In Patients with Severe COVID-19, the Profound Decrease in the Peripheral Blood T-Cell Subsets Is Correlated with an Increase of QuantiFERON-TB Gold Plus Indeterminate Rates and Reflecting a Reduced Interferon-Gamma Production.

Increased rates of indeterminate QuantiFERON-TB Gold Plus Assay (QFT-Plus) were demonstrated in patients hospitalized with Coronavirus Disease (COVID)-19. We aimed to define the prevalence and characteristics of hospitalized COVID-19 patients with indeterminate QFT-Plus. A retrospective study was performed including hospitalized COVID-19 patients, stratified in survivors and non-survivors, non-severe and severe according to the maximal oxygen supply required. Statistical analysis was performed using JASP ver0.14.1 and GraphPad Prism ver8.2.1. A total of 420 patients were included, median age: 65 years, males: 66.4%. The QFT-Plus was indeterminate in 22.1% of patients. Increased rate of indeterminate QFT-Plus was found in non-survivors (p = 0.013) and in severe COVID-19 patients (p < 0.001). Considering the Mitogen-Nil condition of the QFT-Plus, an impaired production of interferon-gamma (IFN-γ) was found in non-survivors (p < 0.001) and in severe COVID-19 patients (p < 0.001). A positive correlation between IFN-γ levels in the Mitogen-Nil condition and the absolute counts of CD3+ (p < 0.001), CD4+ (p < 0.001), and CD8+ (p < 0.001) T-lymphocytes was found. At the multivariable analysis, CD3+ T-cell absolute counts and CD4/CD8 ratio were confirmed as independent predictors of indeterminate results at the QFT-Plus. Our study confirmed the increased rate of indeterminate QFT-Plus in COVID-19 patients, mainly depending on the peripheral blood T-lymphocyte depletion found in the most severe cases.

Impact of HIV-1 tropism on the emergence of non-AIDS events in HIV-infected patients receiving fully suppressive antiretroviral therapy.

OBJECTIVE: The impact of HIV-1 tropism on the emergence of non-AIDS events was evaluated in a cohort of 116 antiretroviral therapy (ART) responder patients. METHODS: The patients were followed for the emergence of hypertension, renal impairment, metabolic and bone disorders (defined as non-AIDS events) each 8 weeks at standard visits. A V3 plasma sequence genotype analysis was performed at the time of ART initiation and the geno2pheno algorithm with the results that defines the false-positive rate (FPR) was used to infer HIV tropism. The associations between the non-AIDS events and the FPR at baseline were evaluated using the χ test for trend. A Cox-regression analysis using the counting process formulation of Andersen and Gill was performed to define whether the emergence of non-AIDS events was correlated to FPR. RESULTS: The prevalence of at least one non-AIDS event resulted higher in patients with a FPR below 10% than in patients with a R5 virus (P = 0.033). Patients with a FPR below 5.0% most frequently developed non-AIDS events during ART (P = 0.01). A higher prevalence of patients with at least two AIDS events was found in the group of patients with a FPR below 5.0% with respect to the others (P < 0.001). At multivariate Cox-regression analysis, having an X4 virus and age were independently associated with a higher probability of non-AIDS event development. CONCLUSION: This study shows that an X4 virus, particularly a FPR less than 5%, is related to non-AIDS events development. Further studies are warranted to understand the mechanisms underlying this phenomenon.

Characterization of drug resistance mutations in naïve and ART-treated patients infected with HIV-1 in Yaounde, Cameroon.

Currently the prevalence of HIV-1 infection in Cameroon is 5.1%, CRF02_AG subtype is responsible for about 50% of infections. Since an HIV-1 drug resistance test is not yet available widely, accurate data on the prevalence of resistant viral strains are missing. The objective of this study was to determine HIV-1 genetic diversity and to characterize HIV-1 mutations conferring drug resistance among antiretroviral therapy (ART)-naïve and ART-treated patients. A cohort of 239 patients infected with HIV were followed-up between January 2007 and July 2010 in Cameroon. Two hundred and sixteen plasma samples were sequenced for phylogenetic analysis and identification of drug resistance mutations in the HIV-1 pol region. A significant genetic diversity was found: Seven pure subtypes (A1, A3, D, F1, F2, G, H), nine circulating recombinant forms (CRFs: 01_AE, 02_AG, 06cpx, 09cpx, 11cpx, 13cpx, 16cpx, 18cpx, 37cpx) and one new unique recombinant form (URF) (G/F2). The rate of transmitted drug resistance (TDR) in naïve patients was 8.2% (4/49). Around 80% of patients failing a first-line ART harbored a virus with at least one resistance mutation to two antiretroviral (ARV) classes, and 36% of those failing a second-line regimen carried a virus with at least one resistant mutation to three ARV classes. The high level of drug resistance observed in the cohort is alarming because this occurred as a result of only few years of treatment. Adherence to therapy, adequate education of physicians, and the appropriate use of genotypic resistance assay are critical points of intervention for the improvement of patient care.

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.

KSHV DNA viremia correlates with low CD4+ cell count in Italian males at the time of diagnosis of HIV infection.

To evaluate the relevance and the virological and immunological markers of Kaposi sarcoma herpesvirus 8 (KSHV) viremia in Italian male patients at the time of diagnosis of infection with HIV-1, 481 men infected with HIV were recruited consecutively. The presence of KSHV DNA was evaluated in peripheral blood mononuclear cells (PBMCs) and in plasma and correlated with demographic and viro-immunological parameters. Seventy-four patients had KSHV DNA detected in PBMCs. By univariate analysis, the presence of KSHV DNA was associated significantly with unprotected homosexual relationships (P=0.003) and it was significantly higher in patients with CD4+ cell <350 (P=0.025). By multivariate analysis, homosexual relationships were associated independently with KSHV DNA in PBMCs (OR: 3.25; 95% CI: 1.1-9.7; P=0.035). Among the 74 patients with KSHV DNA detected in PBMCs, plasma samples from 60 were analyzed and 33 were positive for KSHV DNA. The CD4+ cell counts and percentages were significantly lower in patients with KSHV DNA in both PBMCs and plasma as compared to patients with only KSHV DNA in PBMCs (P=0.006 and P=0.019, respectively). Among the patients with KSHV DNA detected in PBMCs, all 13 patients with CD4+ cells count <200 had detectable levels of KSHV in their plasma. By multivariate analysis adjusted for the epidemiologic and virological parameters, low CD4+ cell count was the only independent variable associated with the presence of KSHV DNA in plasma (OR, 0.001; 95% CI: <0.001-0.001; P=0.03). In HIV-positive antiretroviral therapy-naïve males, KSHV active replication as detected by KSHV DNA in plasma was associated significantly with low CD4+ cell count.

Secondary integrase resistance mutations found in HIV-1 minority quasispecies in integrase therapy-naive patients have little or no effect on susceptibility to integrase inhibitors.

The goal of this study was to explore the presence of integrase strand transfer inhibitor (InSTI) resistance mutations in HIV-1 quasispecies present in InSTI-naïve patients and to evaluate their in vitro effects on phenotypic susceptibility to InSTIs and their replication capacities. The RT-RNase H-IN region was PCR amplified from plasma viral RNA obtained from 49 HIV-1 subtype B-infected patients (21 drug naïve and 28 failing highly active antiretroviral therapy [HAART] not containing InSTIs) and recombined with an HXB2-based backbone with RT and IN deleted. Recombinant viruses were tested against raltegravir and elvitegravir and for replication capacity. Three-hundred forty-four recombinant viruses from 49 patients were successfully analyzed both phenotypically and genotypically. The majority of clones were not phenotypically resistant to InSTIs: 0/344 clones showed raltegravir resistance, and only 3 (0.87%) showed low-level elvitegravir resistance. No primary resistance mutations for raltegravir and elvitegravir were found as major or minor species. The majority of secondary mutations were also absent or rarely present. Secondary mutations, such as T97A and G140S, found rarely and only as minority quasispecies, were present in the elvitegravir-resistant clones. A novel mutation, E92G, although rarely found in minority quasispecies, showed elvitegravir resistance. Preexisting genotypic and phenotypic raltegravir resistance was extremely rare in InSTI-naïve patients and confined to only a restricted minority of secondary variants. Overall, these results, together with others based on population and ultradeep sequencing, suggest that at this point IN genotyping in all patients before raltegravir treatment may not be cost-effective and should not be recommended until evidence of transmitted drug resistance to InSTIs or the clinical relevance of IN minor variants/polymorphisms is determined.

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.

[HIV and related infections in Italian penal institutions: epidemiological and health organization note]. / HIV e infezioni correlate negli Istituti Penitenziari Italiani: note di epidemiologia e di organizzazione sanitaria.

HIV and other infections represent an important health problem in Italian jails. In particular, HIV prevalence is high, due to the characteristics of the prison population, which is constituted by a large proportion of injecting drug users and foreigners. In addition, data from other countries suggest that risky behaviour are not uncommon during imprisonment, and transmission of HIV and other infection in this setting may also occur. Data from surveys conducted by the Penitentiary Authority in Italian jails show a decline of HIV seroprevalence from 9.7% in 1990 to 2.6% in 2001. However, these data are largely incomplete and do not account for possible biases due to self-selection of inmates toward HIV serological testing or to variations in the access to screening activities. More accurate data, possibly obtained through anonymous unlinked surveys, are needed in order to better plan health services and preventive measures.