Human herpesviruses respiratory infections in patients with acute respiratory distress (ARDS).

Acute respiratory distress syndrome (ARDS) is today a leading cause of hospitalization in intensive care unit (ICU). ARDS and pneumonia are closely related to critically ill patients; however, the etiologic agent is not always identified. The presence of human herpes simplex virus 1, human cytomegalovirus and Epstein-Barr virus in respiratory samples of critically ill patients is increasingly reported even without canonical immunosuppression. The main aim of this study was to better understand the significance of herpesviruses finding in lower respiratory tract of ARDS patients hospitalized in ICU. The presence of this group of herpesviruses, in addition to the research of influenza viruses and other common respiratory viruses, was investigated in respiratory samples from 54 patients hospitalized in ICU, without a known microbiological causative agent. Moreover, the immunophenotype of each patient was analyzed. Herpesviruses DNA presence in the lower respiratory tract seemed not attributable to an impaired immunophenotype, whereas a significant correlation was observed between herpesviruses positivity and influenza virus infection. A higher ICU mortality was significantly related to the presence of herpesvirus infection in the lower respiratory tract as well as to impaired immunophenotype, as patients with poor outcome showed severe lymphopenia, affecting in particular T (CD3+) cells, since the first days of ICU hospitalization. In conclusion, these results indicate that herpesviruses lower respiratory tract infection, which occurs more frequently following influenza virus infection, can be a negative prognostic marker. An independent risk factor for ICU patients with ARDS is an impaired immunophenotype.

Detection of JCPyV microRNA in blood and urine samples of multiple sclerosis patients under natalizumab therapy.

Polyomavirus JC (JCPyV) reactivation and development of progressive multifocal leukoencephalopathy is a health concern in multiple sclerosis patients under natalizumab therapy. Here, the JCPyV microRNA-J1-3p and microRNA-J1-5p expressions and genomic variability were investigated in blood and urine samples of multiple sclerosis patients before and under natalizumab therapy and in healthy controls. The two JCPyV microRNAs were detected in the JCPyV-DNA-positive peripheral blood mononuclear cell samples and in the exosomes derived from plasma and urine obtained from JCPyV-DNA-positive and JCPyV-DNA-negative patients. In particular, the increased JCPyV microRNA expression in samples of multiple sclerosis patients under natalizumab therapy was consistent with the high JCPyV-DNA positivity observed in these samples. Moreover, JCPyV microRNA genomic region showed few nucleotide differences in samples obtained from blood and urine of multiple sclerosis patients and healthy controls. Overall, these data suggest a potential role of the JCPyV microRNA expression in counteracting the viral reactivation to maintain JCPyV asymptomatic persistence in the host.

Detection of 12 respiratory viruses by duplex real time PCR assays in respiratory samples.

Different viruses can be responsible for similar clinical manifestations of respiratory infections. Thus, the etiological diagnosis of respiratory viral diseases requires the detection of a large number of viruses. In this study, 6 duplex real-time PCR assays, using EvaGreen intercalating dye, were developed to detect 12 major viruses responsible for respiratory diseases: influenza A and B viruses, enteroviruses (including enterovirus spp, and rhinovirus spp), respiratory syncytial virus, human metapneumovirus, coronaviruses group I (of which CoV 229E and CoV NL63 are part) and II (including CoV OC43 and CoV HKU1), parainfluenza viruses type 1, 2, 3 and 4, human adenoviruses and human bocaviruses. The 2 target viruses of each duplex reaction were distinguishable by the melting temperatures of their amplicons. The 6 duplex real time PCR assays were applied for diagnostic purpose on 202 respiratory samples from 157 patients. One hundred fifty-seven samples were throat swabs and 45 were bronchoalveolar lavages. The results of the duplex PCR assays were confirmed by comparison with a commercial, validated, assay; in addition, the positive results were confirmed by sequencing. The analytical sensitivity of the duplex PCR assays varied from 10(3) copies/ml to 10(4) copies/ml. For parainfluenza virus 2 only it was 10(5) copies/ml. Seventy clinical samples (35%) from 55 patients (30 children and 25 adults) were positive for 1 or more viruses. In adult patients, influenza A virus was the most frequently detected respiratory virus followed by rhinoviruses. In contrast, respiratory syncytial virus was the most common virus in children, followed by enteroviruses, influenza A virus and coronavirus NL63. The small number of samples/patients does not allow us to draw any epidemiological conclusion. Altogether, the results of this study indicate that the 6 duplex PCR assays described in this study are sensitive, specific and cost-effective. Thus, this assay could be particularly useful to identify the main respiratory viruses directly from clinical samples, after nucleic acid extraction, and, also, to screen a large number of patients for epidemiological studies.

Assessment of the risk of polyomavirus JC reactivation in patients with immune-mediated diseases during long-term treatment with infliximab.

Polyomavirus JC (JCV) reactivation causing progressive multifocal leukoencephalopathy is a main concern during biological therapies. Here, JCV reactivation in patients suffering from immune-mediated diseases after a long-term treatment with anti-tumor necrosis factor alpha (TNF-α) inhibitor infliximab was investigated. Peripheral mononuclear blood cells (PBMC), plasma and urine samples were obtained from 61 immune-mediated diseases patients treated or not with infliximab in combination with steroid and other immunomodulators and from 20 healthy donors. JCV DNA was transiently detected in 12 PBMC of 40 patients at different doses of infliximab with a higher prevalence than that of the 21 patients untreated. Conversely, a stable JCV positivity in urine of treated and untreated patients was detected. Sequencing the noncoding control region (NCCR), all samples exhibited the archetype structure with few mutations in transcriptional factor binding regions. The consequence of anti-TNF-α treatment on viral persistence was examined monitoring Torquetenovirus viremia and investigating the TNF-α-induced microRNA regulators of transcriptional factors, with a binding site on NCCR. Although infliximab treatment in this study did not affect directly JCV reactivation, further investigation on host factor(s) regulated by it will be of warranty in the understanding the mechanism(s) that may affect viral persistence.

Lymphocytopenia as a marker for pandemic influenza A/H1N1 2009 virus infection in children.

Lymphocytopenia has been reported in adults with pandemic influenza A/H1N1 2009 infection, but data in children are inconclusive. Data from 76 children presented with flu-like symptoms between July and November 2009 and tested for pandemic influenza A/H1N1 2009 virus and white blood cell (WBC) counts were analyzed. Samples from 37 (48.7%) children resulted in a positive PCR assay for pandemic influenza A/H1N1 2009 virus. When comparing data from these children with data from 39 (51.3%) children with uncomplicated flu-like illness and negative PCR assay for pandemic influenza A/H1N1 2009 virus, no difference in disease duration, median age, red blood cell count, hemoglobin concentration, C reactive protein concentration, and absolute neutrophil count was observed, whereas significant differences were apparent when considering WBC count, relative and absolute lymphocyte count, absolute lymphocyte count z-score, and platelet count. Receiver operating characteristic curve analysis revealed that the best absolute lymphocyte count and absolute lymphocyte count z-score cut-points that simultaneously maximized sensitivity and specificity were 2,256 cells/µl and -0.89, respectively, sensitivity being 0.81 (95% CI: 0.68-0.94), specificity 0.87 (95% CI: 0.77-0.98), positive predictive value 0.85 (95% CI: 0.74-0.97), and negative predictive value 0.83 (95% CI: 0.71-0.94). In conclusion, lymphocytopenia is a marker for influenza A/H1N1 2009 virus infection in children. Absolute lymphocyte count <2,556 cells/µl or absolute lymphocyte count z-score < -0.89 may be useful cut-offs to discriminate against children at higher risk of infection during epidemics. Considering that the pandemic virus is highly likely to continue to circulate in the coming winter season, these findings provide direct and practical implications for the near future.

Ventilatory and ECMO treatment of H1N1-induced severe respiratory failure: results of an Italian referral ECMO center.

BACKGROUND: Since the first outbreak of a respiratory illness caused by H1N1 virus in Mexico, several reports have described the need of intensive care or extracorporeal membrane oxygenation (ECMO) assistance in young and often healthy patients. Here we describe our experience in H1N1-induced ARDS using both ventilation strategy and ECMO assistance. METHODS: Following Italian Ministry of Health instructions, an Emergency Service was established at the Careggi Teaching Hospital (Florence, Italy) for the novel pandemic influenza. From Sept 09 to Jan 10, all patients admitted to our Intensive Care Unit (ICU) of the Emergency Department with ARDS due to H1N1 infection were studied. All ECMO treatments were veno-venous. H1N1 infection was confirmed by PCR assayed on pharyngeal swab, subglottic aspiration and bronchoalveolar lavage. Lung pathology was evaluated daily by lung ultrasound (LUS) examination. RESULTS: A total of 12 patients were studied: 7 underwent ECMO treatment, and 5 responded to protective mechanical ventilation. Two patients had co-infection by Legionella Pneumophila. One woman was pregnant. In our series, PCR from bronchoalveolar lavage had a 100% sensitivity compared to 75% from pharyngeal swab samples. The routine use of LUS limited the number of chest X-ray examinations and decreased transportation to radiology for CT-scan, increasing patient safety and avoiding the transitory disconnection from ventilator. No major complications occurred during ECMO treatments. In three cases, bleeding from vascular access sites due to heparin infusion required blood transfusions. Overall mortality rate was 8.3%. CONCLUSIONS: In our experience, early ECMO assistance resulted safe and feasible, considering the life threatening condition, in H1N1-induced ARDS. Lung ultrasound is an effective mean for daily assessment of ARDS patients.

Human parvovirus PARV4 DNA in tissues from adult individuals: a comparison with human parvovirus B19 (B19V).

BACKGROUND: PARV4 is a new member of the Parvoviridae family not closely related to any of the known human parvoviruses. Viremia seems to be a hallmark of PARV4 infection and viral DNA persistence has been demonstrated in a few tissues. Till now, PARV4 has not been associated with any disease and its prevalence in human population has not been clearly established. This study was aimed to assess the tissue distribution and the ability to persist of PARV4 in comparison to parvovirus B19 (B19V). RESULTS: PARV4 and B19V DNA detection was carried out in various tissues of individuals without suspect of acute viral infection, by a real time PCR and a nested PCR, targeting the ORF2 and the ORF1 respectively. Low amount of PARV4 DNA was found frequently (>40%) in heart and liver of adults individuals, less frequently in lungs and kidneys (23,5 and 18% respectively) and was rare in bone marrow, skin and synovium samples (5,5%, 4% and 5%, respectively). By comparison, B19V DNA sequences were present in the same tissues with a higher frequency (significantly higher in myocardium, skin and bone marrow) except than in liver where the frequency was the same of PARV4 DNA and in plasma samples where B19V frequency was significantly lower than that of PARV4 CONCLUSIONS: The particular tropism of PARV4 for liver and heart, here emerged, suggests to focus further studies on these tissues as possible target for viral replication and on the possible role of PARV4 infection in liver and heart diseases. Neither bone marrow nor kidney seem to be a common target of viral replication.

Increased pathogenicity and shedding in chickens of a wild bird-origin low pathogenicity avian influenza virus of the H7N3 subtype following multiple in vivo passages in quail and turkey.

In order to investigate viral adaptation mechanisms to poultry, we performed serial in vivo passages of a wild bird low pathogenicity avian influenza isolate of the H7N3 subtype (A/mallard/Italy/33/01) in three different domestic species (chicken, turkey, and Japanese quail). The virus under study was administered via natural routes at the dose of 10(6) egg infective dose50/ 0.1 ml to chickens, turkeys, and quails in order to investigate the clinical susceptibility and the shedding levels after infection. Multiple in vivo passages of the virus were performed by serially infecting groups of five naive birds of each species, with samples collected from a previously infected group. Quails and turkeys were susceptible to infection for 10 serial passages, whereas chickens were susceptible to two cycles of infection only. Infection of chicken with the quail- and turkey-adapted viruses showed an increased pathogenicity and/or shedding, causing more severe clinical signs and/or higher levels of viral excretion compared to the original strain. The data obtained herein suggest that infection of selected avian species may facilitate the adaptation of avian influenza viruses originating from the wild bird reservoir to chicken. This is the first time turkey has been shown to act as a species in which a virus from the wild reservoir can increase its replication activity in other domestic species.

Antibody responses to recombinant polyomavirus BK large T and VP1 proteins in young kidney transplant patients.

BK virus (BKV)-specific immunity is critical for polyomavirus-associated nephropathy, but antibody responses are incompletely defined. We compared the hemagglutination inhibition assay (HIA) with immunoglobulin G enzyme immunoassays (EIA) to BKV proteins expressed in baculovirus-infected insect cells. N-terminal, internal, and C-terminal domains of the BKV large T antigen (BKLT) were fused to glutathione S-transferase (GST), yielding GST-BKLTD1, GST-BKLTD2, and GST-BKLTD3, respectively. The BKV capsid VP1 was expressed as a GST fusion (BKVP1) or as a native VP1 assembled into viruslike particles (BKVLP). We tested 422 sera from 28 healthy donors (HD), 99 dialysis patients (DP; median age, 15 years; range, 3 to 32 years), and 46 age-matched kidney transplant patients (KTP; median age, 15 years; range, 2 to 33 years). In HD, HIA and BKVLP EIA both yielded a 91.7% seroreactivity, whereas all other EIA responses were lower (BKVP1, 83.3%; BKLTD1, 25%; BKLTD2, 29%; BKLTD3, 40%). HIA titers significantly correlated with EIA levels for BKVLP, BKVP1, and BKLTD1 but not for BKLTD2 or BKLTD3, which were barely above the cutoff. In DP, the seroreactivities of HIA, BKVLP, and BKLTD1 were lower than that in HD (63.6%, 86.9%, and 10.1%, respectively) and they had lower titers (P < 0.001). In KTP, seropositivities for BKVLP, BKVP1, and BKLTD1 were 78%, 50%, and 17%, respectively, but anti-BKVLP levels increased significantly in KTP with viruria and viremia, whereas anti-BKLTD1 levels increased after clearing sustained BKV viremia. In conclusion, anti-BKVLP is equivalent to HIA in HD but is more sensitive to determine the BKV serostatus in DP and KTP. In KTP, anti-BKVLP responds to recent BKV viruria and viremia, whereas anti-BKLTD1 may indicate emerging BKV-specific immune control.

Human parvovirus B19 (B19V) infection in systemic sclerosis patients.

BACKGROUND: Our previous reports suggested a possible association between parvovirus B19 (B19V) infection and systemic sclerosis (SSc), based on higher prevalence of B19V DNA in SSc patients in respect to controls. METHODS: In the present study, to further evaluate the differences in the pattern of B19 infection in SSc, skin biopsies and bone marrow samples from patients and controls were analysed for B19V DNA detection, genotyping and viral expression. RESULTS: B19V DNA was detected in skin biopsies from 39/49 SSc patients and from 20/28 controls. Bone marrow showed positive in 17/29 SSc patients, 5/21 haematological patients and 0/10 healthy controls. Genotype 1 was more frequent in skin and bone marrow from patients than from controls. Simultaneous persistence of 2 genotypes was detected in SSc skin and bone marrow samples, never in controls. Viral mRNA for capsid protein was detected in the skin of genotype 1-positive patients and not in control skins. CONCLUSION: The results outline some differences in the rate of persistence of B19V DNA, in the simultaneous persistence of 2 genotypes and in the pattern of viral expression among SSc patients and controls.

Oligonucleotides derived from the packaging signal at the 5' end of the viral PB2 segment specifically inhibit influenza virus in vitro.

The development of new antiviral molecules to fight the possible emergence of influenza viruses with pandemic potential is needed. In this study, phosphorothioate oligonucleotides (S-ONs) derived from the packaging signals in the 3' and 5' ends of the viral PB2 RNA were associated with liposomes and tested against influenza virus in vitro. A 15-mer S-ON derived from the 5' end of the viral PB2 RNA, complementary to the 3' end of its coding region (nucleotides 2279-2293) and designated 5-15b, proved markedly inhibitory. The antiviral activity of 5-15b was dose- and time-dependent but was independent of the cell substrate and multiplicity of infection used. Importantly, inhibition of influenza A and B viruses required S-ONs reproducing the respective packaging signals. Furthermore, 5-15b and its antisense derivative S-ON activity did not affect intracellular accumulation of viral RNA. Confocal microscopy showed that 5-15b is clearly nucleophilic. These findings indicate that the packaging signal at the 5' end of the PB2 RNA is an interesting target for the design of new anti-influenza-virus compounds.

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 x 10(4) copies per 10(6) cells, with the exception of two samples of myocardium with about 10(6) copies per 10(6) 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.

Characterization of human H1N1 influenza virus variants selected in vitro with zanamivir in the presence of sialic acid-containing molecules.

Understanding the molecular mechanisms of influenza virus resistance to neuraminidase inhibitors is a main concern for their clinical use. In an attempt to reproduce in vivo selective conditions where influenza virus resistance to neuraminidase inhibitors can occur the zanamivir selection of an A/H1N1 influenza virus strain was carried out in Madin-Darby canine kidney cells performed in the presence or absence of sialic acid-containing inhibitor analogues that act as virus decoy receptors. The zanamivir-selected variants passaged in the presence of sialic acid-containing molecules resembling the human-like virus receptor lost the ability to bind red blood cells. Furthermore, whereas all zanamivir-selected variants exhibited a robust reduction in susceptibility to zanamivir in plaque assays only those obtained after extensive passages acquired a powerful neuraminidase enzyme resistance to zanamivir and oseltamivir. Evidence that balanced neuraminidase and hemagglutinin activities mediated by mutations induced during selection could play a role in the decrease of virus replication susceptibility to zanamivir is reported.

BK virus regulatory region sequence deletions in a case of human polyomavirus associated nephropathy (PVAN) after kidney transplantation.

The pathogenesis of polyomavirus-associated interstitial nephropathy (PVAN) in kidney transplant recipients is likely to depend upon multiple risk factors, which may include viral characteristics. We report a case of interstitial nephropathy in a young kidney allograft recipient, associated with a BK virus (BKV) strain with a rearranged transcription control region (TCR). BKV strains with deletions and nucleotide substitutions in the TCR were present in a kidney biopsy and urine samples. After retransplantation, following loss of renal function, a BKV strain with an archetypal TCR was detected in association with asymptomatic reactivation.

The JCPYV DNA load inversely correlates with the viral microrna expression in blood and cerebrospinal fluid of patients at risk of PML.

BACKGROUND: In light of their regulatory role, changes in the expression of Polyomavirus JC (JCPyV) microRNAs may be relevant for virus reactivation and the development of progressive multifocal leukoencephalopathy (PML). OBJECTIVES: To investigate the presence of JCPyV-DNA and JCPyV microRNA expression in clinical specimens of patients at risk for PML. STUDY DESIGN: The JCPyV-DNA and microRNA status was assessed in peripheral blood mononuclear cells (PBMCs) and plasma from 100 HIV patients, in serum and cerebrospinal fluid (CSF) from 14 HIV PML patients and in PBMCs and plasma from 50 healthy controls using Multiplex real-time PCR and JCPyV miRNA-J1-3p and -5p stem-loop RT-PCR. The JCPyV-DNA microRNA-expressing region was also sequenced. RESULTS: A positive JCPyV-DNA status was more prevalent in HIV patients (67%, 67/100) compared to healthy controls (18%, 9/50). Among these, 46% and 42% of the HIV patients and 18% and 0% of the healthy controls were positive based on PBMC and plasma determinations, respectively. PBMC JCPyV microRNA positivity was observed in 22 out of 46 (48%) JCPyV+ HIV patients and in 3 out of 9 (33%) JCPyV+ healthy controls. Moreover, JCPyV microRNAs in exosomes were found in 6 out of 100 (6%) HIV plasma samples, in 12 out of 50 (24%) healthy samples, in 6 out of 14 (43%) serum samples, and in 3 out of 5 (60%) HIV PML CSF samples. Of note, the JCPyV-DNA load was inversely correlated with expression of the viral microRNA. The JCPyV microRNA genomic expression region showed a different combination of three mutations. CONCLUSIONS: The low levels of JCPyV microRNA expression in HIV patients with high JCPyV-DNA prevalence observed in this study highlight the potential clinical relevance of JCPyV microRNAs in PML risk assessment.

Small RNAs targeting the 5' end of the viral polymerase gene segments specifically interfere with influenza type A virus replication.

Human and avian influenza A viruses, associated with seasonal epidemics and occasionally with pandemics, have a high impact on public health. The development of new antivirals to counteract the emergence of drug resistant influenza virus variants is a main concern. The aim of this study was to develop systems for the efficient and stable expression of small therapeutic RNAs into influenza virus infected cells in order to get further insights on the efficacy of nucleic acid-based antiviral strategies. To this end, lentiviral vectors expressing either microRNAs or antisense-RNAs targeting the 5' end of the PA, PB1 and PB2 influenza virus genomic sequences were generated. Derivative recombinant lentiviral particles were employed to transduce the influenza virus highly susceptible human alveolar basal epithelial A549 cells. The expression of both RNA molecules led to a reduction up to 3 logs of the viral titer when transduced A549 cells were challenged with different human and avian subtypes of influenza type A virus. Importantly, no inhibition of influenza type B virus was observed. Overall our data support the development of nucleic acid-based antiviral strategies to control human and avian influenza A virus infection.

Markers of JC virus infection in patients with multiple sclerosis under natalizumab therapy.

OBJECTIVE: To evaluate the frequency of JC polyomavirus (JCPyV) infection and anti-JCPyV antibodies in patients with multiple sclerosis under natalizumab therapy. METHODS: Presence of anti-JCPyV antibodies and JCPyV DNA was analyzed in 39 patients with relapsing-remitting multiple sclerosis undergoing natalizumab therapy. Anti-JCPyV antibodies were evaluated in serum by a 2-step virus-like particle-based ELISA assay (Stratify), and JCPyV DNA was evaluated in peripheral blood mononuclear cells, plasma, and urine by quantitative PCR. The anti-JCPyV antibodies were evaluated in serum samples collected at the same time or later than those collected for DNA analysis. RESULTS: JCPyV DNA was detected in 59% of patients, and anti-JCPyV antibodies were present in 67%. JCPyV DNA occurred more often in blood than in urine. Anti-JCPyV antibodies were observed in 70% of the JCPyV-infected patients, and JCPyV DNA was detected in 50% of the patients without anti-JCPyV antibodies. When JCPyV DNA was investigated in blood and urine the frequency of infection was higher than previously described. CONCLUSION: Under these experimental conditions, with respect to the observed frequency of JCPyV infection, the sensitivity of the anti-JCPyV antibody assay was lower than expected.

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.

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 Alport's 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.

Multicentre Italian Study Group (MISG) for the standardisation of hepatitis C virus (HCV) PCR.

BACKGROUND: Several studies on standardisation of NAT assays for diagnosis of hepatitis C virus (HCV) infection have been carried out in European countries. In fact the widespreading use of nucleic acid amplification technology (NAT) in diagnostic centres for the evaluation of the HCV infection, requires the application of reference external standards to control laboratory performance; but up to date they are not routinely used. OBJECTIVES: Fifteen diagnostic centres of major Italian Hospitals participated to a quality control study for the standardisation of polymerase chain reaction (PCR)-based HCV-RNA detection, organised by the Committee for the Study of Biotechnology (CoSBio) of the Italian Society of Clinical Microbiology (AMCLI). All the participant centres (PC) used commercial assays, automated or semi-automated. STUDY DESIGN: The study was performed in four rounds. Altogether each centre received 14 reference negative and 22 reference positive sera. The range of copies number per ml of the reference positive sera was 10(4)-10(7). RESULTS AND CONCLUSIONS: Considering the 540 samples tested, 4.54% of false negative (FN) and 4.28% of false positive (FP) results were reported. Thereafter the sensitivity and the specificity were 95.65 and 95.89%, respectively. The errors were distributed among seven out of the 15 PCs. The percentage of FP results was uniformly distributed in each shipment, whereas FN results emerged with the sera at lower HCV genome copies number. The analysis of the data obtained suggests that FP as well as FN results may be attributable to errors or to others problems of laboratories. To improve the performance of Italian, as well as of laboratories throughout the world, the use of external reference standards in multicentre collaborative studies will be required.