Sara Astegiano

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

BACKGROUNDnBK 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.nnnMETHODSnWe 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.nnnRESULTSnSerum 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.nnnCONCLUSIONSnThe 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.

Th1, Th2, Th17 and regulatory T cell pattern in psoriatic patients: modulation of cytokines and gene targets induced by etanercept treatment and correlation with clinical response.

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.

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.

Detection of human rhinoviruses in the lower respiratory tract of lung transplant recipients

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 (pxa0<xa00.0001), and no differences in viral load were observed. Acute respiratory insufficiency was found in 15 cases, three of which were HRV positive (viral load, 6.3x106 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, 103-104 in cases of co-infection). Acute rejection was diagnosed in 12 cases, three of which were HRV positive (viral load, 103 in two cases of co-infection and 105 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 of >105 possibly being associated with clinical symptoms, although lower loads can be detected in both symptomatic and asymptomatic patients.

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

BACKGROUNDnPolyomavirus-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.nnnOBJECTIVESnTo quantify polyomaviruses BK and JC load by real-time TaqMan PCR in tissue specimens (renal and ureteral) from kidney transplant recipients.nnnSTUDY DESIGN AND METHODSnOne-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.nnnRESULTSnBKV-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.nnnCONCLUSIONSnAlthough 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.

Quantitative detection of the new polyomaviruses KI, WU and Merkel cell virus in transbronchial biopsies from lung transplant recipients

Background 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. Aims 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. Methods 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/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. Conclusions 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.

Real Time PCR TaqMan assays for detection of polyomaviruses KIV and WUV in clinical samples.

n Abstractn n 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.n n

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.

Development of a 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

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.