Francesca Falasca

Expression levels of MDR1, MRP1, MRP4, and MRP5 in peripheral blood mononuclear cells from HIV infected patients failing antiretroviral therapy

The aim of the study was to evaluate the mRNA expression of four relevant ABC‐transporter genes [MDR1 (P‐glycoprotein; Pgp), MRP1, MRP4, and MRP5] in HIV‐positive individuals failing treatment and analyze the association between the levels of their expression and viral load, CD4 cell count, and therapeutic history. Ninety‐eight HIV‐positive samples and 20 samples from healthy donors were analyzed, retrospectively. Peripheral blood mononuclear cells (PBMCs) from HIV1‐positive individuals were collected at the time of virological failure. Expression of mRNA of Pgp, MRP1, MRP4, and MRP5 in PBMCs was evaluated by real‐time PCR. A high inter‐individual variability was observed in both HIV‐positive individuals and healthy donors but the expression levels of all mRNA analyzed were significantly higher in the HIV‐infected group (P < 0.05). A weak but significant inverse correlation was observed between CD4 cell counts and expression levels of MRP4 and MRP5. Comparison of mRNA expression between individuals with different therapeutic histories showed that expression of MRP4 and MRP5 genes in patients who were both protease inhibitor (PI) and non‐nucleoside reverse transcriptase inhibitor (NNRTI)‐experienced was significantly higher than in patients who were PI experienced but NNRTI‐naïve. In conclusion, the mRNA expression of Pgp, MRP1, MRP4, and MRP5 varies among HIV‐infected patients and healthy donors but is significantly higher in HIV‐positive patients than in donors. The expression of MRP4 and MRP5 seems to correlate with CD4 cell counts. The same protein seems to be overexpressed in patients receiving NNRTIs. J. Med. Virol. 80:766–771, 2008.

Interleukin-32 isoforms: Expression, interaction with interferon-regulated genes and clinical significance in chronically HIV-1-infected patients

Given the growing evidence for a role of interleukin-32 (IL-32) in the immune response to HIV-1 infection and its interplay with type I and III interferons (IFNs), we studied the gene expression of IL-32 isoforms (α and nonα) in untreated chronically HIV-1-infected patients and in gender- and age-matched healthy individuals. To further characterize both the anti-HIV properties of IL-32 and the cytokine’s relationship with host antiviral innate immune responses, we evaluated whether IL-32 can induce ex vivo the expression of antiviral IFN-induced genes (ISGs), namely myxovirus resistance A (MxA), and apolipoprotein B mRNA-editing enzyme catalytic (APOBEC)3G and APOBEC3F. We also investigated whether in vivo IL-32 (α and nonα) mRNA levels were correlated with those of MxA and APOBEC3G/3F. Results indicated that IL-32 (α and nonα) mRNA levels were significantly higher in HIV-1-infected patients than in healthy individuals. Furthermore, IL-32 (α and nonα) mRNA levels correlated negatively with HIV RNA levels, but not with the CD4+ T-cell count. Our ex vivo studies disclosed that ISGs mRNA levels were increased after IL-32γ treatment of peripheral blood mononuclear cells. Interestingly, significant positive correlations were found between transcript levels of both IL-32α and IL-32nonα and those of MxA and APOBEC3G/3F in untreated chronically HIV-1-infected patients. Overall, our results demonstrated that IL-32 isoforms are highly expressed during chronic HIV-1 infection and that IL-32 could have a central role in the antiviral immune response against HIV-1.

MicroRNA-29 family expression and its relation to antiviral immune response and viro-immunological markers in HIV-1-infected patients

BackgroundSeveral in vitro studies suggested the microRNA-29 (miRNA-29) family is involved in regulating HIV-1 and modulating the expression of interleukin (IL)-32, an anti-HIV-1 cytokine.MethodsTo investigate the contribution of the miRNA-29 family to HIV-1 infection in vivo, we compared miRNA-29 expression in PBMC collected from 58 HIV-1-infected patients, naïve for antiretroviral therapy, and 21 gender- and age-matched HIV-1 seronegative healthy donors, using RT-Taqman assays. The relation between miRNA-29 levels and HIV-1 viro-immunological markers and the activation rate of antiviral immune response were also evaluated. In addition, we profiled miRNA-29 expression in CD4+ T lymphocytes and CD14+ monocytes collected from 5 antiretroviral treated HIV-1 infected patients.ResultsmiRNA-29b levels were higher in HIV-1-infected patients than in the control group (p < 0.001). There were no correlations with either HIV-1 RNA levels or CD4+ T count, whereas a significant correlation was found between miRNA-29-a/c levels and integrated HIV-1 DNA (miRNA-29a: p = 0.009, r = −0.448; miRNA-29c: p = 0.029; r = −0.381). When the HIV-1-infected patients were grouped on the basis of their plasma HIV-1 RNA and CD4+ T cell count, we also found that patients expressing the lowest levels of miRNA-29c showed high viraemia, low CD4+ T cell count and high levels of integrated HIV-1 DNA. Moreover, miRNA-29b levels were correlated with those of IL-32nonα (p = 0.028; r = −0.298). Patients expressing higher levels of miRNA-29b showed lower levels of MxA, an interferon-stimulated gene, also induced by IL-32 (p = 0.006 r = −0.397). Lastly, we found that CD4+ T lymphocytes and CD14+ monocytes shared similar miRNA-29a/b/c expression patterns but the amount of miRNA-29a/b/c, IL-32 isoforms and MxA were highly variable in these two cellular subsets.ConclusionsThe miRNA-29 family could influence the clinical progression of HIV-1 infection, the HIV-1 proviral load and the innate immune response against HIV-1.

Early collection of saliva specimens from Bell's palsy patients: quantitative analysis of HHV-6, HSV-1, and VZV

Bells palsy is the most common cause of facial paralysis. Although it has been associated with diabetes mellitus, hypertension, pregnancy, and preeclampsia, the etiology of Bells palsy remains unknown. The reactivation of latent herpes simplex virus (HSV) or varicella‐zoster virus (VZV) with subsequent inflammation and entrapment of the facial nerve in the narrow labyrinthine segment has been implicated as a cause of facial paralysis, but the active role of these viruses in Bells palsy is still discussed. This study quantified HSV‐1 DNA, VZV DNA, and HHV‐6 DNA in 95 saliva samples collected from patients within 48 hr from the onset of paralysis. HSV‐1, VZV, and HHV‐6 were detected in 13%, 3%, and 61% of patients, respectively. The detection rate did not differ significantly between patients and a control group of healthy donors. Interestingly, however, the value of HHV‐6 DNA copies was significantly higher than that detected in healthy donors. In addition, the mean value of HHV‐6 DNA recorded in patients who had at least a one grade improvement of palsy at the first visit was significantly lower than that detected in patients who showed no change in facial palsy grade or an increase of at least one grade. These findings call into question the role of HSV‐1 and VZV in the etiology of Bells palsy, and suggest that HHV‐6 may be involved in the development of the disease or that the underlying disease mechanism might predispose patients to HHV‐6 reactivation. J. Med. Virol. 86: 1752–1758, 2014.

Mutational Resistance Pattern of HIV Type 1 in CD14+ Monocytes, CD4+ T Cells, and Plasma from Treated Patients

It is necessary to understand the molecular nature of the virus population that persists in cellular reservoirs. To achieve this we planned to characterize the patterns of resistance of HIV-1 in CD14(+) monocytes, CD4(+) T cells, and plasma. Blood samples were collected from 42 patients treated for HIV: 32 were in virological failure and in 10 viremia was undetectable. CD14(+) and CD4(+) T cells were isolated using magnetic beads. Genotyping of the reverse transcriptase and protease gene of HIV-1 was undertaken using the fluorescent dideoxy-terminator method. Of the 32 patients in virological failure, 24 (75%) had resistance mutations in at least one compartment. The numbers and types of mutations from monocytes were the same as those detected in both CD4(+) T cell-associated virus and plasma in only 8% whereas in 71% monocytes exhibited a different mutation pattern. In 21% of patients, the profile of drug-resistant mutations in the virus from blood monocytes was identical to that in plasma but differed from that in CD4. In the 71% of patients with virological suppression, the genotypic resistance pattern differed between monocytes and CD4(+) T cells. Circulating monocytes may harbor a viral dominant population different from those viruses circulating in blood and archived in CD4(+) T cells. Hence, monocytes and other cellular reservoirs might serve as an indirect source of a drug-resistant viral variant.

Trends in drug resistance-associated mutations in a real-life cohort of Italian patients infected with HIV-1

Recent studies support the idea that human immunodeficiency virus type 1 (HIV-1) drug resistance is declining in developed countries. To help assess the current situation in Italy, the dynamics of drug resistance mutations in pol and integrase genes in plasma samples from HIV-1-positive patients attending Sapienza University Hospital, Rome, from 2003 to 2014 were analysed. In total, 1730 genotype resistance tests (GRTs) were retrospectively analysed. The prevalence of major drug resistance mutations (DRMs) was evaluated over time in the global population and in patients with antiretroviral therapy (ART) failure. Population dynamics, changes in ART administration, and HIV-1 RNA levels were analysed in combination with DRM trends. The global population showed a strong reduction in major DRMs to all drug classes. Over the 2003-2014 decade, resistance to nucleoside reverse transcriptase inhibitors (NRTIs), non-nucleoside reverse transcriptase inhibitors (NNRTIs) and protease inhibitors (PIs) declined from 80.0% to 18.7%, from 42.8% to 20.1% and from 74.2% to 8.3%, respectively (P<0.005 for all comparisons). However, only PI-associated mutations showed a significant decrease in patients experiencing ART failure. Interestingly, analysis of the integrase gene disclosed an increased resistance to integrase inhibitors, mainly regarding N155H, detected in 32.6% of raltegravir-treated patients in 2012-2014. In conclusion, in line with previous findings, this study shows that drug resistance is declining in Italy. However, the persistence of DRMs to NRTIs and NNRTIs suggests that despite adherence and treatment optimisation, some patients still experience therapy failure, emphasising the need for GRTs both in naïve and ART-failed patients.

First external quality assurance program of the Italian HLA-B*57:01 Network assessing the performance of clinical virology laboratories in HLA-B*57:01 testing

BACKGROUND Since the HLA-B*57:01 allele is strongly associated with abacavir hypersensitivity reaction, testing for the presence of HLA-B*57:01 is mandatory before administration of abacavir. While HLA-B*57:01 testing is usually provided by pharmacogenetics, genetics or blood transfusion services, clinical virology laboratories can be an optimal opportunity for HLA-B*57:01 testing since they receive blood samples for routine HIV monitoring and have the expertise for convenient and less expensive PCR-based point mutation assays. OBJECTIVES The Italian HLA-B*57:01 Network gathers accredited clinical virology laboratories offering HLA-B*57:01 testing in Italy with the aim to share protocols, test new methods, develop and maintain external quality assurance (EQA) programs. STUDY DESIGN A panel of 9HLA-B*57:01-positive and 16HLA-B*57:01-negative frozen blood samples were blindly distributed to 10 units including 9 clinical virology laboratories and one reference pharmacology laboratory. Each laboratory was free to use its own routine method for DNA extraction and HLA-B*57:01 testing. RESULTS DNA was extracted by automated workstations in 6 units and by manual spin columns in 4. Eight units used the Duplicα Real Time HLA-B*57:01 kit by Euroclone and two units used two different PCR homemade protocols. All the 10 units correctly identified all the 25 samples. CONCLUSIONS The first HLA-B*57:01 EQA program run in Italy showed that clinical virology units are equipped and proficient for providing HLA-B*57:01 testing by inexpensive assays easy to integrate into their routine.

Evaluation of a commercial real-time PCR kit for the detection of the Q80K polymorphism in plasma from HCV genotype 1a infected patients

BACKGROUND Screening of the natural HCV NS3 polymorphism Q80K is required prior to simeprevir administration due to the reduced susceptibility of genotype 1 viruses carrying this amino acid variant. A simple, rapid and robust test for Q80K screening would be advisable in routine diagnostic laboratories. OBJECTIVES The aim of this study was to evaluate a commercial NS3 Q80K real-time PCR kit (Q80K Polymorphism Kit, Clonit srl, Milan, Italy). STUDY DESIGN Forty-three plasma samples obtained from untreated HCV genotype 1a-infected patients and previously sequenced at a reference laboratory, were sent to two public clinical virology laboratories for blinded Q80K screening with the kit under evaluation. The sample panel included 25 cases with the wild type 80Q, 17 with the mutant 80K and 1 with the mutant 80L. RESULTS Laboratory 1 identified 22/25 (88.0%) 80Q and 17/17 (100.0%) 80K cases. Laboratory 2 identified 23/25 (92.0%) 80Q and 16/17 (94.2%) 80K cases. All of the unidentified cases were scored as negative, with no mutant/wild type miscalling. The 80L variant was scored as indeterminate by Laboratory 1 and as negative by Laboratory 2. Overall, sensitivity and specificity for detection of 80K were 97.1% (95% C.I., 82.9-99.8%) and 100.0% (90.2-100.0%), respectively. However, the system did not provide any result for 6/84 cases (7.1% failure rate), not including the 80L variant which is not expected to be detected as stated in the kit package insert. Global inter-laboratory concordance was 93.0%. CONCLUSIONS Despite good specificity, this Q80K detection system needs improvements in amplification success rate and robustness.

IFN-stimulated gene expression is independent of the IFNL4 genotype in chronic HIV-1 infection.

This study aimed to evaluate the association between the IFNL4 rs368234815 (ΔG/TT) dinucleotide polymorphism and the IFN response during chronic HIV-1 infection. We carried out genotyping analysis and measured the expression of IFN-stimulated genes (ISGs) (myxovirus resistance protein A [MxA], ISG15, ISG56, apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like [APOBEC] 3F and APOBEC3G) on peripheral blood mononuclear cells collected from naïve and HAART-treated HIV-1-infected patients. There were no statistically significant differences in endogenous ISGs mRNA levels among HIV-1-positive patients bearing different IFNL4 genotypes, suggesting that ISG expression is independent of the IFNL4 genotype in HIV-1 infection.

Dynamics of HIV DNA and residual viremia in patients treated with a raltegravir-containing regimen.

To the Editors: Current antiretroviral therapy (ART) can reduce plasma HIV-1 RNA levels to below the detection limit of clinical assay (50 copies of HIV-1 RNA), but HIV-1 DNA remains detectable in patients during suppressive ART. HIV-1 persistence despite ART is likely the consequence of a reservoir of latently infected cells established early in infection and cryptic viral replication presumably occurring in anatomical sanctuary sites and facilitated by cell-to-cell transmission. In addition to HIV-1 persistence in T CD4+ cells and other reservoirs, most patients on ART with HIV-1 RNA levels below the detection limit of commercial assays have low-level residual viremia. The origin of residual viremia is uncertain, but it may arise, at least in part, from latently infected cells induced to produce HIV-1. Several studies have addressed the impact of ART on HIV DNA and the relation between the size of the latent reservoir and residual viremia. In particular, the effect of ART on HIV-1 DNA has been studied in patients treated with raltegravir (RAL)-based therapy. Some studies showed that in the presence of RAL, linear HIV-1 cDNA is prevented from integrating into chromatin and is subsequently converted to episomal cDNA. Therefore, an increase in episomal cDNA occurs when active replication is blocked by integrase inhibitors. However, another study found that the switch to an RALcontaining regimen was not associated with a significant change in the levels of HIV-1 2-long terminal repeat (LTR) circles, and no significant changes were observed in HIV-1 total DNA. By contrast, Charpentier et al reported that a RAL-containing regimen in drug-experienced patients was associated with a rapid HIV-1 DNA decrease. All together, these data suggest that the impact of RAL-based therapy on HIV-1 DNA level is still unclear. A number of studies have also evaluated the relationship between HIV1 DNA and residual viremia, finding a positive correlation between these 2 virological parameters. Nevertheless, additional studies are needed to establish whether the association between residual viremia and latent reservoirs persists after long-term suppression of HIV-1 replication. This study analyzed total and integrated HIV-1 DNA levels for up to 196 weeks in patients initiating a RALcontaining regimen, investigating the relationship between residual viremia and the size of the latent reservoir. Specifically, 30 treatmentexperienced HIV-1–infected patients attending Policlinico Umberto I Hospital were retrospectively selected according to the following criteria: (1) receiving a RAL-containing regimen, (2) virological response to RAL-based therapy, (3) follow-up of approximately 196 weeks, and (4) availability of at least 3 peripheral blood mononuclear cell samples collected during follow-up. The main reasons for initiating a RAL-based therapy were virological failure (18 patients) and side effects to the previous therapeutic regimen (12 patients). In addition to RAL, the optimized background therapy contained at least 1 active ARV drug according to the genotypic resistance test. Patients had been infected for a median time of 14 years [interquartile range (IQR), 8–16] and had a long therapeutic history [median: 14 years (IQR, 9–17)]. The median CD4 nadir was 206 cells per microliter (IQR, 113– 700). At baseline, the median CD4 cell count was 380 cells per milliliter (IQR, 152–741). At W196, the median increase in CD4 T-cell count was 124 cells per microliter (IQR, 78–171). At the beginning of the study, the median HIV-1 RNA level was 6777 copies per milliliter (IQR, 1193–14,602). At the end of the follow-up, all participants showed levels of plasma viremia below 37 copies per milliliter (Siemens Healthcare Diagnostic, Tarrytown, NY). Total cell DNA was extracted from frozen peripheral blood mononuclear cells (PBMC) pellets by the DNeasy Blood & Tissue kit (Qiagen, Milan, Italy). Total and integrated HIV1 DNA were quantified by in-house realtime PCR at baseline (W0) and after mean times (6SD) of 52 weeks (68), 96 weeks (612), 144 weeks (616), and 196 weeks (616) from the start of RALbased therapy. Total HIV-1 DNA was quantified using primers (NEC 152 and NEC 131) direct in the LTR gene. Integrated HIV DNA was quantified using an Alu-gag PCR followed by a real-time PCR for LTR gene. At the same time, b-glucuronidase PCR was performed to rule out the presence of inhibitors. To quantify HIV-1 DNA, we used a standard curve (5-fold dilutions of 8E5 cells DNA), and all samples from each patient were tested in triplicate in the same assay. Results were expressed as number of HIV-1 DNA copies/106 PBMC. Plasma HIV-1 RNA loads at W0, W52, and W96 were measured with branched DNA (Siemens Healthcare Diagnostic) showing a dynamic range of 50–500,000 copies per milliliter. At W144 and W196, a more sensitive real-time PCR assay (Versant kPCR; Siemens Healthcare Diagnostic) was used. This assay quantifies the viral load over the range of 37–11,000,000 copies per milliliter and reports qualitative results below the lower limit of quantification as “Target Detected” and Target Not Detected, suggesting the presence or absence of residual viremia. To determine the level of residual viremia, the real-time PCR assay was performed in triplicate for each sample studied to minimize the quantification error associated with the detection of extremely low values. Supported by “Sapienza” University Grant to G.A. Presented at the 24th European Congress of Clinical Microbiology and Infectious Diseases, May 10–13, 2014, Barcelona, Spain. The authors have no conflicts of interest to disclose.