Evaluation of Epstein-Barr virus-specific immunologic response in solid organ transplant recipients with an enzyme-linked ImmunoSpot assay.

Epstein-Barr virus (EBV) is a γ-herpes virus, responsible for infectious mononucleosis in immunocompetent hosts. Cellular immunity appears rapidly during EBV primary infection, keeping it silent despite long-life persistence in B lymphocytes. Defects of the EBV-specific cellular immunity are supposed to be the basis of post-transplantation lymphoproliferative disorders, promoted by high levels of immunosuppression. We retrospectively reviewed 197 solid organ transplant recipients to investigate EBV-specific lymphocyte responsiveness using Enzyme-linked ImmunoSpot assay (EliSpot), which assesses the EBV-specific interferon (IFN)-γ producing peripheral blood mononuclear cells, and kinetics of EBV infection/reactivation post-transplantation using quantitative real-time polymerase chain reaction (PCR) on whole blood. Overall, 102 of the 197 patients (51.8%) showed EBV responsiveness at the EBV-EliSpot assay: 68 (66.6%) showed a persistently positive EBV response in 3 or more determinations and 34 (33.3%) had transient episodes of nonresponsiveness. Ninety-five (48.2%) patients were persistently EBV nonresponders. EBV-DNAemia data were available for 58 patients: 27.6% presented at least one episode of EBV-DNA occurrence. No differences were found in EBV-EliSpot response stratification between the groups of patients who experienced episodes of EBV reactivation and those without EBV-DNAemia. However, EBV DNAemia peak values tended to be higher in the first year post-transplantation in the group of patients with a persistent positive EBV-specific immune response. EBV viral load quantitation in blood and EliSpot EBV-specific immune response determination may represent a powerful tool for monitoring solid organ transplant recipients, guiding immunosuppression modulation in patients with active EBV replication.

Clinical impact of HSV-1 detection in the lower respiratory tract from hospitalized adult patients.

The occurrence and clinical impact of herpes simplex virus (HSV) were evaluated in 342 bronchoalveolar lavage specimens from 237 patients. HSV-1 and HSV-2 were detected in 32.1% and <1% of patients, respectively. A significant difference of HSV-1 prevalence and load was found in relation to admission to intensive care unit, mechanical ventilation and mortality within 28 days; in particular, a viral load ≥10(5) copies/mL bronchoalveolar lavage fluid was significantly associated with critical features. No association was found with immune status or other characteristics. Nine of 21 (42.9%) cases of ventilator-associated pneumonia were positive for HSV-1, with poor outcome in six.

The lack of cytomegalovirus-specific cellular immune response may contribute to the onset of organ infection and disease in lung transplant recipients.

Cellular immune response has been demonstrated to play a role in the control of human cytomegalovirus (HCMV) replication in organ transplant recipients. Herein, HCMV-specific T-cell response and association to the onset of organ infection/disease were prospectively evaluated by EliSPOT assay in a population of 46 lung transplant (LT) recipients at 1, 3, 6, 9 and 12 months post-transplantation. According to our centre?s practice, a combined prolonged antiviral prophylaxis (HCMV-IG for 12 months and ganciclovir or valganciclovir for 3 weeks from postoperative day 21) was given to all LT recipients. HCMV-DNA was concomitantly detected on bronchoalveolar lavage (BAL) and whole blood by real-time PCR. Approximately one third of patients resulted HCMV persistently non-responder; the rate of HCMV infection, as evaluated by HCMV-DNA positivity, tended to be higher in non-responders. Mean viral load on BAL was significantly higher in non-responders vs other patients (p < 0.001). Temporal profile of infections appeared related to the HCMV responder status with a shorter time to onset of infection post-transplantation and a longer duration in non-responders. The occurrence of organ disease (i.e. pneumonia) tended to be higher in non-responders, with poor prognosis, as death occurred in one of three non-responder patients that developed HCMV pneumonia. The lack of HCMV-specific cellular response can contribute to the onset of organ infection and disease also in patients in which antiviral prophylaxis was adopted; this could be due to the potential occurrence of incomplete control of replication in lungs or a delayed priming of T-cell reconstitution.

Rapid shell vial culture for the detection of respiratory viruses from bronchoalveolar lavage in immunocompromised patients.

AIM: Viral lower respiratory tract infections (LRTI) are an important cause of morbidity in immunocompromised patients. The aim of this study was to evaluate the clinical impact of rapid shell vial cultures from bronchoalveolar lavage (BAL). METHODS: Sixty-seven BAL samples from 46 patients have been retrospectively examined: 51 from 31 transplant recipients and 16 from 15 immunocompromised patients. BAL were inoculated on human embryonic lung fibroblasts and VERO cells to isolate the following viruses: cytomegalovirus (CMV), herpesviruses, varicella-zoster virus, respiratory syncytial virus, adenovirus, Influenza viruses A and B and Parainfluenza viruses. Clinical, microbiological, laboratory, and radiological data were collected. RESULTS: A LRTI was present in 56.7% of cases: viral 40.3%, bacterial and/or fungal 23.9%, and mixed 7.5%. CMV accounted for 92.6% of viral LRTI. The prevalence of viral infections did not differ between symptomatic and asymptomatic patients; only bacterial and/or fungal infections were significantly more prevalent in symptomatic patients. No clinical, radiological or laboratory feature was significantly associated with the presence of a viral LRTI. In lung transplant recipients the rate of CMV infection was 50%. The result of BAL suggested commencement of antiviral chemotherapy in 25/67 episodes. CONCLUSION: Rapid shell vial culture and immunofluorescence techniques from BAL could play an important role in the clinical management of immunocompromised subjects.

Human polyoma virus BK DNA detection by nested PCR in renal transplant recipients.

Several studies report a correlation between the human polyomavirus BK (BKV) and interstitial nephritis in renal transplant recipients in whom immunosuppressive treatment is thought to allow or induce reactivation of the virus. Furthermore, it is described that nephropathy may result from the use of newly introduced immunosuppressive drugs. In the present study, we evaluated the presence of BKV DNA by nested-PCR (n-PCR) in urine and serum samples from 35 renal transplant patients related to the immunosuppressive regimens and renal function.

HCMV pp67-mRNA detection by nucleic acid sequence-based amplification in renal transplant patients.

CMV infection is a major cause of morbidity and mortality following renal transplantation. Clinical diagnosis is difficult, and rapid and sensitive diagnostic methods are needed since antiviral therapy is available. The determination of the presence of viral transcripts is considered a direct marker of HCMV replication in vivo. In particular, it seems that the expression of late transcripts might better reflect active HCMV replication, dissemination and disease, and should cease upon effective blockage of viral polymerase by antiviral agents, such as gancyclovir. The unspliced pp67-mRNA can be specifically amplified using nucleic acid sequence-based amplification (NASBA) in a background of DNA. In the present study blood samples of forty-two renal transplant patients with active HCMV infection, demonstrated by pp65-antigenaemia, were investigated to detect pp67-mRNA using the NASBA technique. Thirty-one pp65-antigenaemia positive patients resulted NASBA positive (73.8%) also in case of very low levels of antigenaemia; in 9/42 (21.4%) pp67-m-RNA was detected between 6 and 15 days before antigenaemia. Our results indicate that pp67-mRNA NASBA is a useful tool for the early diagnosis of active HCMV infection and for starting and modulating antiviral therapy, in addition to quantitative techniques such as antigenaemia, in renal transplant patients.

Quantitative PCR in EBV-infected renal transplant patients.

In this study we investigated the levels of Epstein Barr virus (EBV) DNA by quantitative polymerase chain reaction (Q-PCR) in serum, whole blood and peripheral blood mononuclear cells (PBMC) from anti-EA IgG seropositive or anti-EA IgG seronegative EBV infected renal transplant recipients. We compared serological data with the viral load to monitor the risk of developing post-transplant lymphoproliferative disorders (PTLD). All patients were asymptomatic and none of them developed PTLD at the time of the study. EBV DNA quantitation for each patient varied in whole blood and PBMC samples probably due to different numbers of mononuclear cells present in samples from which DNA was extracted (whole blood vs. purified PBMC). In 92% of the serum samples EBV DNA was undetectable probably due to absence of free genomes since the number of DNA copies detected in samples from whole blood and PBMC does not reach very high levels. The correlation between the presence of EA-antibody, considered serological evidence of EBV reactivation, and the viral load showed that 60% of EA-positive patients had quantifiable EBV DNA, whereas in 40% of EA-positive patients EBV DNA was undetectable, showing serological reactivity but no viral replication. Of the remaining EA-negative patients, EBV DNA could be detected in 71% of them, whereas 29% did not show EBV DNA, indicating no EBV replication. In conclusion, our results confirm that the presence of serum IgG anti-EA antibody is not a reliable marker of active EBV infection whereas the evaluation of the viral load in blood samples is a useful diagnostic tool to monitor and to better understand the course of EBV infection in immunocompromised renal transplant patients at risk of developing PTLD.

[Epstein-Barr virus infection and lymphoproliferative disorders in patients after renal transplantation]. / Infezione da virus di Epstein-Barr e disordini linfoproliferativi in portatori di trapianto renale.

We have studied EBV infection in renal transplant patients during the first year after transplantation. At trasplantation all patients were EBV seropositive and reactivation of EBV infection was demonstrated in 54% cases after one year. CMV active infection was also demonstrated in 42% of patients with EBV reactivation. No correlation was observed between EBV reactivation and age, sex, immunosuppressive treatment, degree of immunosuppression or donor/recipient HLA matching. A correlation between immunosuppressive treatment, EBV infection and lymphoproliferative disorders (LD) is described in literature, however none of our patients developed LD so far, probably due to the different immunosuppressive protocol employed.

[Early diagnosis of HCMV infections in patients undergoing transplantation of the kidney]. / Diagnosi precoce di infezione da HCMV in pazienti sottoposti a trapianto di rene.

HCMV infection is a major cause of morbidity and mortality following kidney transplantation. Clinical diagnosis is difficult, and rapid and sensitive diagnostic methods are needed since antiviral therapy is available. One hundred-forty-five consecutive kidney-transplanted patients were studied during a period of three months after transplantation. For laboratory diagnosis of HCMV infection, we looked for the presence of pp-65 antigen in polymorphonuclear leukocytes, HCMV-DNA and IgM. Demonstration of HCMV pp-65 antigen by immunofluorescence and HCMV DNA by PCR in leukocytes were efficient methods for early diagnosis of infection.

[Anti-CMV antibody affinity in renal transplant recipients]. / Affinità degli anticorpi anti-CMV in portatori di trapianto renale.

Variations in anti-CMV antibody affinity have been studied in 106 renal transplant patients. Maturation of immune response has been followed during two years after transplantation evaluating antibody affinity by ELISA before and after urea denaturation treatment. In primary infections while the antibody titer rises, the resistance to denaturing treatment rises as well, indicating an increased antibody affinity. In patients already seropositive at transplantation, the increase of antibody affinity has also been found: comparing the affinity index (AI) at transplantation and one year later, only 16.5% of patients showed an AI value between 80 and 100 at transplantation, whereas after one year 62.3% of patients reached such AI values (p = 0.0001).

[Infection by human herpesvirus type 6 (HHV-6) and renal transplantation]. / Infezione da virus erpetico umano di tipo 6 (HHV-6) e trapianto di rene.

Human herpes virus type 6 (HHV-6) infection was serologically investigated in renal transplant recipients. Before transplantation, 75.5% of patients was seropositive for HHV-6 and no correlation with age, sex and time on dialysis was found. During the first month after transplantation 66% of patients showed a variation in serological status against HHV-6 (seroconversion or fourfold increase of antibody titer). All patients who seroconverted had received the kidney from a HHV-6 seropositive donor, furthermore, in 11/13 (84.6%) pairs of patients receiving the kidney form the same seropositive donor, both members or had HHV-6 active infection or had no infection. The frequency of HHV-6 active infection in seropositive patients is almost the same in case of seronegative or seropositive donor. Comparing HHV-6 and CMV infections, they resulted independent as CMV infection in these patients occurs in a following period (II-III month). Notwithstanding a higher frequency of kidney rejection in patients with active HHV-6 infection, no significative correlation was found.