Immunogenicity and Safety of BNT162b2 Homologous Booster Vaccination in People Living with HIV under Effective cART.

Data on COVID-19 boosting vaccination in people living with HIV (PLWH) are scant. We investigated the immunogenicity and safety of the BNT162b2 homologous boosting vaccination. Anti-SARS-CoV-2 spike antibodies (LIAISON® SARS-CoV-2 S1/S2 IgG test, DiaSorin®), CD4+, CD8+ and viraemia were monitored at T0 (pre-vaccination), T1 (4 weeks after the second dose), T2 (pre-booster) and T3 (4 weeks after the booster dose). Humoral responses were evaluated according to sex, age, BMI, nadir and baseline CD4+ counts, as well as type of cART regimen. Forty-two subjects were included: the median age was 53 years (IQR: 48−61); the median time since HIV was 12.4 years (IQR: 6.5−18.3); the median nadir and baseline CD4+ counts were 165 (IQR: 104−291) and 687 cells/mm3 (IQR: 488−929), respectively. The booster dose was administered at a median of 5.5 months after the second dose. Median anti-SARS-CoV-2 IgG concentration had significantly decreased at T2 compared to T1 (107 vs. 377, p < 0.0001). Antibody levels elicited by the booster dose (median: 1580 AU/mL) were significantly higher compared with those of all the other time points (p < 0.0001). None of the investigated variables significantly affected antibody response induced by the booster dose. Local and systemic side-effects were referred by 23.8% and 14.3% of the subjects, respectively. One patient developed sensorineural hearing loss (SNHL) 24 h after boosting. He recovered auditory function upon endothympanic administration of corticosteroids. The BNT162b2 boosting vaccination in PLWH is safe and greatly increased the immune response with respect to the primary vaccination.

Unequal Access to Testing and Vaccination Services for the Homeless and Undocumented Population During COVID-19 Pandemic.

Objectives: To furnish a model to ensure access and use of healthcare services to the undocumented and homeless population. Methods: Between March 2020 and October 2021, public and third sector actors in Rome implemented an accessible COVID-19 screening service and vaccination program targeting the homeless and undocumented population. Results: 95.6% of the patients tested negative to both rapid and molecular tests. 0.9% tested positive to both. 0.7% were false negatives, while 2.8% were false positives. None of the participants refused the diagnostic treatment. From July to October 2021, 1384 people received a complete cycle of the COVID-19 vaccine through the program. 632 (45.6%) also agreed to perform the antibodies testing before inoculation. 318 (50.31%) of these were positive at the time of vaccination. Conclusion: We present a cost-effective model for reducing structural barriers to access diagnostic and preventive services for the homeless and undocumented population that can be applied to different public health settings.

Rapid Detection of Methicillin-Resistant Staphylococcus aureus Directly from Blood for the Diagnosis of Bloodstream Infections: A Mini-Review.

Staphylococcus aureus represents a major human pathogen able to cause a number of infections, especially bloodstream infections (BSI). Clinical use of methicillin has led to the emergence of methicillin-resistant S. aureus (MRSA) and MRSA-BSI have been reported to be associated with high morbidity and mortality. Clinical diagnosis of BSI is based on the results from blood culture that, although considered the gold standard method, is time-consuming. For this reason, rapid diagnostic tests to identify the presence of methicillin-susceptible S. aureus (MSSA) and MRSA isolates directly in blood cultures are being used with increasing frequency to rapidly commence targeted antimicrobial therapy, also in the light of antimicrobial stewardship efforts. Here, we review and report the most common rapid non-molecular and molecular methods currently available to detect the presence of MRSA directly from blood.

Expression and Function of NKG2D Is Impaired in CD8+ T Cells of Chronically HIV-1-Infected Patients Without ART.

OBJECTIVES: Increasing line of evidence indicates that the NKG2D-activating receptor plays a relevant role in the effector functions of cytotoxic lymphocytes. In this study, we investigated the expression and function of NKG2D in CD8⁺ T cells from chronically HIV-1-infected patients with or without antiretroviral therapy (ART). METHODS: We measured by flow cytometry the expression of NKG2D on CD8⁺ T-cell subsets of ART-naive and ART patients as well as seronegative healthy subjects (HIV-neg). An intrapatient analysis before and after ART initiation was also performed. Results were correlated with viral load, CD4⁺ T-cell counts, markers of immune activation (CD38, sCD14), and soluble NKG2D ligands (sMICA and sULBP2). The function of NKG2D on CD8⁺ T cell cytotoxicity was tested by ex vivo degranulation assays. RESULTS: We showed that NKG2D was downregulated on all CD8⁺ T-cell subsets of ART-naive patients. The expression of NKG2D on CD8⁺ T cells inversely correlated with viral load and CD38 expression but not with plasma levels of sMICA and sULBP2. Importantly, we found that NKG2D-mediated costimulation of CD8⁺ T-cell lytic activity was strongly reduced in ART-naive patients if compared with HIV-neg and ART subjects. Finally, intrapatient analysis demonstrated that effective anti-HIV-1 therapy restores NKG2D expression and NKG2D-induced cytotoxicity by CD8⁺ T cells. CONCLUSIONS: These data underscore that NKG2D downregulation contributes to impaired CD8⁺ T-cell responses in untreated HIV-1 infection and have implications for monitoring immune functions and response to treatments, and for the development of novel anti-HIV-1 strategies combining ART with drugs that stimulate NKG2D expression and function.

Maraviroc as intensification strategy in HIV-1 positive patients with deficient immunological response: an Italian randomized clinical trial.

BACKGROUND: Immunological non-responders (INRs) lacked CD4 increase despite HIV-viremia suppression on HAART and had an increased risk of disease progression. We assessed immune reconstitution profile upon intensification with maraviroc in INRs. METHODS: We designed a multi-centric, randomized, parallel, open label, phase 4 superiority trial. We enrolled 97 patients on HAART with CD4+<200/µL and/or CD4+ recovery ≤ 25% and HIV-RNA<50 cp/mL. Patients were randomized 1:1 to HAART+maraviroc or continued HAART. CD4+ and CD8+ CD45+RA/RO, Ki67 expression and plasma IL-7 were quantified at W0, W12 and W48. RESULTS: By W48 both groups displayed a CD4 increase without a significant inter-group difference. A statistically significant change in CD8 favored patients in arm HAART+maraviroc versus HAART at W12 (p=.009) and W48 (p=.025). The CD4>200/µL and CD4>200/µL + CD4 gain ≥ 25% end-points were not satisfied at W12 (p=.24 and p=.619) nor at W48 (p=.076 and p=.236). Patients continuing HAART displayed no major changes in parameters of T-cell homeostasis and activation. Maraviroc-receiving patients experienced a significant rise in circulating IL-7 by W48 (p=.01), and a trend in temporary reduction in activated HLA-DR+CD38+CD4+ by W12 (p=.06) that was not maintained at W48. CONCLUSIONS: Maraviroc intensification in INRs did not have a significant advantage in reconstituting CD4 T-cell pool, but did substantially expand CD8. It resulted in a low rate of treatment discontinuations. TRIAL REGISTRATION: ClinicalTrials.gov NCT00884858 http://clinicaltrials.gov/show/NCT00884858.

The potential impact of routine testing of individuals with HIV indicator diseases in order to prevent late HIV diagnosis.

BACKGROUND: The aim of our work was to evaluate the potential impact of the European policy of testing for HIV all individuals presenting with an indicator disease, to prevent late diagnosis of HIV. We report on a retrospective analysis among individuals diagnosed with HIV to assess whether a history of certain diseases prior to HIV diagnosis was associated with the chance of presenting late for care, and to estimate the proportion of individuals presenting late who could have been diagnosed earlier if tested when the indicator disease was diagnosed. METHODS: We studied a large cohort of individuals newly diagnosed with HIV infection in 13 counselling and testing sites in the Lazio Region, Italy (01/01/2004-30/04/2009). Considered indicator diseases were: viral hepatitis infection (HBV/HCV), sexually transmitted infections, seborrhoeic dermatitis and tuberculosis. Logistic regression analysis was performed to estimate association of occurrence of at least one indicator disease with late HIV diagnosis. RESULTS: In our analysis, the prevalence of late HIV diagnosis was 51.3% (890/1735). Individuals reporting at least one indicator disease before HIV diagnosis (29% of the study population) had a lower risk of late diagnosis (OR = 0.7; 95%CI: 0.5-0.8) compared to those who did not report a previous indicator disease. 52/890 (5.8%) late presenters were probably already infected at the time the indicator disease was diagnosed, a median of 22.6 months before HIV diagnosis. CONCLUSIONS: Our data suggest that testing for HIV following diagnosis of an indicator disease significantly decreases the probability of late HIV diagnosis. Moreover, for 5.5% of late HIV presenters, diagnosis could have been anticipated if they had been tested when an HIV indicator disease was diagnosed.However, this strategy for enhancing early HIV diagnosis needs to be complemented by client-centred interventions that aim to increase awareness in people who do not perceive themselves as being at risk for HIV.

Soluble ligands for the NKG2D receptor are released during HIV-1 infection and impair NKG2D expression and cytotoxicity of NK cells.

In humans, the interaction of the natural killer group 2 member D (NKG2D)-activating receptor on natural killer (NK) and CD8(+) T cells with its major histocompatibility complex class I-related chain (MIC) and UL16 binding protein (ULBP) ligands (NKG2DLs) promotes recognition and elimination of stressed cells, such as tumor or infected cells. Here, we investigated the capacity of HIV-1 to modulate NKG2DL expression and escape NGK2D-mediated immunosurveillance. In CD4(+) T lymphocytes, both cell surface expression and release of MICA, MICB, and ULBP2 were up-regulated >2-fold by HIV-1 infection. In HIV-infected CD4(+) T lymphocytes or Jurkat T-cell lines, increased shedding of soluble NKG2DLs (sNKG2DLs) was impaired by a matrix metalloproteinase inhibitor (MMPI). Moreover, naive HIV(+) patients displayed increased plasma sMICA and sULBP2 levels and reduced NKG2D expression on NK and CD8(+) T cells compared to patients receiving highly active antiretroviral therapy (HAART) or healthy donors. In individual patients, HAART uptake resulted in the drop of sNKG2DL and recovery of NKG2D expression. Finally, sNKG2DLs in patients' plasma down-regulated NKG2D on NK and CD8(+) T cells and impaired NKG2D-mediated cytotoxicity of NK cells. Thus, NKG2D detuning by sNKG2DLs may promote HIV-1 immune evasion and compromise host resistance to opportunistic infections, but HAART and MMPI have the potential to avoid such immune dysfunction.

HIV-1 dual/mixed tropic isolates show different genetic and phenotypic characteristics and response to maraviroc in vitro.

Dual/mixed-tropic HIV-1 strains are predominant in a significative proportion of patients, though few information is available regarding the genetic characteristics, quasispecies composition, and susceptibility against CCR5-antagonists of the primary-isolates. For this reason, we investigated in deep details, both phenotypically and genotypically, the characteristics of 54 HIV-1 primary-isolates obtained from HIV-infected patients. Tropism was assessed by multiple-cycles phenotypic-assay on U87MG-CD4(+)-CCR5(+)-/CXCR4(+)-expressing cells. In vitro selection in PBMCs of X4-tropic viral strains following maraviroc-treatment was also performed. Phenotypic-assay reported pure R5-tropic viruses in 31 (57.4%) isolates, dual/mixed-tropic viruses in 22 (40.7%), and pure X4-tropic virus in only 1 (1.8%). Among dual/mixed-tropic isolates, 12 showed a remarkably higher replication-efficacy in CCR5-expressing cells (R5(+)/X4), and 2 in CXCR4-expressing cells (R5/X4(+)). Genotypic-tropism testing showed a correlation between PSSM-scores, geno2pheno false-positive-rate, and V3-net-charge with both CCR5-usage and syncytium-inducing ability. Moreover, specific gp120- and gp41-mutations were significantly associated with tropism and/or syncytium-inducing ability. Ultra-deep V3-pyrosequencing showed the presence of a swarm of genetically distinct species with a preference for CCR5-coreceptor not only in all pure R5-isolates, but also in 6/7 R5(+)/X4-tropic isolates. In both pure-X4 and R5/X4(+)-isolates, we observed extensive prevalence of X4-using species. In vitro selection-experiments with CCR5-inhibitor maraviroc (up to 2 months) showed no-emergence of X4-tropic variants for all R5- and R5(+)/X4-isolates tested (while X4-virus remained fully-resistant). In conclusion, our study shows that dual/mixed-tropic viruses are constituted by different species, whereby those with characteristics R5(+)/X4 are genotypically and phenotypically similar to the pure-R5 isolates; thus the use of CCR5-antagonists in patients with R5(+)/X4-tropic viruses may be a therapeutic-option that deserves further investigations.

Cellular HIV-1 DNA quantitation in patients during simplification therapy with protease inhibitor-sparing regimens.

Simplified regimens containing protease-inhibitors (PI)-sparing combinations were used in patients with virological suppression after prolonged highly active antiretroviral therapy. This study evaluated the total HIV-1 DNA quantitation as a predictor of long-term success for PI-sparing simplified therapy. Sixty-two patients were enrolled in a prospective non-randomized cohort. All patients have been receiving a triple-therapy regimen, two nucleoside reverse transcriptase inhibitors (NRTIs) plus one PI, for at least 9 months and were characterized by undetectable plasma HIV-1 RNA levels (<50 cp/ml) for at least 6 months. Patients were changed to a simplified PI-sparing regimen to overcome PI-associated adverse effects. HIV-DNA levels in peripheral blood mononuclear cells (PBMCs) were evaluated at baseline and at the end of follow-up. Patients with proviral DNA levels below the median value (226 copies/10(6) PBMCs) had a significant higher CD4 cell count at nadir (P = 0.003) and at enrolment (P = 0.001) with respect to patients with HIV-DNA levels above the median value. At month 18, 53 out of 62 (85%) patients on simplified regimen showed virological success, 4 (6.4%) patients experienced virological failure and 5 (8%) patients showed viral blip. At logistic regression analysis, HIV-DNA levels below 226 copies/10(6) PBMCs at baseline were associated independently to a reduced risk of virological failure or viral blip during simplified therapy (OR 0.002, 95% CI 0.001-0.46, P = 0.025). The substitution of PI with NRTI or non-NRTIs may represent an effective treatment option. Indeed, treatment failure or viral blip were experienced by 6% and 8% of the patients on simplified therapy, respectively. In addition, sustained suppression of the plasma viral load was significantly correlated with low levels of proviral DNA before treatment simplification.

In vitro fluconazole susceptibility of 1565 clinical isolates of Candida species evaluated by the disk diffusion method performed using NCCLS M44-A guidelines.

We determined the in vitro activity of fluconazole against 1565 clinical Candida spp. isolates collected from different specimens of non-AIDS outpatients and inpatients in 3 different regions of Italy. Susceptibility testing was performed by agar disk diffusion using the NCCLS document M44-A guidelines. Candida albicans was the most frequently isolated yeast (68%) followed by C. glabrata (15%), C. tropicalis (5%), C. parapsilosis (5%), and C. krusei (5%). Other yeasts represented 4% of all isolates. Of the 1565 isolates tested, 1449 (92.6%) were susceptible (S) to fluconazole, 43 (2.7%) were susceptible dose-dependent (S-DD) and 73 (4.7%) were resistant (R). Almost all (98.2%) of the C. albicans isolates were classified as S or S-DD. Despite its widespread use, fluconazole displayed good activity against the isolates we tested, and the disk diffusion method was confirmed as a reliable approach to the evaluation of in vitro susceptibility of yeasts to this antimycotic agent.

Viral growth assay to evaluate the replicative capacity of HIV-1 isolates.

The replicative capacity of HIV is studied by carrying out replication-competition experiments with the insertion of the gene of interest. These assays cannot capture the complicated patterns of mutations of different genes.A cross sectional study was carried out on 10 HIV-infected nai;ve patients and on 15 patients failing HAART. The CD8-depleted PBMCs, with known proviral DNA and cellular HIV-RNA copy numbers, were cultured. A reference curve was determined using the data obtained from 10 nai;ve patients. The replicative capacity was calculated as the ratio multiplied by 100 of the p24 antigen level of isolates over the p24 antigen level determined on the reference curve.A linear correlation between p24 antigen level and the infectious doses of HIV-DNA alone or plus cellular RNA copy number of PBMCs was found in naive patients (r=0.63, P<0.001 and r=0.67, P<0.001, respectively). Although all patients failing therapy had strains with impaired replicative capacity, a wide range of values (0.1-74.5%) was detected. All strains with a replicative capacity above 10% had non-nucleoside reverse transcriptase inhibitors related mutations.A viral assay to evaluate the HIV replicative capacity is described. The high variability of replicative capacity confirms the need to undertake replicative capacity assay using the whole virus.