Expression dynamics of human cytomegalovirus immune evasion genes US3, US6, and US11 in the blood of lung transplant recipients.

Delayed elimination of human cytomegalovirus (HCMV)-infected cells by the host immune system may contribute to viral dissemination and pathogenesis of HCMV infection. The mRNA expression dynamics of HCMV-encoded immune evasion genes US3, US6, and US11 expressed after active HCMV infection were analyzed in blood samples of lung transplant recipients by means of quantitative nucleic acid sequence-based amplification. The results were compared with the expression dynamics of IE1 mRNA and pp67 late mRNA, levels of pp65 antigenemia, and antiviral treatment. During acute infection, high levels of US3 and US6 RNA were detected before antigenemia, which were detected simultaneously with IE1 RNA. US11 RNA was detected simultaneously with antigenemia but before late pp67 RNA. These data suggest an active role of viral immune evasion during HCMV infection in vivo. Interestingly, immune evasion RNA remained detectable after clinical recovery, often independently of IE1 RNA expression, indicating persistent viral activity, which may have implications for long-term control of HCMV.     

Comparative analysis of a double primer PCR assay with plasma, leukocytes and antigenemia for diagnosis of active human cytomegalovirus infection in bone marrow transplant patients

The aim of the study was to determine the prognostic value of a double primer PCR assay to detect human cytomegalovirus (HCMV) infection or disease in bone marrow transplant (BMT) recipients. A total of 209 blood samples including peripheral blood mononuclear cells (PBMN), polymorphonuclear (PMN) leukocytes and plasma from 26 BMT recipients were tested by PCR assay. To discriminate between latent and active HCMV infection, 177 blood samples were also tested by a quantitative antigenemia assay. HCMV serology status of donors and recipients was determined before transplantation by an enzyme immunosorbent assay method. Using the double primer PCR assay, the number of positive samples increased by an average of 11.6%. Symptomatic active HCMV infection was diagnosed in 14 (53.8%) out of 26 BMT patients. There was a good association between double primer PCR assay of PMN leukocytes and antigenemia assays for detection of active HCMV infection in all patients. Detection of HCMV DNA in PMN leukocytes of BMT patients by double primer PCR assay can be an alternative method for antigenemia assay. However, quantitative PCR methods will be necessary for monitoring antiviral treatment.     

Human Cytomegalovirus and Autoimmune Diseases: Where Are We?

Human cytomegalovirus (HCMV) is a ubiquitous double-stranded DNA virus belonging to the β-subgroup of the herpesvirus family. After the initial infection, the virus establishes latency in poorly differentiated myeloid precursors from where it can reactivate at later times to cause recurrences. In immunocompetent subjects, primary HCMV infection is usually asymptomatic, while in immunocompromised patients, HCMV infection can lead to severe, life-threatening diseases, whose clinical severity parallels the degree of immunosuppression. The existence of a strict interplay between HCMV and the immune system has led many to hypothesize that HCMV could also be involved in autoimmune diseases (ADs). Indeed, signs of active viral infection were later found in a variety of different ADs, such as rheumatological, neurological, enteric disorders, and metabolic diseases. In addition, HCMV infection has been frequently linked to increased production of autoantibodies, which play a driving role in AD progression, as observed in systemic lupus erythematosus (SLE) patients. Documented mechanisms of HCMV-associated autoimmunity include molecular mimicry, inflammation, and nonspecific B-cell activation. In this review, we summarize the available literature on the various ADs arising from or exacerbating upon HCMV infection, focusing on the potential role of HCMV-mediated immune activation at disease onset.     

Successful re‐transplantation in patient with recurrent parvovirus B19 pure red cell aplasia

Impaired cell‐mediated, as well as antibody‐mediated immunity predisposes a renal transplant recipient to a wide variety of atypical infection. With an increasing number of re‐transplant, the balance between immunosuppression and the risk of recurrent disease poses a clinical and therapeutic challenge. Here, we report a successful re‐transplantation in a case of parvovirus B19 infection leading to anaemia and collapsing glomerulopathy in the allograft managed with intravenous immunoglobulin (IVIG) and reduction of immunosuppression. This case emphasizes re‐consideration to renal transplant after clearance of the virus in a previous renal allograft lost to PVB19 infection.     

Monitoring of human cytomegalovirus infections and ganciclovir treatment in heart transplant recipients by determination of viremia, antigenemia, and DNAemia.

Fourteen heart transplant recipients were monitored for human cytomegalovirus (HCMV) infection based on determination of antigenemia, viremia, and DNAemia (by polymerase chain reaction [PCR]) in peripheral blood polymorphonuclear leukocytes (PMNL). Three patients had symptomatic primary, 10 had recurrent (3 asymptomatic), and 1 (seronegative) had no HCMV infection. Severe clinical symptoms appeared when levels of viremia/antigenemia were greater than 50 infected PMNL/2 x 10(5) cells examined. Of 200 blood samples examined, 93 (46.5%) were positive for viremia/antigenemia and DNAemia, whereas 48 (24.0%) were positive for DNAemia only; 59 (29.5%) were negative in all assays. Follow-up of HCMV infections in heart transplant recipients showed that PCR can detect viral appearance in blood 7-10 days earlier than assays for antigenemia/viremia. On the other hand, viral disappearance from blood, as assessed by PCR, occurred weeks or months later than revealed by other assays. Detection of virus by PCR only was never associated with overt HCMV-related clinical symptoms. Of the 8 symptomatic patients treated with ganiclovir, 2 became PCR-negative at the end of treatment and 1 cleared virus from blood in the following weeks, whereas 5 showed persistent or recurrent infection.     

beta-Herpesvirus (human cytomegalovirus and human herpesvirus 6) reactivation in at-risk lung transplant recipients and in human immunodeficiency virus-infected patients

Human herpesvirus (HHV)-6 is a beta-herpesvirus-like human cytomegalovirus (HCMV) with the potential to reactivate in immunocompromised persons. HHV-6 and HCMV were assessed in the peripheral blood leukocytes of 26 lung transplant recipients and of 37 human immunodeficiency virus (HIV)-infected patients receiving highly active antiretroviral therapy, to determine the degree of concordance between HHV-6 and HCMV reactivation in different biologic settings. In the lung transplant recipients (145 samples), HHV-6 was not detected, even though 44 (30%) of 145 samples were from 9 HCMV DNA-positive patients (13 episodes of HCMV pneumonitis). Among the HIV-infected patients (172 samples), HCMV DNA was detected in 29 (17%) of 172 samples from 10 patients (4 episodes of HCMV disease). HHV-6 DNA was detected in 2 HIV-infected patients who did not have HCMV detected at that time. These findings suggest that the pathobiologic control mechanisms for these 2 beta-herpesviruses may be significantly different.     

Human cytomegalovirus load in plasma and bronchoalveolar lavage fluid: a longitudinal study of lung transplant recipients

BACKGROUND: In lung transplant recipients (LTRs), the measurement of human cytomegalovirus (HCMV) load dynamics in the local environment of the lung allograft may offer distinct advantages over their assessment in the peripheral blood compartment. METHODS: We compared HCMV load in paired bronchoalveolar lavage (BAL) and plasma samples in a prospective cohort of LTRs. RESULTS: In all, 182 paired samples were collected from 41 LTRs. Qualitative findings were concordant for 79.7% of the paired samples (virus load detected or absent in 8.1% and 71.5% of paired samples, respectively). In 35 paired samples (20.3%), HCMV was detected in the BAL but not the plasma sample; the converse was not found for any of the paired samples. HCMV inclusions were histologically detected in the lungs of 8 patients on 9 occasions. HCMV was detected in both BAL and plasma samples on 7 of the 9 occasions and in only the BAL sample on 2 occasions. Application of a threshold virus load in BAL samples, to predict histologically proven HCMV infection, was associated with improved diagnostic sensitivity and specificity. CONCLUSIONS: Quantification of HCMV in the lung allograft correlates with histological detection and, in addition, offers the potential to monitor subclinical viral replication in the lung allograft.     

CASE REPORT: Post-Liver Transplant Crohn's Disease: Graft Tolerance but Not Self-Tolerance?

Inflammatory bowel disease (IBD) rarely occurs de novo after liver transplantation, and when it does usually presents as ulcerative colitis in patients transplanted for primary sclerosing cholangitis. We present two patients who developed de novo Crohn's colitis two and three years after liver transplant for primary biliary cirrhosis (PBC) and chronic hepatitis B infection, respectively. Both were on maintenance immunosuppression with a calcineurin inhibitor and azathioprine with no evidence of allograft rejection. Investigations for enteric and opportunistic infection were negative. In the nontransplant setting, the development of IBD is likely multifactorial with an immune origin. In our cases, the immunosuppression was titrated to minimal levels when IBD developed. Clearly, although a significant degree of allograft tolerance can occur, autoimmune diseases can still develop elsewhere, suggesting selective immune tolerance.     

Validation of a DNAemia cutoff for preemptive therapy of cytomegalovirus infection in adult hematopoietic stem cell transplant recipients

A randomized trial comparing a DNAemia cutoff of 10 000 copies per ml whole blood and first pp65 antigenemia positivity for initiation of preemptive therapy of human cytomegalovirus (HCMV) infection in adult hematopoietic stem cell transplant recipients was completed. DNAemia was chosen for cutoff definition since it is more automatable and standardizable than antigenemia, and more closely reflects the actual viral replication. The primary end point of the study was to compare the number of patients treated in the two arms. A total of 83 patients (42 in the DNAemia, and 41 in the antigenemia arm) were enrolled in the study. The incidence of HCMV infection, as detected by the relevant randomization assay (76% in the DNAemia versus 85% in the antigenemia arm), was comparable in the two arms, whereas the number of patients treated was significantly lower in the DNAemia arm (63 versus 80%, P=0.02). A single patient in the DNAemia arm suffered from biopsy-proven HCMV gastric disease diagnosed in the absence of detectable virus in blood. The incidence of graft-versus-host disease, and transplantation-related mortality did not differ between the two arms. In conclusion, our study shows that the use of a cutoff significantly reduces the number of patients requiring antiviral treatment, thus sparing unnecessary drug administration.     

Human cytomegalovirus (HCMV) replication dynamics in HCMV-naive and -experienced immunocompromised hosts

Human cytomegalovirus (HCMV) can infect both HCMV-naive and -experienced transplant patients. In this study, the growth rate of HCMV in HCMV-naive hosts (1.82 units/day; 95% confidence interval [CI], 1.44-2.56 units/day) was shown to be significantly faster than the growth rate of virus in HCMV-experienced hosts undergoing recurrent infection (0.61 units/day; 95% CI, 0.55-0.7 units/day; P<.0001). The basic reproductive number (R(0)) for HCMV-naive liver transplant patients was 15.1 (95% CI, 8.9-44) but was only 2.4 (95% CI, 2.35-2.8) for HCMV-experienced transplant recipients, corresponding to an anti-HCMV immune efficacy of approximately 84%, despite immunosuppressive therapy. The R(0) values suggest that an anti-HCMV drug or vaccine with an efficacy of >93% (95% CI, 89%-98%) is required to eliminate viral growth during infection of HCMV-naive liver transplant recipients, whereas lower efficacy levels are sufficient to reduce the R(0) value to <1 in hosts with prior HCMV immunity.     

Uninfected and cytomegalic endothelial cells in blood during cytomegalovirus infection: effect of acute rejection

After transplantation, human cytomegalovirus (HCMV) infections can cause vascular damage to both the graft and the host. To study a possible relationship between the degree of vascular injury, clinical symptoms of HCMV infection, and transplant rejection, the appearance and numbers of endothelial cells (ECs) in blood of 54 kidney transplant recipients were investigated in a prospective clinical study. Two types of endothelial cells were identified: cytomegalic ECs (CECs) were detected in patients with moderate or high HCMV antigenemia, and uninfected ECs were observed in patients with and without HCMV infection. The incidence of either CECs, ECs, or the combination of both was associated with HCMV-related clinical symptoms (P<.01). Remarkably, the occurrence of rejection episodes before HCMV infection was an important risk factor for the occurrence of ECs in blood (ECs, CECs, or both) during HCMV infection (P<.001).     

Suspected delayed immune recovery against cytomegalovirus after reduced-intensity stem cell transplantation using anti-thymocyte globulin

A reduced-intensity hematopoietic stem cell transplantation (RIST) regimen was developed to induce immunosuppression to facilitate the engraftment of donor cells. However, there have been concerns that the incidence of opportunistic infection may increase after this procedure. To address this problem, we retrospectively analyzed the medical records of 24 RIST recipients who were treated over a recent 16-month period for comparison with 31 recipients of conventional allogeneic transplantation (CST). The RIST regimen consisted of cladribine (0.66 mg/kg), busulfan (8 mg/kg), and rabbit anti-thymocyte globulin (ATG; 5-10 mg/kg). All of the patients received allogeneic peripheral blood stem cells from an HLA-identical or one-locus mismatched related donor. Although the incidence of positive CMV antigenemia was comparable between the two groups (58% vs 68%), RIST patients developed positive antigenemia significantly sooner than did CST patients (P = 0.01) and showed higher initial and maximum antigenemia values (P = 0.026 and P = 0.003, respectively). These findings may suggest that immune recovery against CMV was delayed after our RIST procedure, but this did not directly translate into an increase in clinically significant CMV disease. Early therapeutic intervention with ganciclovir might play a role in preventing the progression of early CMV infection to CMV disease.     

Cytomegalovirus (CMV)-specific T cell immunity after renal transplantation mediates protection from CMV disease by limiting the systemic virus load.

The role of cytomegalovirus (CMV)-specific cytotoxic T lymphocytes (CTLs) and T helper cells (Th) in controlling CMV infection, as detected by antigenemia assay and polymerase chain reaction (PCR) in blood leukocytes, and CMV disease was investigated in 20 renal transplant recipients. Within 3 months after transplant, CMV-specific CTL and Th responses were demonstrable in 11 (55%) and 15 (75%) patients, respectively; CMV infection was detected by antigenemia and PCR in 19 (95%) patients each. During the month of first CMV detection, there was an inverse correlation between CTL response and antigenemia at >/=20 positive cells/105 leukocytes (P=.007) but no association with lower antigenemia levels or PCR positivity. CMV disease developed in 7 (35%) patients and was associated with high-level antigenemia but was inversely correlated with detection of CTLs (P=.04). After renal transplantation, CMV-specific CTLs limit the systemic virus load as reflected by antigenemia levels and thereby mediate protection from CMV disease.     

Human Immunodeficiency Virus Infection Does Not Alter Serum Transaminases and Hepatitis B Virus (HBV) DNA in Homosexual Patients with Chronic HBV Infection

The influence of human immunodeficiency virus (HIV) infection on the clinical course of chronic hepatitis B virus (HBV) infection is controversial. We followed a cohort of 64 homosexual men with persistent HBs antigenemia for a median of 24 months in the hepatitis clinic of a large urban public hospital. We divided the patients into three groups according to their immune status. Group 1 (n = 13) consisted of HIV-seropositive men with evidence of immunosuppression; group 2 (n = 17), HIV-positive patients without evidence of immunosuppression; and group 3 (n = 34), HIV-negative patients. We followed serum ALT and HBV DNA determinations. There was no difference in the demographic characteristics of the three groups. Group 1 had significantly lower levels of circulating T4 lymphocytes. We found no differences in the number and severity of episodes of HBV reactivation, serum ALT levels, or HBV DNA scores among the three groups. In each group, the percentage of patients with circulating HBV DNA was the same. We conclude that HIV infection apparently does not influence the markers of liver inflammation or HBV replication in homosexual men.     

Dendritic cell vaccination in allogeneic stem cell recipients: induction of human cytomegalovirus (HCMV)-specific cytotoxic T lymphocyte responses even in patients receiving a transplant from an HCMV-seronegative donor

Vaccination with peptide-loaded dendritic cells (DCs) has been shown to be potent immunostimulatory therapy for the management of serious infections. After allogeneic stem cell transplant (SCT), a prolonged and severe immune deficiency often leads to infectious complications. Human cytomegalovirus (HCMV) infection is one such life-threatening complication after allogeneic SCT. A phase 1/2 study including 24 allogeneic SCT recipients at high risk for HCMV disease was performed to analyze the feasibility and efficacy of vaccination with HCMV peptide-loaded DCs. No acute adverse effects were observed, and a significant clinical benefit could be demonstrated in comparison to our historical control group. An induction or expansion of HCMV-specific cytotoxic T lymphocytes was observed in 5 patients after DC vaccination.     

Antigenemia in the diagnosis and monitoring of active cytomegalovirus infection after liver transplantation.

In 45 liver transplant recipients, the value of weekly monitoring of cytomegalovirus (CMV) antigenemia for early diagnosis of active CMV infection was compared with serology and rapid viral isolation. Active CMV infection occurred in 23 patients. The sensitivities of the antigenemia assay and serology (of blood) and rapid viral isolation (from blood or urine) were 96%, 96%, 57%, and 70%, respectively. First diagnostic results of these methods were obtained a median of 25, 36, 31, and 49 days, respectively, after transplant. CMV infection was symptomatic in 20 patients; antigenemia was present at the onset of disease in 13 of these. Maximum CMV antigenemia levels were higher in patients with severe disease than in those with mild or asymptomatic infection. CMV antigenemia is a sensitive, early, quantitative marker of active CMV infection after liver transplantation.     

Cytomegalovirus antigen detection in peripheral blood leukocytes after allogeneic marrow transplantation.

Detection of cytomegalovirus (CMV) antigenemia was compared with shell vial centrifugation cultures for rapid detection of CMV infection. In a prospective study, 59 CMV seropositive patients were monitored weekly during the first 100 days after allogeneic marrow transplantation for virus excretion from urine, throat, and blood and for antigenemia by direct staining of peripheral leukocytes using an antibody pool directed against pp 65. Antigenemia was present in 21 of 22 patients with culture-proven CMV infection and in 3 of 37 without culture-proven CMV infection (sensitivity 95%, specificity 91%). The median time of onset of antigenemia and shell vial cultures was day 47 and 55 after transplant, respectively (P = .0006). Among patients who developed CMV disease without preceding cultures, antigenemia was detected in all patients with CMV pneumonia (N = 6) and in two of three patients with gastrointestinal disease by a median of 10 and 7 days, respectively, before the onset of disease (P = .0002). Levels of antigenemia were significantly higher in patients with disease or viremia than in patients with excretion from urine or throat (P less than .05). Whether the antigenemia assay is more sensitive than rapid culture methods to focus antiviral prophylaxis in marrow transplant patients must be determined in controlled studies.     

Human cytomegalovirus immediate-early mRNAemia versus pp65 antigenemia for guiding pre-emptive therapy in children and young adults undergoing hematopoietic stem cell transplantation: a prospective,randomized, open-label trial

In the search for better protocols of preemptive therapy of human cytomegalovirus (HCMV) infection in hematopoietic stem cell transplant (HSCT) recipients, we conducted a randomized trial comparing antigenemia with the nucleic acid sequence-based assay (NASBA) for determination of HCMV immediate-early messenger RNA (IEmRNA) as the guiding assay for initiation of pre-emptive antiviral treatment. In the IEmRNA arm, antiviral therapy was started upon IEmRNA positivity confirmed the following day, whereas in the antigenemia arm, therapy was started in the presence of either at least 2 pp65-positive leukocytes/2 x 105 examined or a single positive leukocyte confirmed the following day. In both arms, treatment was stopped upon 2 consecutive negative results. All patients were monitored for 3 months after HSCT. The primary end point of the study was duration of anti-HCMV therapy. On the whole, 80 children (41 in the IEmRNA and 39 in the antigenemia arm), recipients of transplants from either a relative or an unrelated donor, completed the study. No patient developed HCMV disease. In the IEmRNA arm, the incidence of HCMV infection was higher compared to the antigenemia arm (80% vs 51%, respectively, P =.0069), as well as the percentage of treated patients (66% vs 44%, respectively, P =.045). However, the percentage of relapses and treated relapses was comparable in the 2 arms. There was no significant difference in median duration of therapy per patient. Although these data indicate that IEmRNA determination does not offer advantages in terms of treatment duration, it can safely replace antigenemia, while semiautomation is the major advantage of the NASBA procedure.     

Infusion of autologous Epstein-Barr virus (EBV)-specific cytotoxic T cells for prevention of EBV-related lymphoproliferative disorder in solid organ transplant recipients with evidence of active virus replication

Epstein-Barr virus (EBV)-associated posttransplantation lymphoproliferative disorders (PTLDs) are a well-recognized complication of immunosuppression in solid organ transplant recipients. The reported therapeutic approaches are frequently complicated by rejection, toxicity, and other infectious pathologies, and overall mortality in patients with unresponsive PTLD remains high. Thus, low-toxicity treatment options or, preferably, some form of prophylactic/preemptive intervention are warranted to improve PTLD outcome in this setting. We assessed whether transfer of EBV-specific cytotoxic T lymphocytes (CTLs) generated in vitro from the peripheral blood of allograft recipients receiving immunosuppression could increase EBV-specific killing in vivo without augmenting the probability of graft rejection. Autologous EBV-specific CTLs were generated for 23 patients who were identified as being at risk of developing PTLD through the finding of elevated EBV DNA load. Of the 23 patients, 7 received 1 to 5 infusions of EBV-specific CTLs. CTL transfer was well tolerated, and none of the patients showed any evidence of rejection. An increase of the EBV-specific cytotoxicity was observed after infusion, notwithstanding continuation of immunosuppressive therapy. EBV DNA levels had a 1.5- to 3-log decrease in 5 patients, whereas in the other 2 graft recipients CTL transfer had no apparent stable effect on EBV load. Our data suggest that the infusion of autologous EBV-specific CTLs obtained from peripheral blood mononuclear cells recovered at the time of viral reactivation is able to augment virus-specific immune response and to reduce viral load in organ transplant recipients. This approach may, therefore, be safely used as prophylaxis of EBV-related lymphoproliferative disorders in these patients, following a strategy of preemptive therapy guided by EBV DNA levels.     

Adenovirus causing fever,upper respiratory infection,and allograft nephritis complicated by persistent asymptomatic viremia

A 20‐year‐old woman, with renal transplant complicated by recurrence of focal segmental glomerulosclerosis and post‐transplant lymphoproliferative disorder, presented nearly 2 years after transplantation with fever, conjunctivitis, and sinus congestion. She was found to have severe adenovirus (ADV)‐induced granulomatous interstitial nephritis, confirmed by immunohistochemical staining for ADV in the renal biopsy, without urinary symptoms, hematuria, or laboratory evidence of a change in allograft function. Fever, upper respiratory tract symptoms, and evidence of adenoviral infection in the allograft resolved with decreased immunosuppression and treatment with cidofovir and intravenous immunoglobulin. Creatinine rose during treatment and remained elevated, possibly related to cidofovir nephrotoxicity. Despite therapy and continued reduction in immunosuppression, asymptomatic low‐level viremia persisted for a year. In renal transplant patients with ADV infection, allograft involvement should be highly suspected even without overt urinary symptoms or laboratory evidence of allograft dysfunction. Demonstration of allograft involvement may prompt alternative management that could limit continued allograft infection. No clear recommendations exist for management of asymptomatic ADV viremia in solid organ transplant patients.