Alberta Azzi

Prospective monitoring of polyomavirus BK replication and impact of pre-emptive intervention in pediatric kidney recipients.

Polyoma BK virus (BKV)‐associated nephropathy (PVAN) is a relevant cause of poor renal allograft survival. In a prospective analysis, we monitored BKV DNA in blood and urine samples from 62 consecutive pediatric kidney recipients. In patients with BKV replication, we analyzed the impact of reduction of maintenance immunosuppression on viral load kinetics and PVAN in patients with BKV replication. BKV‐specific immunity was concomitantly evaluated on blood samples of viremic patients, by measuring the frequency of BKV‐specific interferon‐γ‐producing and cytotoxic T cells, and BKV IgG antibody levels. At a median follow‐up of 24 months, BK viruria was observed in 39 of 62 patients, while BK viremia developed in 13 patients (21%). In all viremic patients, immunosuppression reduction resulted in the clearance of viremia, and prevented development of PVAN, without increasing the rate of acute rejection or causing graft dysfunction. As a consequence of immunosuppression adjustment, an expansion of BKV‐specific cellular immunity was observed that coincided with viral clearance. We conclude that treating pediatric kidney transplant patients pre‐emptively with immunosuppression reduction guided by BKV DNA in blood is safe and effective to prevent onset of PVAN. BKV‐specific cellular immunity may be useful to guide this intervention.

Human polyomavirus BK (BKV) load and haemorrhagic cystitis in bone marrow transplantation patients

Several observations suggest an association between long-lasting haemorrhagic cystitis (HC) in bone marrow transplantation (BMT) recipients and human polyomavirus BK (BKV) reactivation, but no conclusive evidence has been obtained so far. The amount of BKV measured in the urine of BMT patients during an episode of HC was compared with that detected in the urine of BMT patients without HC and of immunocompetent individuals in order to better assess the association of BKV reactivation with HC. For this purpose a quantitative competitive PCR was developed. The application of this assay to clinical samples allowed us to distinguish asymptomatic reactivation both in healthy individuals and in immunocompromised patients from reactivation associated with HC, in almost all cases. Low levels, below the sensitivity of the quantitative assay, were shown in asymptomatic healthy individuals and in about 50% of immunocompromised patients. A significantly higher viral load than in the urine of asymptomatic immunocompromised patients was detected in the urine of patients with HC. These data strengthen the hypothesis that BKV reactivation can cause, together with other factors, the majority of late HC in BMT recipients as well as in patients treated for acute refractory lymphoblastic leukemia.

Polyomavirus BK-specific immunity after kidney transplantation.

Failure to mount or maintain a protective immune response may influence the development of polyomavirus BK (BKV)-associated nephropathy (PVAN). However, limited data are so far available on BKV-specific immunity after kidney transplantation. BKV-specific cellular immune response was retrospectively analyzed in kidney recipients with or without BKV infection/reactivation by measuring the frequency of interferon (IFN)-gamma-secreting cells in peripheral blood. Patients with BKV-active infection and good renal function (n=6) had a mean BKV-specific lymphocyte frequency 2 log lower than healthy controls and in the same range as BKV-seropositive recipients without active infection (n=7). Patients with PVAN (n=5) revealed undetectable levels of BKV-specific cells. However, two patients from the latter cohort treated with immunosuppression reduction showed the emergence of specific immunity, with IFN-gamma production in the same range as healthy controls. Our preliminary data suggest that lack of protective immunity toward BKV may favor the occurrence of BKV active infection and influence the progression to PVAN.

Transmission of parvovirus B19 by coagulation factor concentrates exposed to 100°C heat after lyophilization

BACKGROUND: Double inactivation by solvent/detergent treatment plus heating at 100°C for 30 minutes after lyophilization has been adopted to improve viral safety of factor VIII and factor IX concentrates, particularly with respect to non‐lipid‐enveloped viruses. The aim of this study was to evaluate the safety of concentrates exposed to these virucidal methods.

Human polyomaviruses DNA detection in peripheral blood leukocytes from immunocompetent and immunocompromised individuals

Peripheral blood leukocytes from immunocompetent and immunocompromised individuals were analyzed for human polyomarivus BK and JC DNA presence. A nested polymerase chain reaction which amplify the transcriptional control region of the genome of both viruses was employed. The immunocompromised patients included bone marrow transplantation recipients and AIDS patients. BKV sequences were detectable in 52.8-62.5% of the individuals included in this study, whereas the percentage of individuals with JCV sequences in peripheral blood lymphocytes varied from 38.8% to 50%. The frequency of reactivations of BKV and JCV were also determined by detection of shedding in urine of viral DNA. The highest frequency of reactivations of either BKV or JCV was demonstrable in the group of bone marrow transplantation recipients, but reactivations occurred also in immunocompetent individuals. JCV sequences amplified from urine samples showed a restriction pattern similar to the archetype one, whereas sequences obtained from lymphocytes showed rearranged pattern as well as archetype pattern. Finally all JCV sequences from cerebrospinal fluid seemed to be rearranged. These observations suggest that peripheral blood lymphocytes have a fundamental role in the persistence of polyomaviruses infection and in the dissemination at least of JCV within the organism allowing that rearranged variants, better adapted to grow in brain tissue, emerge.

Dendritic cells pulsed with polyomavirus BK antigen induce ex vivo polyoma BK virus-specific cytotoxic T-cell lines in seropositive healthy individuals and renal transplant recipients

Polyoma BK virus (BKV)-associated interstitial nephritis has emerged as a relevant complication of immunocompromise after kidney transplantation, leading to reduced survival of the renal allograft. The limitations of current antiviral treatment and the high probability of rejection in kidney graft recipients when control of viral replication is attempted by reduction of immunosuppression warrant further efforts to develop alternative therapeutic tools. Cellular immunotherapy has proved to be a successful approach for prevention and/or treatment of other viral complications in the immunocompromised host. For assessing the feasibility of translating this strategy to the prevention of BKV-associated disease, a procedure for ex vivo reactivation of BKV-specific cytotoxic T cells (CTL) was developed from BKV-seropositive healthy donors and allograft recipients through stimulation with dendritic cells pulsed with inactivated BKV. The CTL lines thus obtained showed BKV specificity, as an efficient lysis of BKV-infected targets was accompanied by little or no reactivity against mock-infected autologous or allogeneic targets. In vitro killing of allogeneic BKV-infected targets, likely as a result of populations of TCRgammadelta+/CD3+ displaying MHC class I unrestricted cytotoxicity, was also displayed. Application of this culture system may allow a preemptive therapy approach to BKV-related complications in transplant recipients, based on CTL treatment guided by BKV DNA levels.

Tissue Persistence of Parvovirus B19 Genotypes in Asymptomatic Persons

Parvovirus B19 (B19V) can persist in immunocompetent symptomatic and non‐symptomatic individuals, as demonstrated by the finding of viral DNA in different tissues, in absence of viremia and of anti‐B19V IgM. The spread and the nature of this phenomenon have not been clearly determined. In order to investigate the frequency of persistence and the tissue distribution of the three genotypes of B19V, the viral load of the persistent virus and its expression in the affected tissues, 139 tissue samples and 102 sera from 139 asymptomatic individuals have been analyzed by consensus PCRs and genotype specific PCRs for B19V detection and genotyping. Viral load was measured by real time PCR and viral mRNAs were detected by RT‐PCR. Altogether, 51% individuals carried B19V DNA, more frequently in solid tissues (65%) than in bone marrow (20%). Genotype 1 was found in 28% tissue samples, genotype 2 in 68% and genotype 3 in 3% only. Viral load ranged from less then 10 copies to 7 × 104 copies per 106 cells, with the exception of two samples of myocardium with about 106 copies per 106 cells. mRNA of capsid proteins was present in two bone marrow samples only. In conclusion, in asymptomatic individuals B19V persistence is more common in solid tissues than in bone marrow, and genotype 2 persists more frequently than genotype 1. The results suggest that the virus persists without replicating, at sub‐immunogenic levels. J. Med. Virol. 80:2005–2011, 2008.

Retransplantation After Kidney Graft Loss Due to Polyoma BK Virus Nephropathy: Successful Outcome Without Original Allograft Nephrectomy

Although polyoma BK virus (BKV)-associated interstitial nephritis has received increasing attention because of its clinical relevance in kidney allograft recipients, data on risk for repeated renal transplantation after BKV-related allograft loss are limited, and the need to perform an original graft nephrectomy is the object of debate. A 15-year-old boy with renal failure secondary to Alports syndrome underwent renal transplantation. His posttransplantation course was complicated by acute rejection episodes and the presence of circulating anti-glomerular basement membrane antibodies that required aggressive immunosuppressive treatment. Graft failure caused by BKV-associated interstitial nephropathy occurred despite a reduction in immunosuppression and cidofovir treatment. The patient received a second transplant without an original graft nephrectomy, and 15 months after retransplantation, he persists with optimal graft function and is constantly BKV DNA negative in both urine and plasma. Our report indicates that an original allograft nephrectomy may not be mandatory for successful retransplantation after graft loss caused by BKV nephropathy.

Investigation of an imported case of Middle East Respiratory Syndrome Coronavirus (MERS-CoV) infection in Florence, Italy, May to June 2013

On 31 May 2013, the first case of Middle East Respiratory Syndrome Coronavirus (MERS-CoV) infection in Italy was laboratory confirmed in a previously healthy adult man, who developed pneumonia with moderate respiratory distress after returning from a holiday in Jordan. Two secondary cases were identified through contact tracing, among family members and colleagues who had not previously travelled abroad. Both secondary cases developed mild illness. All three patients recovered fully.

Detection of B19 parvovirus infections by a dot-blot hybridization assay using a digoxigenin-labelled probe

A non-radioactive dot-blot hybridization assay for the detection of B19 parvovirus infections was developed using a digoxigenin-labelled probe both on nylon and nitrocellulose filters. A 700 bp BamHI HindIII fragment of B19 DNA was used to construct the probe. Probe labelling was carried out by incorporating deoxyuridine triphosphate labelled with digoxigenin. The dot-blot hybridization assay was visualized by an immunoenzymatic reaction using antidigoxigenin Fab fragments labelled with alkaline phosphatase. The specificity and sensitivity of digoxigenin-labelled B19 DNA probe was compared with the results obtained with 32P-labelled B19 DNA probe. Out of the 504 serum samples tested, 3 samples were positive in all the hybridization assays performed and 494 were negative, 7 serum samples gave a weak positive reaction when Dig-B19 probe was used on nitrocellulose filters. The 77 pharyngeal swabs tested were negative in all the hybridization assays performed. Our hybridization assay showed a high sensitivity and reproducibility and it appears to be a rapid, practical and reliable test for routine screening of B19 parvovirus DNA in large numbers of clinical specimens.