Francesca Sidoti

Monitoring of BK virus replication in the first year following renal transplantation

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 x 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.

Epstein-Barr Virus in Cutaneous T-Cell Lymphomas: Evaluation of the Viral Presence and Significance in Skin and Peripheral Blood

The importance of viral agents in the development of cutaneous T-cell lymphomas (CTCL) is still debated. For this purpose, we retrospectively evaluated the Epstein-Barr virus (EBV) presence in Sézary syndrome (SS), mycosis fungoides (MF), inflammatory dermatoses (ID), and healthy donors (HD) using different approaches: EBV-DNA was quantified in skin biopsies and peripheral blood using real-time PCR, EBV-encoded small RNA (EBER) transcripts were detected by in situ hybridization (ISH), and latent membrane protein1-2 antigens were detected by immunohistochemistry. Skin biopsies were EBV-DNA-positive in 8/30 (27%) SS, 7/71 (10%) MF, and 2/18 (11%) ID patients and in none of the 25 normal skin samples. Positive mRNA (EBER) signals, always confined to cerebriform T lymphocytes, were found in 5/30 SS patients (17%), whereas signals in all MF and ID patients were negative. The presence of EBV-DNA in skin and blood samples was associated with a significantly lower survival in MF/SS patients. In evaluating EBV serological status, most (>70%) SS, MF, and ID patients showed a serological reactivation demonstrated by the presence of anti-EA IgG. In conclusion, although the finding of EBV-DNA in CTCL does not prove its etiopathogenetic role and may be related instead to immunosuppression, our study demonstrates that it has prognostic relevance.

Quantitative detection of Epstein-Barr virus in bronchoalveolar lavage from transplant and nontransplant patients.

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<0.05). There was no significant difference of EBV positivity considering the type of transplanted organ. Viral load did not significantly differ comparing specimens of TR versus NT, specimens of solid organ transplant versus bone marrow transplant recipients. EBV was frequently positive in patients with a diagnosis of pneumonia (28.6%), respiratory insufficiency (24.5%), and exacerbation of underlying bronchopneumopathies (30.8%); however, there was no difference comparing TR and NT. EBV was mostly detected in concomitance with other infectious pathogens. Mortality within 28 days of BAL sampling was not related to EBV-DNA positivity and load. Conclusions. EBV is frequently detected in BAL from TR and NT; however, its pathogenic role in lower respiratory tract remains poorly known, also because of the frequent detection of concomitant infectious pathogens. Further studies are needed to better elucidate this issue and the underlying local conditions favoring viral replication.

Polyomaviruses BK- And JC-DNA quantitation in kidney allograft biopsies.

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 <3% of the cases. 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 (>10(4) genome equivalents/cell) were found in two renal samples with histopathologically confirmed PVAN; while JC viral load was >10(4) 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.

Development of real time RT-PCR assays for detection of type A influenza virus and for subtyping of avian H5 and H7 hemagglutinin subtypes.

Rapid detection and subtyping of H5 and H7 subtypes influenza A viruses are important for disease control in poultry and potential transmission to humans. Currently, virus isolation and subsequent HA and NA subtyping constitute the standard for avian influenza viruses detection and subtype identification. These methods are highly accurate and sensitive but are also laborious and time-consuming. Reverse transcription PCR and real time reverse transcription PCR assays, suitable tests for rapid detection, have previously been used for the specific diagnosis of H5 and H7 viruses, however, at present, no primer and probe sets are available for the identification of all H5 and H7 strains. Herein, we have developed specific and sensitive real time reverse transcription PCR assays for the detection of type A influenza virus and for subtyping of avian H5 and H7 hemagglutinin subtypes and we have also compared these molecular assays with viral isolation in terms of sensitivity. Our results demonstrate that the real time reverse transcription PCR assays are more sensitive, specific, less expensive compared to viral isolation. In conclusion, molecular assays could represent an useful tool for rapid detection and screening of H5 and H7 isolates during influenza A virus outbreaks alternatively to viral isolation.

Development of a RT Real-time PCR for the detection and quantification of human rhinoviruses

Human Rhinoviruses (HRV) are the most common viral agents, being responsible for upper as well as lower respiratory tract infections. Evidence demonstrating that 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 including HRV in virological diagnostics of acute lower respiratory tract illness. This article describes the development and optimization of a reverse transcription (RT) real-time PCR assay for quantification of HRV RNA in clinical samples. Efficiency, sensitivity, specificity, inter- and intra-assay variability, and dynamic range have been determined. Subsequently, the assay has been validated on bronchoalveolar lavage (BAL) specimens obtained from immunocompetent and immunocompromised patients.

Detection of Mimivirus in Bronchoalveolar Lavage of Ventilated and Nonventilated Patients

Background/Aims: We investigated the prevalence of Mimivirus in bronchoalveolar lavage (BAL) specimens from ventilated versus nonventilated patients. Methods: The occurrence of Mimivirus DNA was evaluated by two previously developed real-time PCR assays in 69 BAL specimens: 30 from patients on mechanical ventilation for at least 48 h and 39 from nonventilated patients from different clinical settings, including lung transplant recipients. Results: None of the BAL specimens from ventilated and nonventilated patients resulted positive for Mimivirus. Conclusion: This study, similarly to other studies that used molecular assays to detect Mimivirus, found no occurrence of the virus in the lower respiratory tract, thus being in contrast to serological investigations which reported a significant association between Mimivirus and the development of pneumonia. Gene polymorphism could explain these results or, alternatively, it could be hypothesized that Mimivirus does not represent a common cause of lower respiratory tract infection in either ventilated or nonventilated patients. Further studies on a larger population of patients from a different clinical setting evaluating both serology and DNA detection in lower respiratory tract specimens, including BAL and possibly tissue samples, could allow a better definition of the epidemiological and pathological role of Mimivirus in the development of pneumonia.

Human herpesvirus 7 detection by quantitative real time polymerase chain reaction in primary cutaneous T-cell lymphomas and healthy subjects: lack of a pathogenic role.

Background  Primary cutaneous T‐cell lymphomas (CTCLs) are a heterogeneous group of lymphomas where the tumour population emerges within a multiple subclone pattern. Mycosis fungoides (MF) and Sézary syndrome (SS) are characterized by the expansion of clonal CD4+/CD45RO+ memory T cells. Lymphomatoid papulosis (LyP) is a chronic, lymphoproliferative disorder included in the CD30+ primary CTCL spectrum. Several studies have suggested a role of viral infection for super‐antigenic activation of T lymphocytes; however, evidence of their association with CTCLs is still lacking. Human herpesvirus (HHV) 7 is a CD4+ T‐lymphotropic herpesvirus; its restricted cellular tropism and the ability to induce cytokine production in infected cells could make it an important pathogenic cofactor in lymphoproliferative disorders.

Alternative Molecular Tests for Virological Diagnosis

Several nucleic acid amplification techniques (NAATs), particularly PCR and real-time PCR, are currently used in the routine clinical laboratories. Such approaches have allowed rapid diagnosis with a high degree of sensitivity and specificity. However, conventional PCR methods have several intrinsic disadvantages such as the requirement for temperature cycling apparatus, and sophisticated and costly analytical equipments. Therefore, amplification at a constant temperature is an attractive alternative method to avoid these requirements. A new generation of isothermal amplification techniques are gaining a wide popularity as diagnostic tools due to their simple operation, rapid reaction and easy detection. The main isothermal methods reviewed here include loop-mediated isothermal amplification, nucleic acid sequence-based amplification, and helicase-dependent amplification. In this review, design criteria, potential of amplification, and application of these alternative molecular tests will be discussed and compared to conventional NAATs.

Evaluation of CMV-specific cellular immune response by EliSPOT assay in kidney transplant patients.

BACKGROUND Immunological monitoring for CMV can be useful in transplant patients; however, few centers perform it on a routine basis. OBJECTIVES In this study, CMV-specific cellular response was evaluated in a population of kidney transplant recipients and related to viral infection/reactivation and other demographic and clinical features. STUDY DESIGN Three hundred and twenty-eight patients were studied by EliSPOT assay: 201 prospectively monitored in the first year posttransplantation, 127 with a single determination at >1 year. Clinical features, including occurrence of CMV-DNAemia, CMV serostatus, anti-viral strategies and immunosuppressive protocols, were evaluated. RESULTS Overall, 66.5% of patients were CMV-responders at EliSPOT assay. No episode of infection occurred at follow-up (mean 24.5 months) in 73.4% responders versus 55.5% non-responders (p<0.005); CMV-free period was significantly longer in responders (p<0.001). Although no significant difference of peak viral load was found, prevalence of CMV-DNAemia values >10(5)copies/mL was significantly higher in non-responders versus responders (8.2% and 2.3%, p<0.05). Non-responder status was significantly associated to CMV-seronegativity (p<0.0001), anti-viral prophylaxis use (p<0.0001), and immunosuppression induction with basiliximab (p<0.005). No significant association was found for other clinical features and immunosuppressive protocols. CONCLUSIONS Immunological data for CMV could be used in the clinical evaluation and decision-making process, in combination with virological monitoring, in kidney transplant recipients.