-genotypes, particularly with the presence of polymorphisms at NNI-resistance positions, could affect their responsiveness to NS5B-inhibitors. A pre-therapy HCV-NS5B sequencing could help to individualize patients with full efficacy of NNIs-containing regimens.
Antimicrobial Agents and Chemotherapy 03/2014; · 4.57 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Background. We evaluated reliability and clinical usefulness of genotypic-resistance- testing (GRT), in patients failing combination-antiretroviral- therapy (cART) with viremia levels 50-1000 copies/mL, for whom GRT is generally not recommended by current guidelines. Methods. Genotyping-success-rate was evaluated in 12828 HIV-1 plasma-samples with viremia >50 copies/mL, tested using the commercial ViroSeq HIV-1 Genotyping-System or a homemade-system. Samples were stratified in 6 groups according to different viremia-levels (50-200; 201-500; 501-1000; 1001-10000; 10001-100000; >100000 copies/mL). Phylogenetic analysis was performed to test the reliability and reproducibility of the GRT also at low-viremia-levels. Drug-resistance was evaluated in 3895 samples from 2200 treatment-failing-patients (viremia >50copies/mL) by considering the resistance-mutations panelled in the IAS list (2013). Results. Overall, success rate of amplification/sequencing was 96.4%. Viremia-levels of 50-200 and 201-500 copies/mL afforded success rates of 67.2% and 88.1%, respectively, reaching 93.2% at 501-1000 copies/mL and ≥97.3% above 1000 copies/mL. Phylogenetic analysis revealed a high homology among sequences belonging to the same subject for 96.4% of patients analyzed. The overall resistance prevalence was 74%. Drug-resistance was commonly found also at low-viremia-levels. Detection of at least one resistance-mutation was: 50-200 copies/mL=52.8%; 201-500=70%; 501-1000=74%; 1001-10000=86.1%; 10001-100000=76.7%; >100000=63% (P<0.001). Similar bell-shaped results were found when the GRT-analysis was restricted to 2008-2012, though at slightly lower prevalence. Conclusions. In patients failing cART with viremia-levels 50-1000 copies/mL, HIV-1 genotyping provides reliable and reproducible results, that are informative about emerging drug-resistance also at low-viremia-levels. Results may be helpful for the therapy optimization in patients under virological-failure, to decrease the risk of virological failures with drug-resistance accumulation.
[Show abstract][Hide abstract] ABSTRACT: During these last two years less drug regimens (LDRs) in HIV, and in particular protease inhibitor (PI)/r-based strategies, have been explored both in clinical trials and in clinical practice. Many results are now available and more is known about how to use them safely and effectively. Understanding that an LDR strategy represents a real tailored therapeutic approach for the patient is crucial for the long-term success and positive management of HIV infection. Trust between patients and HIV specialists and a real focus on the patient's life are key factors for long life treatment success, in particular when using a LDR strategy. This is clearly shown by the HIV patient's journey (HPJ) methodology, used in an Italian national workshop to better define the criteria and challenges of LDR strategies. This paper shows the results of this complex process.
[Show abstract][Hide abstract] ABSTRACT: Novel respiratory viruses have been identified as possible agents of upper and lower respiratory tract infections. Multiplex real-time PCRs have been developed to identify clinically relevant respiratory pathogens. In this study, 178 respiratory samples already screened for influenza virus types A and B by Flu A/B ASR real-time PCR kit were retrospectively analyzed with the Respiratory Multi Well System (MWS) r-gene™ real-time PCR kit which detects a wide spectrum of respiratory pathogens. The goal was to demonstrate the importance of a wide spectrum screening compared to a single diagnostic request. The Flu A/B ASR kit detected influenza B virus in 1.7% of the samples (3/178) and no influenza A virus. The MWS r-gene™ kit detected influenza virus in 6.7% (12/178) of samples (0.6% influenza A, and 6.2% influenza B), while the overall detection rate for respiratory pathogens was 54% (96/178). Co-infections were detected in 8/178 (4.5%) samples. Adenovirus was the infectious agent detected most frequently, followed by respiratory syncytial virus. The risk of being infected by respiratory syncytial virus is almost threefold higher in patients older than 65 years compared to the younger age group (OR:2.7, 95% CI: 1.2-6.2). Wide spectrum screening of respiratory pathogens by real-time PCR is an effective means of detecting clinically relevant viral pathogens.
[Show abstract][Hide abstract] ABSTRACT: We have developed a sequencing assay for determining the usage of the genotypic HIV-1 co-receptor using peripheral blood mononuclear cell (PBMC) DNA in virologically suppressed HIV-1 infected patients. Our specific aims were to (1) evaluate the efficiency of V3 sequences in B versus non-B subtypes, (2) compare the efficiency of V3 sequences and tropism prediction using whole blood and PBMCs for DNA extraction, (3) compare the efficiency of V3 sequences and tropism prediction using a single versus a triplicate round of amplification.
The overall rate of successful V3 sequences ranged from 100 % in samples with >3,000 copies HIV-1 DNA/10(6) PBMCs to 60 % in samples with <100 copies total HIV-1 DNA /10(6) PBMCs. Analysis of 143 paired PBMCs and whole-blood samples showed successful V3 sequences rates of 77.6 % for PBMCs and 83.9 % for whole blood. These rates are in agreement with the tropism prediction obtained using the geno2pheno co-receptor algorithm, namely, 92.1 % with a false-positive rate (FPR) of 10 or 20 % and of 96.5 % with an FPR of 5.75 %. The agreement between tropism prediction values using single versus triplicate amplification was 98.2 % (56/57) of patients using an FPR of 20 % and 92.9 % (53/57) using an FPR of 10 or 5.75 %. For 63.0 % (36/57) of patients, the FPR obtained via the single amplification procedure was superimposable to all three FPRs obtained by triplicate amplification.
Our results show the feasibility and consistency of genotypic testing on HIV-1 DNA tropism, supporting its possible use for selecting patients with suppressed plasma HIV-1 RNA as candidates for CCR5-antagonist treatment. The high agreement between tropism prediction by single and triple amplification does not support the use of triplicate amplification in clinical practice.
[Show abstract][Hide abstract] ABSTRACT: HIV resistance affects virological response to therapy and efficacy of prophylaxis in mother-to-child-transmission. The study aims to assess the prevalence of HIV primary resistance in pregnant women naive to antiretrovirals.
Cross sectional baseline analysis of a cohort of HIV + pregnant women (HPW) enrolled in the study entitled Antiretroviral Management of Antenatal and Natal HIV Infection (AMANI, peace in Kiswahili language). The AMANI study began in May 2010 in Dodoma, Tanzania. In this observational cohort, antiretroviral treatment was provided to all women from the 28th week of gestation until the end of the breastfeeding period. Baseline CD4 cell count, viral load and HIV drug-resistance genotype were collected.
Drug-resistance analysis was performed on 97 naive infected-mothers. The prevalence of all primary drug resistance and primary non-nucleoside reverse-transcriptase inhibitors resistance was 11.9% and 7.5%, respectively. K103S was found in two women with no M184V detection. HIV-1 subtype A was the most commonly identified, with a high prevalence of subtype A1, followed by C, D, C/D recombinant, A/C recombinant and A/D recombinant. HIV drug- resistance mutations were detected in A1 and C subtypes.
Our study reports an 11.9% prevalence rate of primary drug resistance in naive HIV-infected pregnant women from a remote area of Tanzania. Considering that the non-nucleoside reverse-transcriptase inhibitors are part of the first-line antiretroviral regimen in Tanzania and all of Africa, resistance surveys should be prioritized in settings where antiretroviral therapy programs are scaled up.
[Show abstract][Hide abstract] ABSTRACT: To investigate the possible relationship between human papillomavirus (HPV) infection and recurrent miscarriage (RM).
In this retrospective case-control study, 49 women with unexplained RM (Group 1 - cases) and 475 women without any miscarriage and with at least one pregnancy at term (Group 2 - controls) were checked for cervical HPV infection through Hybrid Capture(®) II (HC 2) or polymerase chain reaction (PCR).
HPV+ DNA tests were detected in 13 (26.53%) RM women and in 294 (61.89%) control women (P < 0.001). The prevalence rate in HPV+DNA tests was significantly different in the 30-39 years age range. No differences between groups were detected in HPV types, nor in the cytological and histological findings.
Women with RM have a lower prevalence of HPV+DNA tests than controls. This suggests that immune reactivity potentially leading to RM could be in some way protective against genital HPV infection.
American Journal Of Reproductive Immunology 09/2013; · 3.32 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The main objective is to evaluate the efficacy and durability of Lopinavir-ritonavir monotherapy (LPV/r-MT) in virologically-controlled HIV-positive individuals switching from combination antiretroviral therapy (cART).
Criteria to be included in this observational study were to have initiated for the first time LPV/r-MT after >=2 consecutive HIV-RNA ≤50 copies/mL achieved on a >=3 drugs-including regimen. The main end-points were time to virological rebound (VR, defined in two ways: time of first of two consecutive viral load (VL)>50 and >200 copies/ml); time to discontinuation/intensification and time to experience either a single VL >200 copies/ml or discontinuation/intensification (=treatment failure-TF). Individuals' follow-up accrued from the date of starting LPV/r-MT to event or last available VL. Kaplan-Meier curves and Cox regression analysis were used.
228 individuals were included; median age: 46 (IQR: 40-50) years, 36% females, 36% IDU, 25% HCV-co-infected. Median CD4 at nadir: 215 cell/mm3 (IQR:116-336); at baseline: 615 cell/mm3 (IQR: 436-768). By 36 months from switching to LPV/r-MT, the proportion of individuals with VR (confirmed VL>200 cp/ml) was 11% and with TF was 35%. In the multivariable Cox model the factors associated with a lower risk of TF was the duration of viral suppression <50 copies/mL prior to baseline (ARH=0.92, 95% CI:0.85-0.99, p=0.024, per 6 months longer) and having LPV/r as part of last cART (ARH=0.45, 95% CI:0.21-0.95, p=0.037) .
In daily clinical practice, we confirm a relatively safe approach of simplification to LPV-MT in selected population with long-lasting virological control.
[Show abstract][Hide abstract] ABSTRACT: Substantial improvements in the safety of blood and plasma products for the management of bleeding disorders have been achieved in recent decades. This has led some clinicians to believe that the infectious threat is over and that inhibitor formation is the foremost complication of hemophilia therapy. On the contrary, elimination of all microbes from blood is difficult, potentially impossible, and there are always threats from emerging pathogens. The risk of infection transmission is also increasing due to greater exposure to products, increasing prophylaxis and high-dose regimens for immune tolerance, and longevity of hemophilia patients. Current products can be considered "reasonably safe," but pathogen testing is not all-inclusive, and manufacturing and purification techniques are often not standardized. Although safer nonplasma-derived products are widely used, they are not available for all bleeding disorders, and so there is an ongoing need for plasma-derived products. This review will discuss the evolving risk from emerging pathogens in the context of the issues described. Reducing the risk from emerging infections requires global collaboration to devise ways to monitor and continue to improve blood safety.
Seminars in Thrombosis and Hemostasis 09/2013; · 4.22 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The identification of novel reverse-transcriptase (RT) drug-resistance mutations is critical in predicting the probability of success to anti-HBV treatment. Furthermore, due to HBV-RT/HBsAg gene-overlap, they can have an impact on HBsAg-detection and quantification.
356 full-length HBV-RT sequences from 197 drug-naive patients and 159 patients experiencing virological-breakthrough to nucleoside/nucleotide-analogs (NUCs) were analyzed. Mutants and wild-type HBs-antigens were expressed in HuH7-hepatocytes and quantified in cell-supernatants and cell-lysates by Architect HBsAg-assay.
Ten novel RT-mutations (rtN53T-rtS78T-rtS85F-rtS135T-rtA181I-rtA200V-rtK212Q-rtL229V/F-rtM309K) correlated with specific NUC-treatments and classical drug-resistance mutations on divergent evolutionary pathways. Some of them reduced RT-binding affinity for anti-HBV drugs and altered S-antigen structure. Indeed, rtS78T (prevalence: 1.1% in drug-naïve and 12.2% in adefovir-failing patients) decreased the RT-affinity for adefovir more than the classical adefovir-resistance mutations rtA181T/V (WT:-9.63Kcal/mole, rtA181T:-9.30Kcal/mole, rtA181V:-7.96Kcal/mol, rtS78T:-7.37Kcal/mol). Moreover, rtS78T introduced a stop-codon at HBsAg-position 69, and completely abrogated HBsAg-quantification in both supernatants and cell-lysates, indicating an impaired HBsAg-secretion/production. Furthermore, the HBsAg-mutation sP217L, silent in RT, significantly correlated with M204V/I-related virological-breakthrough and increased HBsAg-quantification in cell-lysate.
Mutations beyond those classically known can affect drug-binding affinity of mutated HBV-RT, and may have potential effects on HBsAg. Their cumulative effect on resistance and HBV-pathogenicity indicates the importance of preventing therapeutic failures.
The Journal of infection 06/2013; · 4.13 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: In 2007, two novel polyomaviruses KI and WU were uncovered in the respiratory secretions of children with acute respiratory symptoms. Seroepidemiological studies showed that infection by these viruses is widespread in the human population. Following these findings, different biological specimens and body compartments have been screened by real-time PCR in the attempt to establish a pathogenetic role for KI polyomavirus (KIPyV) and WU polyomavirus (WUPyV) in human diseases. Although both viruses have been found mainly in respiratory tract samples of immunocompromised patients, a clear causative link with the respiratory disease has not been established. Indeed, the lack of specific clinical or radiological findings, the frequent co-detection with other respiratory pathogens, the detection in subjects without signs or symptoms of respiratory disease, and the variability of the viral loads measured did not allow drawing a definitive conclusion. Prospective studies carried out on a large sample size including both immunocompromised and immunocompetent patients with and without respiratory symptoms are needed. Standardized quantitative real-time PCR methods, definition of a clear clinical cutoff value, timing in the collection of respiratory samples, are also crucial to understand the pathogenic role, if any, of KIPyV and WUPyV in human pathology.
[Show abstract][Hide abstract] ABSTRACT: Determination of hepatitis C virus genotype is crucial for establishing the duration of antiviral therapy and predicts response to treatment. In this study, consecutive serum samples collected from two patients with chronic hepatitis C infection were tested by two assays used widely, the Abbott RealTime HCV Genotype II and the Versant HCV Genotype 2.0 assays, in order to assign a genotype to the virus. The obtained results were verified by phylogenetic analysis of the NS5B region and sequencing of the 5'-UTR of the viral genome. Testing of the serum samples from both patients gave an indeterminate result with the Abbott assay. By contrast, The Versant assay gave an indeterminate result for one patient and identified an HCV-2b subtype in the other patient. Phylogenetic analysis of the NS5B region confirmed the presence of HCV-2b in this latter patient and disclosed the presence of HCV-3h in the other patient. Sequencing of the 5'-UTR revealed the presence of nucleotide changes at position -166 and -119 of HCV-2b, and at position -138, -108 and -99 of HCV-3h. Nucleotide mutations located in the 5'-untraslated region of hepatitis C virus may impair the ability of commercial assays to assign an HCV genotype.
Journal of virological methods 06/2013; · 2.13 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: OBJECTIVES: The HIV reverse transcriptase (RT) mutation K65R confers resistance to nucleos(t)ide reverse transcriptase inhibitors (NRTIs). Here, analysing a large database, we report the selection of another rare K65E mutation in patients failing on NRTI-containing regimens. METHODS: Clinical and virological characteristics of patients harbouring the K65E mutation were analysed using a large RT sequence database from treatment-experienced individuals. Structural analysis of the K65E RT mutant complex was performed by means of docking simulations. The replication capacity was assessed using viruses harbouring the K65E mutation introduced by site-directed mutagenesis (SDM) in pNL 4-3. RESULTS: Overall, in 23 530 sequences from patients failing on antiretroviral therapy, the prevalence of substitutions at position K65 in RT was 2.4%. In addition to K65R (n = 395) and K65N (n = 9), another mutation, K65E, was found in 15 patients. In 11 out of 15 cases, tenofovir, abacavir, didanosine or stavudine were present at the time of K65E selection. The molecular recognition of RT containing K65E supports evidence for the role of this mutation in resistance to tenofovir. The SDM pNL4-3 K65E variant harboured a very low replicative capacity (5% versus wild-type). CONCLUSIONS: We investigated the role of a novel rare NRTI mutation located at position Lys65 of RT (K65E), found in drug-experienced patients failing on NRTIs. The low frequency of this mutation is probably related to the high impairment of replicative capacity induced by this mutation. This study should have significant clinical implications, as these findings warn clinicians that other minor substitutions at Lys65 (such as K65E) play a role in NRTI resistance.
Journal of Antimicrobial Chemotherapy 06/2013; · 5.34 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Here we summarize the discussions and conclusions from an expert workshop held in October 2012 to consider the implications of HIV drug resistance in the context of scale-up of access to antiretroviral therapy and prophylaxis in resource-limited settings. Topics considered during the workshop included the implications of drug resistance for the selection of first-line regimens and sequencing of treatments, optimal surveillance strategies and prevention of mother-to-child transmission.