Maria Cristina Rossi

Both Human Immunodeficiency Virus Cellular DNA Sequencing and Plasma RNA Sequencing Are Useful for Detection of Drug Resistance Mutations in Blood Samples from Antiretroviral-Drug-Naive Patients

ABSTRACT Genotypic antiretroviral testing is recommended for newly infected drug-naive subjects, and the material of choice is plasma RNA. Since drug resistance mutations (DRMs) may persist longer in cellular DNA than in plasma RNA, we investigated whether the use of peripheral blood mononuclear cell (PBMC) human immunodeficiency virus (HIV) DNA increases the sensitivity of genotypic testing in antiretroviral-drug-naive subjects. We compared the rate of primary drug resistance in plasma RNA and PBMC DNA in 288 HIV type 1-infected drug-naive persons tested at a single clinical virology center from June 2004 to October 2006. Resistance in the plasma compartment to at least one drug was detected for 64 out of 288 (22.2%) naive patients and in the PBMC compartment for 56 (19.4%) patients. Overall, DRMs were found in 80 out of 288 (27.8%) patients. PBMC DRMs were present in plasma RNA from 16 subjects with wild-type virus infections. Another nine patients had additional DRMs in PBMCs with respect to those detected in plasma RNA. On the other hand, extra plasma DRMs were detected in PBMCs for 24 and 8 subjects with wild-type and drug-resistant virus, respectively. Resistance to more than one class of antiretroviral drug was detected by plasma and PBMC analysis for 25.0% and 36.2% of the subjects, respectively. Our data support the potential utility of genotypic resistance testing of PBMC DNA in conjunction with the currently recommended plasma RNA analysis.

Tumor Necrosis Factor-α Increased Production during Thalidomide Treatment in Patients with Tuberculosis and Human Immunodeficiency Virus Coinfection

To the Editor—We read with interest the article by Bekker et al. [1] on the role of thalidomide-induced antigen-specific immune stimulation in patients with human immunodeficiency virus (HIV) and Mycobacterium tuberculosis coinfection. In their report, it is suggested that thalidomide treatment of HIVinfected patients does not reduce plasma tumor necrosis factor (TNF)‐a levels. The observation is explained by a differential effect of thalidomide on monocyte and T cell TNF-a production. In particular, the authors report that thalidomide inhibited TNF-a production by lipopolysaccharide-stimulated monocytes but failed to inhibit TNF-a production by activated T cells [2‐5]. Finally, the authors found an increase in TNF-a production at day 21 of therapy in the thalidomide group, thus suggesting that the drug could be responsible for this increase by stimulation of T cell activation. As Bekker et al. observed, these data seem to be in contrast with the findings of previous studies, mainly performed in vitro, which reported an anti-inflammatory effect of thalidomide, mediated by an inhibition of TNF-a production [4, 6]. Nevertheless, the data confirm the most recent in vivo reports showing an increase in TNF-a concentrations and soluble TNF-a receptors during thalidomide treatment [7, 8]. These data suggest that thalidomide is not a systemic TNFa inhibitor. Moreover, it must be underlined that increased TNF-a production in the thalidomide-treated patients was associated with unexplained deaths when thalidomide was used in the treatment of toxic epidermal necrolysis [7]. We conducted a study, similar to the one performed by Bekker and colleagues, using thalidomide to treat HIV- and tuberculosis-coinfected patients. We studied 6 HIV- and M. tuberculosis‐infected patients, characterized by a poor response to the antituberculosis treatment, who were subsequently treated with thalidomide as adjuvant therapy. Immunological evaluations prior to thalidomide introduction suggested that these patients had significantly lower levels of TNF-a production than did the control subjects. Following thalidomide treatment, we observed a progressive increase in TNF-a production with a peak after about day 35 of therapy, thus confirming the observations made by Bekker and colleagues [1]. The increase in TNF-a concurred with a significant recovery of Th1 cytokine production, such as interleukin2 and interferon-g, and was followed by a significant improvement in clinical conditions (reduction of fever, increase in body weight, improvement in radiological findings, and negativization of M. tuberculosis cultures) in all patients. Our data, along with the results of Bekker et al., emphasize the usefulness of thalidomide treatment in patients infected by M. tuberculosis and demonstrate the complexity of the immunomodulating effects mediated by this drug. In agreement with the more recent in vivo reports, we confirm that thalidomide did not reduce TNF-a levels, and this is probably due to an activation of T cell activity, as hypothesized by Bekker and colleagues. However, these data raise questions as to the nature of the interaction between TNF-a and thalidomide. In the light of these results, we suggest extreme caution in undertaking studies that support the clinical use of thalidomide, on the basis of the assumption of its contradictory role in TNF-a inhibition.

The topical administration of bisphosphonates in implant surgery: a randomized split-mouth prospective study with a follow-up up to 5 years.

OBJECTIVE To evaluate the efficacy of the topical administration of bisphosphonates in implant therapy. MATERIALS AND METHODS Thirty-nine consecutive patients were selected for a split-mouth study. Inclusion criteria were: presence of a bilateral or total edentulism, ability to tolerate conventional implant procedures, older than 18 years. Ten patients were smokers. Ten patients were fully edentulous in both maxilla and mandible, 12 patients had fully edentulous maxilla or mandible, and 17 were bilaterally partially edentulous (9 in the mandible and 8 in the maxilla). A one-stage procedure was adopted in all cases. The prosthetic phase started 10 weeks after implant insertion. Each patient received implants on the control side and the test side, with insertion performed in the conventional way on the control side; on the test side, a 3% clodronate solution mixed with a surfactant (Tween-20) at a 1:3 ratio was topically administered both at the implant surface and at the implant site. RESULTS One hundred fifty-five implants were inserted. The test and control groups included 75 and 80 implants, respectively. The implant insertion torque was no less than 30 Ncm. A total of 7 implants failed in the control group (6 before loading and one after 12 months of loading). No failure occurred on the test side. By the 5-year follow-up, no further implant failure had been recorded. Overall, implant survival rates at 5 years for the test and control groups were, respectively, 100% and 91.3%, the difference being significant (p < .01). Mean marginal bone loss was 0.85 ± 0.71 mm in the test group and 1.12 ± 0.85 mm in the control group after 1 year of loading and stable thereafter. The difference was not significant. CONCLUSIONS The topical administration of bisphosphonates may positively affect implant survival in the preloading and postloading phases in partially and fully edentulous patients. However, a larger study population is needed to verify these promising clinical results.

PCR-Hybridization Assay for Mycobacterium avium Complex: Optimization of Detection in Peripheral Blood from Humans

ABSTRACT We evaluated the sensitivity of a DNA amplification test for the detection of Mycobacterium avium in blood samples using different blood components and different DNA extraction methods.M. avium-inoculated blood samples were processed to obtain separate blood components: peripheral blood mononuclear cells (PBMCs), polymorphonuclear cells (PMNCs), and whole-blood sodium dodecyl sulfate (SDS)-lysate pellets. The sensitivity for the detection of the lowest mycobacterial load (1 CFU/ml) was significantly greater (P < 0.01) with DNA extracted from SDS-lysate pellets than with DNA extracted from PBMCs or PMNCs. Subsequently, DNA extraction methods based on guanidine NaOH, and proteinase were compared. The sensitivity of the guanidine-based method was significantly greater (P < 0.01) than those of the others.

Decreasing trends of drug resistance and increase of non-B subtypes amongst subjects recently diagnosed as HIV-infected over the period 2004–2012 in the Veneto Region, Italy

The present study was designed to prospectively monitor transmitted drug resistance mutations (TDRMs) in the Veneto Region, Italy. Genotypic resistance testing was conducted on the plasma of 1882 patients consecutively enrolled at the time of diagnosis of human immunodeficiency virus (HIV) infection from 2004 to 2012. TDRMs were defined according to the Stanford HIV database algorithm. In total, 214 (16.1%) B subtype-infected and 58 (10.5%) non-B subtype-infected individuals were identified as having a primary or recent HIV-1 infection. In subtype B-infected subjects in 2004-2006, the prevalence of TDRMs was 20.0% in chronic infections and 25.5% in recent infections; in 2007-2009 the rates were 11.5% and 5.3%, respectively; and in 2010-2012 they were 11.3% and 15.2%, respectively. In non-B subtype-infected subjects in 2004-2006, the prevalence of TDRMs was 18.0% in chronic infections and 16.5% in recent infections; in 2007-2009 the rates were 5.7% and 0%, respectively; and in 2010-2012 they were 6.2% and 8.7%, respectively. Protease inhibitor resistance and combined resistance to two or three classes of drugs declined during the three study periods. The observed decrease in TDRMs and a simplification of the resistance patterns may reflect a change over time in the characteristics of the infecting subjects who are often unaware of their infection and transmit a wild-type strain.

Drug resistance in B and non-B subtypes amongst subjects recently diagnosed as primary/recent or chronic HIV-infected over the period 2013–2016: Impact on susceptibility to first-line strategies including integrase strand-transfer inhibitors

OBJECTIVES To characterize the prevalence of transmitted drug resistance mutations (TDRMs) by plasma analysis of 750 patients at the time of HIV diagnosis from January 1, 2013 to November 16, 2016 in the Veneto region (Italy), where all drugs included in the recommended first line therapies were prescribed, included integrase strand transfer inhibitors (InNSTI). METHODS TDRMs were defined according to the Stanford HIV database algorithm. RESULTS Subtype B was the most prevalent HIV clade (67.3%). A total of 92 patients (12.3%) were expected to be resistant to one drug at least, most with a single class mutation (60/68-88.2% in subtype B infected subjectsand 23/24-95.8% in non-B subjects) and affecting mainly NNRTIs. No significant differences were observed between the prevalence rates of TDRMs involving one or more drugs, except for the presence of E138A quite only in patients with B subtype and other NNRTI in subjects with non-B infection. The diagnosis of primary/recent infection was made in 73 patients (9.7%): they had almost only TDRMs involving a single class. Resistance to InSTI was studied in 484 subjects (53 with primary-recent infection), one patient had 143C in 2016, a total of thirteen 157Q mutations were detected (only one in primary/recent infection). CONCLUSIONS Only one major InSTI-TDRM was identified but monitoring of TDRMs should continue in the light of continuing presence of NNRTI-related mutation amongst newly diagnosed subjects, sometime impacting also to modern NNRTI drugs recommended in first-line therapy.

PrEP in Italy: The time may be ripe but who’s paying the bill? A nationwide survey on physicians’ attitudes towards using antiretrovirals to prevent HIV infection

Several studies have demonstrated the efficacy of the oral pre-exposure prophylaxis (PrEP) with tenofovir (with or without emtricitabine) on preventing HIV-negative partners of HIV infected patients to become infected through sexual contacts. PrEP is already available in the United States and now is approved by European Medicine Agency. In this setting we would like to gauge physicians’ knowledge, acquaintance with and attitude to include PrEP in their clinical practice. A cross sectional survey was conducted among Italian physicians expert on antiretroviral therapy. Out of 146 physicians, 35% of participants declared to be familiar with PrEP but only 46% of them believed that, currently, there are not enough reasons to make it available in Italy. 51% of physicians have already been attracted to prescribe it and 63.4% have been openly asked about PrEP. The main concerns noticed were: the risk of acquire other sexual transmitted diseases (STDs) (70% of physicians feared that PrEP could favor STDs spread), the potential harmful of PrEP if not adequately implemented and, especially the risk of possible side effects if not properly used. Nevertheless, 55.9% of participants believed that Health Authorities face an ethical obligation to make PrEP available as part of the strategies to protect from HIV transmission and half of the respondents asked for further researches to better define the role for PrEP. Attitudes regarding PrEP impact on Italian National Health Organization were also very interesting: 57.5% of participants did not believe that investing in PrEP would be an appropriate use of healthcare resources, while 70.6% affirmed that PrEP’s financial coverage should not be funded by the Italian National System of Health (SSN). This survey showed a high awareness of PrEP potential among Italian physicians coupled with a great deal of skepticism about how and if implementing it in clinical practice.