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Background. The ultimate goal of organ transplantation is the reestablishment of organ function and the restoration of a solid immunity to prevent the assault of potentially deadly pathogens. T cell immunity is crucial in controlling cytomegalovirus (CMV) infection. It is still unknown how preexisting antiviral T cell levels, prophylaxis, or preemptive antiviral strategies and pharmacological conditioning affect immune reconstitution. Methods. Seventy preemptively treated CMV-seropositive recipients, 13 prophylaxis-treated CMV-seronegative recipients of seropositive donor transplants, 2 seropositive recipients of seronegative donor kidneys, and 27 pretransplant subjects were enrolled in a cross-sectional study and analyzed for CMV viremia (DNAemia) and CMV-specific T cell response (interferon-gamma enzyme-linked immunospot assay) before transplantation and at 30, 60, 90, 180, and 360 days after transplantation. Results. CMV-seropositive transplant recipients displayed a progressive but heterogeneous pattern of immune reconstitution starting from day 60 after transplantation. CMV-seronegative recipients did not mount a detectable T cell response throughout the prophylaxis regimen. A single episode of CMV viremia (CMV copy number, 7000-170,000 copies/mL) was sufficient to prime a protective T cell immune response in CMV-seronegative recipients. Antithymocyte globulin treatment did not significantly affect CMV-specific T cell response. Conclusions. Baseline immunity, antiviral therapy but not antithymocyte globulin treatments profoundly influence T cell reconstitution in kidney transplant recipients. [ABSTRACT FROM AUTHOR]

Objectives: We present an updated picture (1/1/2017-31/08/2019) of the frequency of carbapenemase producing Klebsiella pneumoniae (CPKP) in surveillance rectal swabs (SRS) and in clinical samples (CS) of patients admitted to a tertiary level hospital, focusing on longitudinal evolution of CPKP detected in SRS and on colistin resistant strains., Methods: Retrospective longitudinal analysis. Only the first positive CPKP strain isolated from each patient was included., Results: 638 CPKP strains were identified (471 in SRS and 167 in CS). SRS frequency increased over time in the medical department, remained high in the surgical department (SD) and decreased in the intensive care department. Most SRS-71.3%-and 49.1% of CS had nosocomial origin; about half of the SRS were identified in the SD. Regarding SRS evolution, carriage was confirmed in 39.5% of patients, no more testing in 25.5%, clinical involvement in 24.8 %, and negative result in 10.2%. Rates of colistin resistance were 20.1% in 2017, 31.2% in 2018 and 26.9% in 2019., Conclusions: CPKP diffusion is still an important issue despite the surveillance program. It is vital to enhance medical staff's awareness on this because most CPKP first detections in SRS occurred during hospital stay due to a nosocomial acquisition with a comparable picture over time. Colistin resistance is increasing., (Copyright © 2020 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Background: This longitudinal study described Cytomegalovirus (CMV) DNA, Epstein-Barr (EBV) DNA and human herpesvirus 8 (HHV-8) DNA asymptomatic salivary shedding in HIV-positive men who have sex with men (MSM). We aimed to 1-analyze frequency and persistence of herpesvirus shedding, 2-correlate herpesvirus positivity and HIV viroimmunological parameters and 3-assess the association between HIV-RNA suppression and herpesvirus replication., Methods: Herpesvirus DNA was tested with an in-house real-time PCR in 2 salivary samples obtained at T0 and T1 (24 months after T0). HIV-RNA was evaluated in the 24 months prior to T0 and in the 24 months prior to T1; MSM were classified as successfully suppressed patients (SSPs), viremic patients (VPs) and partially suppressed patients (PSPs). EBV DNA load was classified as low viral load (EBV-LVL, value ≤10,000 copies/ml) and as high viral load (EBV-HVL,> 10,000 copies/ml). Mann-Whitney U test tested the difference of the median between groups of patients. Chi-squared test and Fisher's exact test compared categorical variables according to the frequencies. Kruskal-Wallis test compared continuous data distributions between levels of categorical variables., Results: Ninety-two patients (median CD4+ count 575 cells/mm3, median nadir 330 CD4+ cells/mm3) were included: 40 SSPs,33 VPs and 19 PSPs. The more frequently single virus detected was EBV, both at T0 and at T1 (in 67.5 and 70% of SSPs, in 84.8 and 81.8% of VPs and in 68.4 and 73.7% of SPSs) and the most frequently multiple positivity detected was EBV + HHV-8. At T1, the percentage of CMV positivity was higher in VPs than in SSPs (36.4% vs 5%, p < 0.001), the combined shedding of HHV-8, CMV and EBV was present only in VPs (15.1%, p = 0.01 respect to SSPs) and no VPs confirmed the absence of shedding found at T0 (vs 17.5% of SSPs, p = 0.01). EBV-HVL was more frequent in VPs than in SSPs: 78.6% at T0 (p = 0.03) and 88.9% at T1 (p = 0.01)., Conclusions: The relationship between uncontrolled plasma HIV viremia and CMV, EBV, and HHV-8 shedding is multifaceted, as demonstrated by the focused association with EBV DNA load and not with its frequency and by the persistent combined detection of two oncogenic viruses as EBV and HHV-8 regardless of HIV virological control.

Plasmodium knowlesi is a simian parasite responsible for most human cases of malaria in Malaysian Borneo. A timely recognition of infection is crucial because of the risk of severe disease due to the rapid increase in parasitemia. We report a case of P. knowlesi infection in a traveller who developed fever and thrombocytopenia after returning from the Philippines in 2016. Rapid antigen test was negative, microscopy examination showed parasites similar to Plasmodium malariae, with a parasite count of 10,000 parasites per μL blood, while molecular testing identified P. knowlesi infection. Treatment with atovaquone-proguanil led to resolution of fever and restoration of platelet count in two days. P. knowlesi infection should be suspected in febrile travellers returning from South East Asia. Due to the low sensitivity of rapid antigen tests and the low specificity of microscopy, confirmation by molecular tests is recommended.

Objectives: Very few data exist on viral meningitis and encephalitis in elderly patients (>65 years old)., Methods: This study investigated the detection of herpes simplex virus (HSV), varicella zoster virus (VZV), human herpes virus 6 (HHV-6), HHV-7, HHV-8, cytomegalovirus (CMV), Epstein-Barr virus (EBV), enterovirus (EV), human adenovirus (HAdV), human parechoviruses (HPeVs), and tick-borne encephalitis virus (TBEV) through real-time PCR (RT-PCR) in patients >65 years old who had cerebrospinal fluid (CSF) tested for a suspected central nervous system infection., Results: A total of 2868 RT-PCRs were performed on 502 CSF samples. Overall, 65 positive RT-PCRs were found: 23 for HSV (35.4% of positives), 15 for EV (23.1% of positives), 14 for EBV (21.5% of positives), 12 for VZV (18.5% of positives), and one for CMV (1.5% of positives). A positive RT-PCR in CSF was detected in 24 (17.4%) patients aged ≥ 80 years and in 35 (9.6%) patients aged 65-79 years (p=0.02). VZV was more frequently detected in the oldest subjects (5.9% vs. 1.6%, p=0.03)., Conclusions: HSV was the most common viral aetiology identified in the study, with VZV infection being recognized more frequently in those patients aged ≥ 80 years., (Copyright © 2016 The Authors. Published by Elsevier Ltd.. All rights reserved.)

The novel real-time PCR assay developed as described here was able to detect bla(KPC1/2-12) (bla(KPC-1/2) to bla(KPC-12)) from easily available clinical specimens in less than 2 h. The genotypic assay was highly sensitive (100%) and specific (98%). In some cases, it was able to detect bla(KPC) 48 h before positive detection by standard phenotypic assay on patients who were monitored daily. The high sensitivity and rapidity of the molecular method make it the method of choice for KPC surveillance and, thus, containment purposes.

Background: In pediatric kidney transplant recipients, viral infections occur soon after transplant and may be transmitted from the graft., Methods: This study of 75 pediatric kidney transplants investigated whether genome sequences of parvovirus B19, Epstein-Barr virus (EBV), human cytomegalovirus (HCMV), and BK polyomavirus (BKV) could be detected in kidney graft samples (graft biopsy samples and preservation and washing solutions) collected before implantation and whether their presence was a risk factor for infections in the recipient., Results: B19 DNA was detected in approximately 30% of graft biopsy samples, preservation solutions, and washing solutions; EBV DNA was detected in approximately 20% of preservation and washing solutions but rarely in biopsy samples; and HCMV DNA and BKV DNA were rarely detected in graft biopsy samples. Seronegative recipients of B19 DNA-positive and EBV DNA-positive grafts had a significantly higher risk of infection during the early posttransplant period than did recipients of negative grafts. In particular, none of the B19-seronegative recipients of B19 DNA-negative grafts experienced infection soon after transplant, whereas most recipients of B19 DNA-positive grafts experienced infection within the first month after transplant., Conclusions: Molecular testing of donor grafts for viruses that infect circulating and resident cells in the graft-such as B19 in the kidney-could be useful (in association with donor/recipient serostatus) for identifying recipients at high risk for posttransplant infections.

Recombinant Semliki Forest virus (SFV) is an attractive viral vector system owing to its ability to allow high efficiency of viral protein expression. To produce recombinant pseudotyped human immunodeficiency virus type 1 (HIV-1) virions, we designed a chimeric SFV/HIV vector system that contains both the HIV-1 cis- and trans-acting elements under the transcriptional control of the SFV replicase and investigated the ability of the hybrid SFV/HIV system to produce lentiviral particles capable of transducing target cells. Co-transfection of target cells with the two helper SFV packaging system RNAs along with each SFV/Gag-Pol, SFV/VSV(G) as well as SFV/HIV-1 vector unit replicon led to the generation of efficient transducing competent recombinant SFV/HIV particles. In contrast, co-transduction of target cells with the SFV/HIV chimeric virions produced recombinant particles with low transducing ability. Our data suggest that both the genomic and the subgenomic RNAs containing the HIV-1 vector unit were negatively selected for incorporation into recombinant particles, despite the fact that the SFV-driven HIV-1 vector replicon was the only one containing a lentiviral packaging sequence. The results of this study provide insights relevant to the design of chimeric lentiviral vectors.

Background: The relevance of viral infections in the development of allograft lesions is still unclear, although some viruses have been implicated. The present study investigated systemic and intrarenal viral infections in kidney transplant recipients and their association with the risk of acute rejection and chronic allograft injuries that are predictive of long-term dysfunction., Methods: The presence of DNA sequences of human herpesviruses, polyomaviruses, and parvovirus B19 was analyzed in renal allograft biopsy specimens obtained at baseline, after acute renal dysfunction, and during follow-up evaluation in 69 transplant recipients who were children or young adults. Results were correlated with clinical data, viral DNAemia, and results of renal function tests and allograft histology analyzed at the same time points., Results: Overall, viral DNA was detectable in 46% of baseline and 70% of follow-up biopsy specimens of kidney allografts, where it generally persisted. The most frequently detected viruses were B19 and human herpesvirus 6, already present in donor kidneys, and BK virus and Epstein-Barr virus, usually involving the allograft during follow-up. Among viruses, only the intrarenal persistence of B19 DNA and B19 DNAemia was associated with the development of chronic allograft injury, whereas human cytomegalovirus DNAemia was a risk factor for acute rejection., Conclusions: Parvovirus B19 seems to target the kidney electively. Its intrarenal persistence is associated with chronic kidney allograft injury.

We identified a 6-aminoquinolone compound, WC5, that inhibits human cytomegalovirus (HCMV) replication with a selectivity index of approximately 500. WC5 also showed activity against drug-resistant HCMV strains. In contrast, it did not significantly affect the replication of human herpesvirus 6 and 8 and was approximately 10-fold less active against murine cytomegalovirus. Thus, WC5 may represent a lead for the development of new, potent, and selective anti-HCMV compounds.

To determine the prevalence of human herpesvirus 8 (HHV-8) infection, the rate of HHV-8 seroconversion, and the presence of serum HHV-8 DNA after bone marrow transplantation (BMT), we evaluated sera from 187 Italian BMT donor-recipient pairs. Antibodies to lytic and latent HHV-8 antigens were detected by immunofluorescence. Sera of donor-recipient pairs who seroconverted were examined by real-time polymerase chain reaction (RT-PCR). Before BMT, 24 (13%) of 187 donors and 20 (11%) of 187 recipients were seropositive; after BMT, 28 (15%) of 187 recipients were seropositive. Seroconversion occurred in 19 (11%) of 167 recipients seronegative at baseline: 14 (9%) from 149 seronegative donors and five (28%) from 18 seropositive donors (relative risk of seroconversion with BMT from a seropositive donor = 2.96, 95% confidence interval = 1.21 to 7.25; P = .02, two-sided Fisher's exact test). One donor and two recipients who seroconverted after BMT were positive for HHV-8 by RT-PCR. No HHV-8-related complications were observed after a median follow-up of 6 years. BMT-associated HHV-8 seroconversion is relatively common in seronegative recipients from seropositive donor, but factors other than BMT may also contribute to seroconversion.

Different strategies proposed in the literature to attempt gene therapy of AIDS are based mainly on the intracellular production of RNA and protein therapeutics. This report describes the construction and the anti-human immunodeficiency virus type 1 (HIV-1) activity of a new type of antisense tRNA directed against a nucleotide region in the first coding exon of HIV-1 tat (nucleotides 5924 to 5943; Los Alamos data bank) which is conserved among many HIV-1 clones. The anti-tat antisense sequence was inserted into a tRNA(Pro) backbone by replacement of the anticodon loop, without altering the tRNA canonic tetraloop structure. The antisense tRNA was able to interact effectively with its target in vitro. Jurkat cells that constitutively expressed the anti-tat tRNA following retroviral vector transduction exhibited significant resistance to HIV-1 de novo infection. Resistance seemed to correlate with the level of antisense expression. This is the first time that such a tRNA antisense strategy has been shown to be effective as a genetic treatment of HIV-1 infection in tissue culture. The construct design proposed in this report has some intrinsic advantages: the transcript is driven by a polymerase III promoter, the short length of the RNA minimizes effects of intramolecular base pairing that may impair target recognition, and the antisense RNA has the stability and intracellular fate of a native tRNA molecule.

Endothelin-1 stimulates aldosterone secretion by interacting with specific receptors. Accordingly, we wished to investigate endothelin-1, endothelin-A (ETA) receptor, and endothelin-B (ETB) receptor gene expression, localization, and properties in aldosterone-producing adenomas and in the normal human adrenal cortex. We carried out 125I-endothelin-1 displacement studies with cold endothelin-1, endothelin-3, the specific ETA antagonist BQ-123, and the specific ETB weak agonist sarafotoxin 6 C and coanalyzed data with the nonlinear iterative curve-fitting program LIGAND. We also studied gene expression with reverse transcription-polymerase chain reaction with specific primers for endothelin-1, ETA, and ETB complementary DNA. Normal adrenal cortices from consenting kidney cancer patients (n = 2) and aldosterone-producing adenomas (n = 4) were studied; for the latter, surrounding normal cortex and kidney biopsy tissue served as controls. To further localize the receptor subtypes, tissue sections were studied by autoradiography in the presence and absence of 500 nmol/L BQ-123, 100 nmol/L sarafotoxin 6 C, and 1 mumol/L cold endothelin-1. In all tissues examined, endothelin-1, ETA, and ETB messenger RNAs were easily detected. However, in aldosterone-producing adenomas, both receptors' genes were expressed at a higher level than in the kidney.(ABSTRACT TRUNCATED AT 250 WORDS)

In this study we evaluated modifications of various structural and functional properties of the plasma membrane of HeLa S3 cells following infection by the lytic virus herpes simplex virus type 1 (HSV-1). Na+/K(+)-ATPase activity considerably decreased during the first few hours post-infection (p.i.), whereas Na+ and K+ concentrations were not significantly affected until a much later period. By 8 h p.i., a partial membrane depolarization in infected cells had occurred, as indicated by a small change in the transmembrane potential. HSV infection induced a time-dependent lipid peroxidation of HeLa cell plasma membranes temporally correlated with the progressive reduction in Na+/K(+)-ATPase activity. Moreover, a significant decrease of membrane fluidity appeared at a late phase of the viral replicative cycle probably representing cumulative membrane damage. These results demonstrate that HSV-1 infection induced the production of free radicals in non-phagocytic cells. Since lipid peroxidation begins at an early stage of the virus replicative cycle, it may be directly related to viral cytopathicity.

Compelling evidence indicates that the endothelium-derived potent vasoconstrictor endothelin-1 (ET-1) stimulates aldosterone secretion by interacting with specific receptors. Although two different ET-1 receptors have been identified and cloned, the receptor subtype involved in mediating aldosterone secretion is still unknown. Accordingly, we wished to investigate whether the genes of ET-1 and of its receptors A and B are expressed in the normal human adrenal cortex. We designed specific primers for ET-1 and the ETA and ETB receptors genes and developed a reverse transcription polymerase chain reaction (RT-PCR) with chemiluminescent quantitation of the cDNA. In addition, we carried out 125I ET-1 displacement studies with cold ET-1, ET-3 and the specific ETA and ETB ligands BQ123 and sarafotoxin 6C. Localization of each receptor subtype was also investigated by autoradiography. Binding experiments were first individually analyzed by Scatchard and Hofstee plot and then coanalyzed by the nonlinear iterative curve fitting program Ligand. Histologically normal adrenal cortex tissue, obtained from kidney cancer patients (n = 7), and an aldosterone-producing adenoma (APA), which is histogenetically derived from the zona glomerulosa (ZG) cells, were studied. Results showed that the ET-1, ETA and ETB mRNA can be detected by RT-PCR in all adrenal cortices as well as in the APA. The best fitting of the 125I ET-1 displacement binding data was consistently provided by a two-site model both in the normal adrenal cortex (F = 22.1, P < 0.0001) and in the APA (F = 18.4, P < 0.0001). In the former the density (Bmax) of the ETA and ETB subtype was 2.6 +/- 0.5 pmol/mg protein (m +/- SEM) and 1.19 +/- 0.6, respectively. The dissociation constant (Kd) of ET-1, ET-3, S6C, and BQ-123 for each receptor subtype resulted to be within the range reported for human tissue for the ETA and ETB receptors. In the APA tissue the Bmax tended to be lower (1.33 and 0.8 pmol/mg protein, for the ETA and ETB, respectively) but the Kd were similar. Autoradiographic studies confirmed the presence of both receptor subtypes on the ZG as well as on APA cells. Thus, the genes of ET-1 and both its receptor subtypes ETA and ETB are actively transcribed in the human adrenal cortex. Furthermore, both receptor subtypes are translated into proteins in ZG and APA cells.