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In the absence of efficient preventive vaccines, topical microbicides offer an attractive alternative in the prevention of Herpes simplex type 1 (HSV-1) and type 2 (HSV-2) infections. Because of their recognized anti-adhesive activity against bacterial pathogens, cranberry (Vaccinium macrocarpon Ait.) extracts may represent a natural source of new antiviral microbicides. However, few studies have addressed the applications of cranberry extract as a direct-acting antiviral agent. Here, we report on the ability of the novel cranberry extract Oximacro(®) and its purified A-type proanthocyanidins (PACs-A), to inhibit HSV-1 and HSV-2 replication in vitro. Analysis of the mode of action revealed that Oximacro(®) prevents adsorption of HSV-1 and HSV-2 to target cells. Further mechanistic studies confirmed that Oximacro(®) and its PACs-A target the viral envelope glycoproteins gD and gB, thus resulting in the loss of infectivity of HSV particles. Moreover, Oximacro(®) completely retained its anti-HSV activity even at acidic pHs (3.0 and 4.0) and in the presence of 10% human serum proteins; conditions that mimic the physiological properties of the vagina - a potential therapeutic location for Oximacro(®). Taken together, these findings indicate Oximacro(®) as an attractive candidate for the development of novel microbicides of natural origin for the prevention of HSV infections.

Antimicrobial resistance is a global issue currently resulting in the deaths of hundreds of thousands of people a year worldwide. Data present in the literature illustrate the emergence of many bacterial species that display resistance to known antibiotics; Acinetobacter spp. are a good example of this. We report here that Acinetobacter radioresistens has a Baeyer-Villiger monooxygenase (Ar-BVMO) with 100% amino acid sequence identity to the ethionamide monooxygenase of multidrug-resistant (MDR) Acinetobacter baumannii . Both enzymes are only distantly phylogenetically related to other canonical bacterial BVMO proteins. Ar-BVMO not only is capable of oxidizing two anticancer drugs metabolized by human FMO3, danusertib and tozasertib, but also can oxidize other synthetic drugs, such as imipenem. The latter is a member of the carbapenems, a clinically important antibiotic family used in the treatment of MDR bacterial infections. Susceptibility tests performed by the Kirby-Bauer disk diffusion method demonstrate that imipenem-sensitive Escherichia coli BL21 cells overexpressing Ar-BVMO become resistant to this antibiotic. An agar disk diffusion assay proved that when imipenem reacts with Ar-BVMO, it loses its antibiotic property. Moreover, an NADPH consumption assay with the purified Ar-BVMO demonstrates that this antibiotic is indeed a substrate, and its product is identified by liquid chromatography-mass spectrometry to be a Baeyer-Villiger (BV) oxidation product of the carbonyl moiety of the β-lactam ring. This is the first report of an antibiotic-inactivating BVMO enzyme that, while mediating its usual BV oxidation, also operates by an unprecedented mechanism of carbapenem resistance.

The defense against influenza virus (IV) infections still poses a series of challenges. The current antiviral arsenal against influenza viruses is in fact limited; therefore, the development of new anti-influenza strategies effective against antigenically different viruses is an urgent priority. Bioactive compounds derived from medicinal plants and fruits may provide a natural source of candidates for such broad-spectrum antivirals. In this regard, cranberry (Vaccinium macrocarpon Aiton) extracts on the basis of their recognized anti-adhesive activities against bacteria, may provide potential compounds able to prevent viral attachment to target cells. Nevertheless, only few studies have so far investigated the possible use of cranberry extracts as an antiviral tool. This study focuses on the suitability of a cranberry extract as a direct-acting anti-influenza compound. We show that the novel cranberry extract Oximacro® inhibits influenza A and B viruses (IAV, IBV) replication in vitro because of its high content of A-type proanthocyanidins (PAC-A) dimers and trimers. Mechanistic studies revealed that Oximacro® prevents attachment and entry of IAV and IBV into target cells and exerts a virucidal activity. Oximacro® was observed to interact with the ectodomain of viral hemagglutinin (HA) glycoprotein, thus suggesting the interference with HA functions and a consequent loss of infectivity of IV particles. Fluorescence spectroscopy revealed a reduction in the intrinsic fluorescence of HA protein after incubation with purified dimeric PAC-A (PAC-A2), thus confirming a direct interaction between HA and Oximacro® PAC-A2. In silico docking simulations further supported the in vitro results and indicated that among the different components of the Oximacro® chemical profile, PAC-A2 exhibited the best binding propensity with an affinity below 10 nM. The role of PAC-A2 in the anti-IV activity of Oximacro® was eventually confirmed by the observation that it prevented IAV and IVB replication and caused the loss of infectivity of IV particles, thus indicating PAC-A2 as the major active component of Oximacro®. As a whole, these results suggest Oximacro® as a potential candidate to create novel antiviral agents of natural origin for the prevention of IV infections.

Purpose The aim of this vitro study was to reproduce and evaluate the response of bone and bacteria to traditional and innovative implant surfaces with difference wettability. Materials and methods Two hundred fifty-two samples made of grade 4 titanium with different coating (machined [MAC]; double-etched, Ti-AE; zirconium nitride [Ti-ZrN]) were used for this in vitro study. Disks were divided into test (bioactivated using plasma of argon) and control group (untreated). To assess the surface morphology of the specimens, representative images were acquired via scanning electron microscopy (SEM). Murine preosteoblasts (MC3T3-E1) were used to study the biologic response in vitro, while the quantification of protein adsorption was achieved through the incubation of the titanium samples in a 2% solution of fetal bovine serum (FBS) in phosphate-buffered saline (PBS). The sterilized titanium disks were then colonized by bacterial species from a single sputum sample obtained from a healthy volunteer. For every analysis, 24 disks were used (12 for each group). Results SEM and topographic analyses demonstrated a Sa value of 0.33 (Ti-ZrN), 0.34 (MAC), and 0.62 (Ti-AE). Compared with the control groups, plasma treatment significantly increased the protein adsorption level on all the different titanium surfaces (5.88 ± 0.21 vs 7.85 ± 0.21, 7.13 ± 0.14 vs 9.74 ± 0.65, 4.41 ± 0.62 vs 6.13 ± 0.52, respectively, for MAC, Ti-treated, and Ti-ZrN). Similar behavior was described for cell adhesion (27.67 ± 2.03 vs 58.00 ± 20.13, 116.67 ± 12.02 vs 159.33 ± 8.09, 52.00 ± 4.73 vs 78.33 ± 4.67, respectively, for MAC, Ti-treated, and Ti-ZrN). Plasma treatment significantly augmented the number of CFU only in MAC and ZrN samples. Conclusion With the limitations of this in vitro study, the following conclusions could be drawn: (1) rough implant surfaces present a higher adhesion and proliferation of preosteoblastic cells and bacterial biofilm; (2) rough implant surfaces benefited the most by the plasma of argon treatment.

Urinary tract infections (UTIs) are one of the most common pathological conditions in both community and hospital settings. It has been estimated that about 150 million people worldwide develop UTI each year, with high social costs in terms of hospitalizations and medical expenses. Among the common uropathogens associated to UTIs development, UroPathogenic Escherichia coli (UPEC) is the primary cause. UPEC strains possess a plethora of both structural (as fimbriae, pili, curli, flagella) and secreted (toxins, iron-acquisition systems) virulence factors that contribute to their capacity to cause disease, although the ability to adhere to host epithelial cells in the urinary tract represents the most important determinant of pathogenicity. On the opposite side, the bladder epithelium shows a multifaceted array of host defenses including the urine flow and the secretion of antimicrobial substances, which represent useful tools to counteract bacterial infections. The fascinating and intricate dynamics between these players determine a complex interaction system that needs to be revealed. This review will focus on the most relevant components of UPEC arsenal of pathogenicity together with the major host responses to infection, the current approved treatment and the emergence of resistant UPEC strains, the vaccine strategies, the natural antimicrobial compounds along with innovative anti-adhesive and prophylactic approaches to prevent UTIs.

Evidence demonstrating that human rhinovirus (HRV) disease is not exclusively limited to the upper airways and may cause lower respiratory complications, together with the frequency of HRV infections and the increasing number of immunocompromised patients underline the need for rapid and accurate diagnosis of HRV infections. In this study, we developed the first quantitative real-time nucleic acid sequence-based amplification assay with an internal control using molecular beacon probes for selective and sensitive detection of human rhinovirus serotypes. We described a simple method to accurately quantify RNA target by computing the time to positivity (TTP) values for HRV RNA. Quantification capacity was assessed by plotting these TTP values against the starting number of target molecules. By using this simple method, we have significantly increased the diagnostic accuracy, precision, and trueness of real-time NASBA assay. Specificity of the method was verified in both in silico and experimental studies. Moreover, for assessment of clinical reactivity of the assay, NASBA has been validated on bronchoalveolar lavage (BAL) specimens. Our quantitative NASBA assay was found to be very specific, accurate, and precise with high repeatability and reproducibility.

The genes encoding glycoprotein complexes of human cytomegalovirus are often polymorphic; in particular, glycoprotein B (gB), which is essential for both in vivo and in vitro replication, is encoded by the highly polymorphic gene UL55. In this study, the distribution of gB genotypes was investigated in 44 bronchoalveolar lavage specimens from adult patients positive for human cytomegalovirus DNA by a multiplex nested fast PCR able to amplify 5 gB genotypes (gB1–gB5). The distribution of gB genotypes was as follows: 12 (27.3%) gB1, 11 (25%) gB2, 9 (20.4%) gB3, 4 (9.1%) gB4, 0 gB5, and 8 (18.2%) mixed genotypes. No difference in prevalence was found in relation to clinical features, including immunological status, non-transplant or transplant condition, and type of transplanted organ, or in follow-up specimens; while gB4 and gB3 were shown to be significantly more prevalent in patients with respiratory insufficiency, and gB4 and gB2 in those with pneumonia. The prevalence of gB genotypes in the lower respiratory tract was similar to that previously reported using other specimen types and patients, with gB1 found to be the most prevalent. The association of gB genotypes with specific clinical features should be further investigated.

The presence of non-organ-specific (NOSA) and anti-endothelial antibodies (AECAs) and the onset of rejection in relation to cytomegalovirus (CMV) infection was investigated in 96 renal transplant recipients: 48 CMV pp65-antigenemia-negative (group 1) and 48 positive (group 2). The presence of autoantibodies (autoAbs) was evaluated before and following renal transplantation (first three months) by indirect immunofluorescence. Before transplantation, none of the patients was positive to AECAs, while eight (8.3%) were positive to NOSAs. Post-transplantation, AECA were found in none of patients from group 1 vs. 15/48 (31.2%) from group 2 (p

BackgroundRecently, three new polyomaviruses—KI, WU and Merkel cell (MCV)—have been discovered and their detection has been reported in different types of specimens, including respiratory samples, suggesting their shedding in the airways. In lung graft recipients, viral agents are associated with events that may limit the success of transplantation, including organ infection/disease and allograft rejection.AimsTo evaluate the prevalence of KI, WU and MCV in transbronchial biopsies from lung transplant recipients and investigate the association with clinical and histopathological features.MethodsThe quantitation of new polyomaviruses DNA by real-time PCR and association with clinical and histopathological findings were evaluated in 66 transbronchial biopsies from lung transplant recipients.ResultsKI, WU and MCV were detected in 9.2%, 12.3% and 33.8% of specimens, respectively; with mean viral load ranging from 81 copies/104 cells for WU to 258 for MCV, thus not differing from that previously reported in native lungs. No significant association with clinical and histopathological findings (including acute respiratory insufficiency, interstitial and organising pneumonia, acute and chronic rejection) was found.ConclusionsResults showed a relatively high frequency of detection of the novel polyomaviruses in transbronchial biopsies from lung transplant recipients. It is likely that this accounted for the positive results found in some cases with different pathological background, although no significant association with a specific clinical and/or histopathological pattern was found.

The newly discovered polyomaviruses KI and WU (KIV and WUV) were isolated from secretions of patients with respiratory symptoms as well as in blood, spleen, lymphoid tissues, and stools, especially in immunocompromised conditions. The aim of this work was to evaluate the prevalence of KIV and WUV in bronchoalveolar lavage (BAL) from lung transplant recipients. We also examined potential correlations between these viruses and occurrences of pneumonia, acute respiratory insufficiency, or other acute respiratory conditions and acute rejection episodes. Discharge diagnosis was based on the International Classification of Diseases-Italian version 2002, based on the 9th-revision clinical modification. A rejection episode was diagnosed by transbronchial lung biopsy in accordance with the 2007 International Society for Heart and Lung Transplantation Working Formulation. Overall, we analyzed 53 BALs obtained from 24 transplant recipients. Positive polymerase chain reaction results were observed in 6 samples (11.3%) from 6 patients (25%), versus 7 samples (13.2%) from 7 patients (29.2%) for KIV and WUV, respectively. Regarding the diagnosis of pneumonia, the prevalence was 22.2% and 33.3% for KIV and WUV, respectively. In cases of acute respiratory insufficiency or other acute respiratory conditions, 2 out of 9 samples were positive for KIV (22.2%) and 4 out of 9 for WUV (44.4%). An Acute rejection episode (ARE) was diagnosed in 7 instances among 6 lung transplant patients: The corresponding BAL specimens showed positive results for KIV in 3 out of 7 (42.8%) cases with ARE vs 3 out of 46 (6.5%) without an ARE (P < .05), and for WUV in 3 out of 7 (42.8%) vs 4 out of 46 (8.7%) (P < .05), respectively. Although the small number of specimens limits the statistical analysis, our results showed a higher prevalence of WUV compared with KIV. The compromised pulmonary environment in the lung allograft may cause reactivation of these viruses. Their roles in this context need to be further evaluated.

Background Human parvovirus B19 infection generally displays a self-limiting course followed by viral clearance; although, in some cases, persistent infection may occur. Few cases of severe pulmonary disease following primary infection in both immunocompetent and immunocompromised patients were reported. Objectives To investigate the prevalence and clinical impact of parvovirus B19 in the lower respiratory tract. Study design The prevalence of parvovirus B19-DNA was evaluated by Real-Time PCR in 264 bronchoalveolar lavages (BAL) from 189 adult patients over a full-year period and related to demographic characteristics, underlying pathologies, immune status, admission to intensive care unit, mortality within 28 days, and discharge diagnosis. Results Parvovirus B19-DNA was detected in 7/189 (3.7%) patients, without significant association to demographic characteristics, immune status, transplant versus non-transplant status, admission to intensive care unit, presence of haematological conditions. In two lung transplant recipients surveillance specimens were positive to B19. Four of the remaining five patients presented respiratory insufficiency. A significant association to mortality was found, as 3/7 (42.9%) positive patients died within 28 days. No patient presented serological evidence of recent or acute infection and viremia. Conclusions Parvovirus B19 may be detected at low frequency in BAL specimens from patients with different pathological backgrounds. This finding could be due to chronic infection with virus persistence in the lower respiratory tract, also in the absence of symptoms unequivocally attributable to B19. The high rate of mortality warrants the need for further studies to evaluate the opportunity to consider parvovirus B19 in the diagnostic work-up of lower respiratory tract infections.

Human parainfluenza viruses (HPIVs) are distributed worldwide and are involved mainly in the pathogenesis of respiratory tract infections. The development and optimization of three quantitative reverse transcription real time polymerase chain reactions (RT Real Time Qt-PCRs) and an indirect immunofluorescence (IFA) for the detection and quantitation of HPIV-1, -2 and -3 in clinical samples are described. Efficiency, sensitivity, specificity, inter- and intra-assay variability and turnaround time of the two methods were compared. These assays have been validated on 131 bronchoalveolar lavage specimens. Based on the results obtained, the molecular methods represent a valid and rapid tool for clinical management and should be included in diagnostic panels aimed to evaluate suspected respiratory tract infections.

Background Polyomavirus-associated nephropathy (PVAN) is one of the most common viral disease affecting renal allograft, with BK being the most frequent causal agent and JCV being considered responsible in Objectives To quantify polyomaviruses BK and JC load by real-time TaqMan PCR in tissue specimens (renal and ureteral) from kidney transplant recipients. Study design and methods One-hundred-thirty-eight specimens (125 kidneys, 13 ureters) obtained from 109 patients were evaluated by quantitative real-time PCR for the detection of BKV- and JCV-DNA. Demographic, virological, and histopathological data were collected. Results BKV-DNA was positive in 32 of 109 patients (29.6%) and JCV-DNA in 20 of 109 patients (18.3%). The highest BK viral loads (>104 genome equivalents/cell) were found in two renal samples with histopathologically confirmed PVAN; while JC viral load was >104 genome equivalents/cell in one ureteral sample. Conclusions Although quantitation of viral DNA on renal allograft biopsies could be complementary to histopathological evaluation and the highest viral load are detectable in renal specimens with PVAN, the identification of a diagnostic cut-off should require further studies.

Background. BK virus-associated nephropathy (BKVAN) is one of the most common viral diseases affecting renal allografts. Screening for viral replication may allow for earlier intervention with reduced allograft loss. A plasma viral load >10 4 copies/mL of BKV DNA is recommended for a presumed diagnosis of BKVAN. Methods. We monitored BKV load on serum and urine samples by Real-Time TaqMan PCR in 229 renal transplant recipients in the first year post-transplantation. Overall, 2025 serum and 2025 urine samples were evaluated. A graft biopsy was performed in 47/229 patients to investigate the declining renal function. Operating characteristics [sensitivity, specificity, negative predictive value (NPV), positive predictive value (PPV)] and receiver operating characteristic (ROC) curve analysis at different viral load values were calculated. Results. Serum BKV viral load was > 10 4 in 5/229 patients (2.2%). A histological diagnosis of BKVAN was made in 3/229 patients (1.3%): 3/5 (60.0%) among those with serum viral load >10 4 and 3/4 (75.0%) in those with >1.6 × 10 4 . Operating characteristics of a serum BK load of 10 4 for the diagnosis of BKVAN were as follows: sensitivity, 100%; specificity, 99.1%; NPV 100%; PPV 59.4%. Specificity and PPV rose to 99.6% and 75.0% when using a cut-off level of 1.6 x 10 4 copies/mL. Conclusions. The recommended level of BK viraemia of 10 4 copies/mL is useful to identify patients at risk of BKVAN, although specificity and PPV increase by using a cut-off level of 1.6 x 10 4 copies/mL. BK replication may occur in the first 3 months post-transplantation and subsequently recede. Therefore, the temporal profile of BKV replication has to be accurately evaluated and occasionally elevated values should prompt a closer monitoring.

Deoxyribonucleic acid (DNA) has been mostly considered as a carrier of genetic information and gene expression. Recently, we have demonstrated that this biomacromolecule is very effective in providing cellulosic fabrics with flame retardant features, thanks to its intrinsic intumescent-like behaviour. Here, we have investigated the role played by different parameters on the overall flame retardant features: DNA molecular size, pH of the DNA aqueous solutions, and number of impregnations. For this purpose, herring DNA, at three different molecular sizes, low (100–200 base pairs, bp), medium (400–800 bp) and high (2000–10,000 bp), has been selected as model biomacromolecule; its effect as fire protective coating on cotton fabrics has been assessed in terms of resistance to a flame application (through horizontal flame spread tests) and to an irradiative heat flux of 35 kW/m 2 (by cone calorimetry). Furthermore, thermogravimetric analyses have been exploited for evaluating thermal and thermo-oxidative stability of the treated fabrics. The results clearly indicate that, despite a similar composition, the coatings containing low molecular size DNA exert a superior fire protection on the fabric substrate, as clearly indicated by horizontal flame spread and cone calorimetry tests. Multiple impregnation treatment turned out to be more performing than the single one, being equal the final add-on achieved. More specifically, the use of multiple impregnations at pH = 4 and 8 allowed achieving self-extinction for 86 and 74% of the tested samples; in addition, 45 and 25% reductions of total heat release and heat release rate peak under 35 kW/m 2 have been found with respect to untreated cotton. These findings may be attributed to themorphology of the low molecular size DNA coating, which shows a greater penetration into the microfibrillar surfaces of the component fibres within the fabric and to its higher thermal stability in air.

Immunofluorescenza diretta (DFA) su cellule HEP2 e R-Mix Too per la rilevazione degli Adenovirus

In lung transplant recipients, cytomegalovirus (CMV) has been associated with direct ie, organ and systemic infection/disease, and indirect effects, including predisposition to develop acute rejection episodes and chronic allograft dysfunction. Cellular immune responses have been demonstrated to play a role in the control of CMV replication. We evaluated CMV-specific cellular responses among lung transplant recipients associated with the onset of organ infection/disease. Cellular responses were evaluated by an Elispot assay of 48 specimens from 24 patients. All samples were evaluated beyond 1 year after transplantation; CMV DNA was concomitantly detected in bronchoalveolar lavage (BAL) and whole blood specimens. Each patient received a combined prolonged antiviral prophylaxis with CMV Ig for 12 months and gancyclovir or valgancyclovir for 3 weeks after postoperative day 21. Nine patients (37.5%) showed transient or persistent CMV nonresponses including donor-recipient negative serologic matching in 2 cases. Positive CMV DNA results were observed in 18/48 BAL specimens (37.5%) from 12 patients (50%). A viral load of >10(4) copies/mL was observed in only 3 cases, 2 of whom were positive also on whole blood. Among these 3 patients, 2 were responders and BAL (as well as whole blood) specimens collected subsequently were negative for CMV DNA; 1 nonresponder patient exhibited a viral load of 426,492 copies/mL BAL (DNAemia

Background: Psoriasis is sustained by pro-inflammatory CD4+ T helper cells mainly belonging to the Th1, Th17 and Th22 lineage. Objective: To identify whether treatment with the anti-tumour-necrosis-factor antagonist etanercept is able to induce significant modulations in transcription factor and cytokine mRNA gene expressions related to the different T cell immune response polarization (Th1, Th2, Th17 and regulatory T cells, Treg) and to correlate them with clinical response. Methods: The study population included 19 psoriasis patients treated with etanercept and 19 healthy subjects. Blood samples were collected at baseline and every 4 weeks during treatment. Taqman quantitative real-time polymerase chain reaction was applied to analyse the expression of: Stat-4, T-bet, IL-12p35 and IFN-γ (Th1-related); GATA-3, IL-4 (Th2-related); Stat-3, RORγt, IL-23p19 (Th17-related); Foxp3, IL-2 (Treg-related). Flow cytometry was applied to analyse CD4+CD25+brightFoxp3+ cells in peripheral blood. Results: Upregulation of Th1 and Th17 and downregulation of Treg subsets was found at baseline. The response to etanercept could be associated with a significant reversal of the Th1/Th17 activation, and a concomitant upregulation of Th2 and Treg subsets. Conclusion: Our data may contribute to a better understanding of the mechanisms underlying the achievement of clinical response in psoriasis and could be helpful for the identification of early predictive markers of response.

The occurrence of human rhinoviruses (HRV) and its relationship to clinical and histopathological findings were investigated in 127 bronchoalveolar lavage specimens from 36 lung transplant recipients by real-time RT-PCR. In addition, 286 samples from 235 other immunocompromised and immunocompetent patients were also studied. HRV was detected in 41.7% of lung transplant recipients vs 14.5% of other patients (p0.0001), and no differences in viral load were observed. Acute respiratory insufficiency was found in 15 cases, three of which were HRV positive (viral load, 6.3 x 10(6) RNA copies/ml in one patient with chronic graft dysfunction). A diagnosis of pneumonia was made in 10 out of 127 cases, two of which were HRV positive (viral load, 10(3)-10(4) in cases of co-infection). Acute rejection was diagnosed in 12 cases, three of which were HRV positive (viral load, 10(3) in two cases of co-infection and 10(5) in a single infection). HRV infection may involve the lower respiratory tract, particularly in the presence of an impaired pulmonary background, such as a transplanted lung. Clinical evaluation should take into account the viral load, with a load of10(5) possibly being associated with clinical symptoms, although lower loads can be detected in both symptomatic and asymptomatic patients.

Real Time RT-PCR developed in recent years represents an useful tool in the diagnosis of RNA viruses. In order to accurately quantify and normalize a RNA target, efficiency of reverse-transcription must be considered. In this study, a cRNA-standard-based quantitative Real Time RT-PCR have been developed for HRV quantification on bronchoalveolar lavage (BAL) specimens. Results has been compared to a quantitative plasmid standard-based Real Time RT-PCR previously developed by us. Large amount of pHRV was linearized and purified. Blunt ends were generated and cRNA production was carried out. Dilutions of cRNA were generated and dynamic range, intra- and inter-test variability, sensitivity, and limit of detection were evaluated. Sixty-seven BAL, previously resulted positive to our plasmid standard-based method, were evaluated using cRNA-standard quantification. cRNA curve showed a broad dynamic range with a good intra- and inter-test variability, with an average of 3.23 threshold cycles more in comparison to plasmid standard-based curve. In terms of specimen quantification, a difference of 1.07 log was found, showing a significant underrate using plasmid standard-based quantification. The method for cRNA-standard construction seems more suitable for quantification of RNA viruses, in order to normalize the quantification in reverse-transcription.

Introduction. Human polyomavirus BK (BKV) is worldwide distributed, with a seroprevalence rate of 70–90% in the adults. Following primary infection, BK remains latent in the renourinary tract as the epidemiologically most relevant latency site, and in B cell, brain, spleen and probably other tissues. Reactivation may occur in both immunocompetent subjects and immunocompromised patients. In renal transplantation, in the context of intense immunosuppression, viral replication may determine BKV-associated nephropathy (BKVAN) with interstitial nephritis and/or ureteral stenosis in 1–10% of the patients and leading to graft failure and return to haemodialysis in 30 to 80% of the cases (5). Screening of BKV replication represents the basic strategy to predict early the onset of BKVAN and may allow for earlier intervention with reduced allograft loss (3, 4). Nowadays, replication of BKV is monitored by quantification of BKV-DNA in serum and urine (2). The aim of this study was to evaluated the role of BKV VP1 mRNA in urine as a marker of viral replication in renal transplant recipients.

Introduction. Human Bocavirus (HBov) is a ubiquitous parvovirus predominantly associated with respiratory tract infections in pediatric patients.The results of the few studies conducted in adult patients are conflicting, with data of prevalence around 0.8%, thus making difficult to evaluate the HBOV clinical role. In this study the prevalence and clinical role of HBoV in adult hospitalized patients were evaluated. Methods. The presence of HBoV was evaluated in 514 bronchoalveolar lavages (BAL), obtained over a period of 24 months from 341 adult patients (mean age, 56.5 ± 16.2 years, range 19-85), by real-time TaqMan PCR.The BAL was performed for the presence of symptoms/signs of suspected infection of the lower airways or for surveillance in the transplanted lung patients (month 1 and every 3 months). Results. 12/341 patients (3.5%) were positive for HBoV; in particular: 1/45 (2.2%) lung transplanted, 1/20 (5%) liver transplanted and 2/26 (7.7%) bone marrow transplanted, while no kidney (n=19) or heart (n=13) transplant patients resulted positive. 8/218 (3.6%) not transplantpatient were positive, in particular 1.8% patients with haematological cancers and 1.8% with other disease.The prevalence of HBoV did not differ between transplanted and not transplanted patients, depending on the type of transplant, and between immunocompetent and immunocompromised individuals. Conclusions. HBoV can be detected at low frequency in BAL of adult patients with acute lower respiratory tract, further studies would show whether the virus plays a role as a single pathogen or whether the altered lung background could help the viral infection.

Introduction: The Cytomegalovirus (CMV) is the major cause of morbidity and mortality in solid organ (SOT) and bone marrow (BMT) transplantation. An early reconstitution of immune response is crucial in limiting CMV replication; on the other hand, a late reconstitution may determine CMV reactivation with possible evolution to symptomatic phase.The Enzyme-Linked Immunosorbent Spot (EliSPOT) assay is a useful tool for monitoring the CMV-specific immune response recovery in transplant patients.This study propose the development and optimization of a interferon-gamma-(IFN-γ)-based EliSPOT assay for the detection of CMV immune response. Methods: CD3 + lymphocytes were separated using Robosep (negative selection, HLA ® EasySep WB Human T Cell Enrichment Kit, Stemcell Technology,Vancouver, Canada).The EliSPOT assay was optimized using the Human IFN gamma ELISPOT Kit and a modified protocol provided by Nanogen Advanced Diagnostics (Buttigliera Alta, Italy). Different parameters were analyzed: number of cells (200,000 - 300,000), antigens (CMV peptide mix and whole CMV antigen), incubation time (18 - 20 - 22 - 24h), number of washes, incubation conditions with secondary antibody and conditions of substrate development. Results: Herein we report the optimal conditions identified. 200,000 CD3+ cells per well were stimulated with CMV peptide mix antigens. Cells were stimulated for 18h at 37 °C in humidified atmosphere; wells were washed and secondary antibody was added and incubated at room temperature for 2h.After incubation a second series of washes were performed. Substrate was added and incubation for 15 minutes was carried out.The reaction was detected by plate reader AID ELISPOT (Strassberg, Germany). Conclusions:We developed an EliSPOT assay for the detection of CMV-immune response and reconstitution.All variables were compared to previous published protocols.The improvement of the EliSPOT assay for the detection of CMV-specific immune response represents the first step for result evaluation in clinical practice.

The Polyomaviruses KI and WU have been identified in 2007 in nasopharyngeal aspirates of patients with acute respiratory tract infections.They have been further isolated in faecal samples, lymphoid tissue, bronchoalveolar lavages and blood, although their pathological associations, molecular epidemiology and tissue tropism are still not widely known.The aim of this study was to determine the presence of KIV and WUV in tonsillar tissue of immunocompetent patients to assess whether the tonsils may represent a site of infection and/or persistence.The presence of Polyomavirus BKV, JCV and SV40 was also evaluated. The presence of Polyomavirus KI,WU, BK, JC and SV40-DNA was evaluated in a prospective study on tonsillar samples obtained from 29 immunocompetent patients (adults and children undergoing tonsillectomy) by a commercially available Taqman Real-time quantitative PCR (BKV Q-PCR Alert Kit, Nanogen) and a home-made system (for KIV,WUV, SV40, JCV).The amplification conditions were optimized and standardized. KIV-DNA was detected in 2/29 (6.9%) tonsil tissue (obtained from an adult and a child), while WU, BK, JC and SV40 were negative in all cases. Our prevalence results are consistent with those reported in the literature on samples from the respiratory tract and lymphoid tissue such as tonsils. Further studies are needed to understand the significance of the presence of KIV in tonsil tissue and assess the possibility that the virus can establish latent and/or persistent infection at this level.

Recently, three new polyomaviruses-KI, WU and Merkel cell (MCV)-have been discovered and their detection has been reported in different types of specimens, including respiratory samples, suggesting their shedding in the airways. In lung graft recipients, viral agents are associated with events that may limit the success of transplantation, including organ infection/disease and allograft rejection.To evaluate the prevalence of KI, WU and MCV in transbronchial biopsies from lung transplant recipients and investigate the association with clinical and histopathological features.The quantitation of new polyomaviruses DNA by real-time PCR and association with clinical and histopathological findings were evaluated in 66 transbronchial biopsies from lung transplant recipients. Results KI, WU and MCV were detected in 9.2%, 12.3% and 33.8% of specimens, respectively; with mean viral load ranging from 81 copies/10(4) cells for WU to 258 for MCV, thus not differing from that previously reported in native lungs. No significant association with clinical and histopathological findings (including acute respiratory insufficiency, interstitial and organising pneumonia, acute and chronic rejection) was found.Results showed a relatively high frequency of detection of the novel polyomaviruses in transbronchial biopsies from lung transplant recipients. It is likely that this accounted for the positive results found in some cases with different pathological background, although no significant association with a specific clinical and/or histopathological pattern was found.

The respiratory syncytial virus (RSV) is the major cause of respiratory infection in children, with bronchopneumonia and bronchiolitis. In the adult the clinical manifestation is lighter than in infant, however, in the case of immunocompromised patients (transplant recipients or patients in immunosuppressive treatment) infection can lead to death. Immunofluorescence is a diagnostic procedures which can detect RSV infection and the presence of viable virus in the biological sample of the patient. In this study, indirect and direct immunofluorescence techniques using monoclonal antibodies directed to RSV have been compared. For the comparison HEP2 shell vials, that are susceptible to RSV, were used.These cells were infected with different virus titres, ranging from 102 to 10-3 TCID50. Subsequently, different primary or Fluorescein isothiocyanate antibody dilutions were tested (1:40, 1:80, 1:160), for indirect and direct immunofluorescence evaluation, respectively, at three incubation periods after infection (48h, 72h, 96h). Indirect immunofluorescence showed a sensitivity of 101 TCID50, with a cells positive detection even at lower dilutions up to 10-1 TCID50 at 72 hours. For direct immunofluorescence, the same sensitivity was observed with 1:40 antibody dilution at 72h, with a detection limit of 10-1 TCID50. Indirect immunofluorescence showed optimal RSV detection at 72h after infection with 1:80 primary antibody dilution, with a sensitivity of 101 TCID50. An equivalent sensitivity has been observed with direct immunofluorescence at 72h, 1:40 antibody dilution, thus representing an advantage in terms of time.

Objectives: Human herpesvirus-7 (HHV-7) is a highly seroprevalent virus that, following primary infection, establishes latency or persistence in some tissues, including lung. The aim of this study was to investigate the prevalence of HHV-7 in the lower respiratory tract of hospitalized adult patients. Methods: The prevalence of HHV-7 DNA was determined by quantitative real-time PCR in 212 bronchoalveolar lavage (BAL) samples obtained from 153 patients. The molecular epidemiology and clinical role of HHV-7 were evaluated. Results: HHV-7 DNA was positive in 44 of 212 specimens (20.7%), obtained from 40 of 153 patients (26.1%), in particular 22/68 (32.35%) and 18/86 (20.9%) in transplant and non-transplant patients, respectively (1 patient evaluated both before and after transplantation). No significant difference according to transplant condition or discharge diagnosis was found. Viral load was >100,000 genome equivalents/ml BAL in 6/22 (27.3%) transplant recipients and 4/18 (22.2%) non-transplant patients (p = n.s.). Conclusions: The evaluation of HHV-7 DNA in BAL may be useful to investigate its potential role in lower respiratory tract infection, alone or in association with other viral and/or non-viral pathogens and to distinguish latency from reactivation.

Human herpesvirus 7 is a highly seroprevalent β-herpesvirus that, following primary infection, remains latent in CD4+ T cells and determines a persistent rather than a latent infection in various tissues and organs, including the lung and skin. This paper describes the development of an in-house light upon extension real-time PCR assay for quantification of human herpesvirus 7 DNA in clinical samples. The efficiency, sensitivity, specificity, inter- and intra-assay variability, and dynamic range have been determined. Subsequently, the assay has been validated by evaluating the human herpesvirus 7 load in bronchoalveolar lavages and skin specimens, chosen as 2 persistency sites, from healthy and pathological individuals. The real-time PCR assay developed in this study could be a useful tool to detect and quantify human herpesvirus 7 DNA in different clinical specimens to elucidate its epidemiological and pathogenic roles.

Recently, polyomaviruses KI and WU were identified in the airways of patients with acute respiratory symptoms. The epidemiology and pathogenesis of these two viruses are not fully understood, and the development of molecular assays, such as Real Time PCR, was useful for examining their biology and role in different clinical syndromes. The evaluation of different target regions for the amplification of polyomaviruses KI and WU, comparing published primer/probe sets and sets designed in the laboratory is described and was used for testing 175 clinical specimens (84 stools and 91 tonsils). The results showed that the laboratory designs were more sensitive for the detection of polyomaviruses KI and WU DNA in clinical samples. The choice of the primer/probe set, and primarily of the region for amplification, may be relevant for understanding the pathogenic role of viruses such as polyomaviruses KI and WU.

Background. The lower respiratory tract is a latency site of Epstein-Barr virus (EBV); however, its pathogenic role is poorly known, particularly in transplant patients. The aim of this study was to evaluate the prevalence and role of EBV in bronchoalveolar lavages (BAL) from transplant recipients (TR) in comparison with nontransplant (NT) patients. Methods. Real-time quantitative polymerase chain reaction for EBV, human herpesvirus-6 (HHV-6), and HHV-7 and rapid shell-vial culture for human cytomegalovirus (HCMV) were performed on 272 consecutive BAL from 194 patients (107 from 59 TR and 165 from 143 NT). Results. EBV-DNA was positive in 65 specimens (23.9%) from 57 patients (29.4%): 24 of 59 (40.7%) TR and 33 of 143 (23.1%) NT (P

This paper describes the development of four Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR) assays devised to evaluate lytic (UL123, immediate-early; UL54, early; UL65, late; and UL99, true late) human cytomegalovirus (HCMV) transcripts. Subsequently, the assays have been validated evaluating the HCMV lytic gene expression in 28 samples (peripheral blood leukocytes, PBLs) from 14 renal transplant recipients. Although the assessment of HCMV transcriptional profile could be useful to evaluate viral reactivation state, further studies on dynamics of viral transcripts in different clinical settings are needed to determine the role of transcriptional profiling in virological monitoring and therapeutic management in immunocompromised patients.

Following primary infection, the polyomavirus BK remains latent in the urinary tract e peripheral blood cells. Reactivation may occur spontaneously in healthy subjects and in immunocompromised conditions. BKV reactivation may determine urinary shedding of infected urothelial cells, hemorrhagic cystitis (particularly in bone marrow transplant recipients), and nephropathy in kidney graft recipients. Moreover, a possible role for BKV in the pathogenesis of systemic lupus erythematosus (SLE) has been hypothesised. SLE is characterised by production of autoantibodies, in particular anti-double stranded (ds)-DNA antibodies. The induction of anti-dsDNA antibodies by BKV has been described in experimental animals and during naturally acquired infection in man. Therefore, it has been proposed that the BKV large T-antigen-chromatin complex may function as a hapten-carrier model with subsequent production of anti-dsDNA antibodies.Aim of this study was to evaluate the relation between BKV and autoimmunity in renal transplant recipients by determining the prevalence of non-organ-specific antibodies (NOSA) by indirect immunofluorescence on serum samples obtained from 95 renal transplant recipients during post-transplantation follow-up. BKV infection was evaluated by competitive-quantitative PCR. NOSA were present in 25/95 patients: 18 ANA and 6 SMA, one patient both ANA and SMA. BKV-DNA was positive in 16/95 patients: 3 NOSA-positive and 13 negative. BKV-DNA was negative in 79/95 patients: 22 NOSApositive and 57 negative. The prevalence of NOSA did not differ between BKV-DNA positive and negative patients. It does not seem to exist a relation between BKV and NOSA in renal transplant recipients.

Mycosis fungoides (MF), the most indolent form of CTCL, originates from a clonal expansion of epidermotropic helper/memory T cells. Sezary syndrome (SS) is a rare primay epidermotropic cutaneous T-cell lymphoma in leukemic. The aetiopathogenesis of MF and SS remains obscure despite several investigations. Infectious, environmental and genetic factors have been implicated as potential aetiological agents. The studies investigating the role of EBV in CTCL present conflicting results. The different sensitivities of the technical methods used in the evaluation of the presence of viral DNA or virus-related antigens make comparison of the results difficult. The aim of this study was to retrospectively evaluate the EBV-DNA load in skin biopsies from MF and SS patients by a highly sensitive (1-10 EBV-DNA copies/reaction) quantitative-competitive PCR (QC-PCR) developed in our lab to better asses the relationship between EBV and CTCL. Skin biopsies were obtained from 21 MF and 10 SS patients; skin biopsies from a 8 patients with inflammatory skin disease were used as controls. EBV-DNA was detected in 70% of biopsies from SS patients vs. 0% of MF patients. No control patients resulted EBV-DNA positive, as expected. In addition, in SS patients, the survival from diagnosis is lesser in EBV-positive patients vs.EBV-negative patients even if not statistically significant.We are going to investigate the presence of EBV-DNA in peripheral blood of a larger number of patients and to evaluate the pattern of viral genes expression, to better assess the aetiopathogenetical role of EB virus in this kind of neoplasies.

Infections of the central nervous system (CNS) represent a difficult diagnostic problem for both clinicians and microbiologists. In particular, the Herpesviridae family plays a central etiological role in CNS viral infections. These diseases have acquired growing importance in the past few years owing to the increasing number of immunocompromised patients and the availability of new antiviral drugs. Prompt detection and diagnosis of CNS viral infections are critical because most infections are treatable, while a delayed recognition may lead to life-threatening conditions or severe sequelae. The traditional methods for detection of herpesviruses in CNS infections exhibit several drawbacks, whereas the polymerase chain reaction (PCR) on cerebrospinal fluid has revolutionized the neurovirology and is becoming an essential part of the diagnostic work-up of patients with suspected CNS viral infections. A sensitive multiplex PCR method was developed for the simultaneous detection of 6 human herpesviruses (human cytomegalovirus, herpes simplex virus 1, herpes simplex virus 2, Epstein–Barr virus, varicella-zoster virus, and human herpesvirus 6) with the aim of simplifying detection and reducing time and costs. The accuracy, reproducibility, specificity, and sensitivity of these assays were established.