Symptomatic Cytomegalovirus Research Articles

Quantitative analysis of cytomegalovirus load using a real-time PCR assay.

A novel real-time PCR assay system was developed to quantify the cytomegalovirus (CMV) genome load. The real-time PCR assay could detect from 6 to over 10(6) copies of CMV-DNA with a wide linear range. The virus load of immunocompromised patients with symptomatic CMV infections was quantified and compared to that of asymptomatic ones. In symptomatic patients, all 17 peripheral blood leukocytes were positive for CMV DNA, and its mean value was 10(3.3) copies/10(6) cells. On the other hand, only 9 of 38 samples (24%) were positive in the asymptomatic patients, and its mean titer was lower (10(2.0) copies/10(6) cells) than that of the symptomatic group (P = 0.002). In plasma, the virus genome was detected in 13 out of 17 samples from symptomatic patients (76%), and its mean value was 10(4.0) copies/ml. In contrast, for the asymptomatic group, only one out of 36 samples were positive (3%). Finally, this system was used to monitor two patients with CMV infections serially. The CMV DNA copy number changed with their clinical symptoms and anti-CMV therapy, and virtually paralleled the result of the pp65 antigenemia assay in both cases. In one patient with the cord blood transplantation, however, the CMV DNA became positive faster than the antigenemia assay. These results indicate that this assay is sensitive and useful for estimating the CMV genome load not only in peripheral blood leukocytes but also in plasma. It can be very helpful for diagnosing CMV-related diseases and monitoring the virus load in patients with CMV infections.

Sensitive detection of cytomegalovirus infection in transplant recipients using nucleic acid sequence-based amplification

THE MAJORITY of the adult human population is seropositive for cytomegalovirus (CMV), a member of the b-herpesvirus family. Normally, infection of immunocompetent individuals does not cause disease. In contrast, infection of immunocompromised individuals, such as patients with AIDS and transplant recipients, can cause severe complications and can even lead to death. Therefore, early detection of active CMV infection is necessary to start effective antiviral treatment. Nucleic Acid Sequence-Based Amplification (NASBA) has been developed for the specific amplification of RNA. In previous studies, we evaluated the diagnostic value of monitoring the expression of the CMV immediate early (IE) 1 and late pp67 mRNA using NASBA, in peripheral blood cells of kidney transplant (Tx) patients. The IE 1 mRNA is expressed directly after entrance of the virus into a cell, while pp67 mRNA is expressed in a late stage in the replication cycle. NASBA results were compared to the techniques that are routinely being used for the detection of CMV: pp65-antigenemia, virus culture, and serology. From this study it was concluded that IE NASBA is a very sensitive assay that detects the onset of CMV infection significantly earlier than the other assays. This is especially valuable for patients that are at risk for symptomatic CMV infection, i.e., seronegative patients with an organ of a seropositive donor and/or intense immunesuppression. The sensitivity and specificity values of pp67 NASBA were comparable to those of the antigenemia assay. We set up a similar study to evaluate the diagnostic value of IE and pp67 NASBA for liver Tx patients. The liver Tx patients are usually severely ill, immunesuppression is more intense compared to the kidney Tx patients and they often have anti-rejection therapy. The results for IE NASBA are presented in this report. The evaluation of pp67 NASBA is still in progress.

DOSING OF INTRAVENOUS GANCICLOVIR FOR THE PROPHYLAXIS AND TREATMENT OF CYTOMEGALOVIRUS INFECTION IN SOLID ORGAN TRANSPLANT RECIPIENTS1

The optimal regimen for the prevention and treatment of cytomegalovirus (CMV) disease in solid organ transplant recipients remains to be defined, particularly for patients with abnormal or changing renal function. A prospective trial was conducted in patients receiving i.v. ganciclovir using a standardized dosing nomogram that corrects for renal function. Steady state peak (P) and trough (T) serum levels were determined by high-performance liquid chromatography and correlated with therapeutic outcomes and toxicities attributable to ganciclovir. Over the study period, 44 individuals received ganciclovir prophylaxis (5 mg(kg/day) and 25 patients were treated (5 mg/kd q12 hr) for symptomatic CMV disease. Ganciclovir levels (microg/ml+/-SD) achieved in prophylaxis were P: 7.98+/-3.34, T: 3.03+/-2.63; and in treatment were P: 9.00+/-3.72, T: 2.65+/-1.82. Despite corrections for renal dysfunction, undialyzed patients with serum creatinine >3.0 mg/dl had trough levels in excess of the population mean (T: range 3-8 microg/ml). Failure of prophylaxis (disease) or therapy (relapse) occurred in 14 patients; 8 of these were at risk for primary infection (donor CMV seropositive, recipient seronegative, P<0.01). Patients at greatest risk for relapse after treatment of CMV disease were liver transplant recipients, patients with ganciclovir-resistant viral isolates, and renal patients with six antigen MHC donor-recipient mismatches. This trial demonstrates the efficacy of a nomogram for ganciclovir dosing during renal dysfunction; reduced doses can be used for prophylaxis for undialyzed patients with renal dysfunction (1.25 mg/kg/day for Cr > or =3.0, 1.25 mg/kg QOD for Cr > or =5.0). Some groups of transplant recipients may require more intensive anti-CMV regimens.

An association between cytomegalovirus infection and chronic rejection after liver transplantation.

Previous studies suggest a link between cytomegalovirus (CMV) infection and chronic rejection. Since these studies, more sophisticated diagnostic methods with high sensitivity and specificity for CMV have been developed and effective therapy/prophylaxis for CMV is now available. We sought CMV prospectively by polymerase chain reaction of serum and urine and by conventional methods in a group of 33 patients undergoing 57 transplants during 1993 or 1994, selected from a larger series. There were 13 grafts lost to chronic rejection. The remaining 44 grafts that did not develop chronic rejection served as controls and comprised 15 successful primary grafts, 15 second transplants, 8 third transplants, and 6 primary grafts that were lost for reasons other than chronic rejection. The combination donor CMV antibody negative with recipient antibody positive and the duration of CMV infection >30 days were associated with an increased relative risk of chronic rejection. In contrast, the presence of CMV infection alone, symptomatic CMV infection, the detection of CMV by PCR of serum or urine, and the peak/cumulative viral load were not predictive. CMV infection occurred earlier in those undergoing a second transplant for chronic rejection than for those undergoing a second transplant for other reasons. In addition, a human leukocyte antigen B mismatch was associated with prolonged CMV infection. These data are consistent with the hypothesis that prolonged subclinical cytomegalovirus infection is associated with an increased risk of chronic rejection.

Evaluation of new quantitative assays for diagnosis and monitoring of cytomegalovirus disease in human immunodeficiency virus-positive patients.

Cobas Amplicor CMV Monitor (CMM) and Quantiplex CMV bDNA 2.0 (CMV bDNA 2.0), two new standardized and quantitative assays for the detection of cytomegalovirus (CMV) DNA in plasma and peripheral blood leukocytes (PBLs), respectively, were compared to the CMV viremia assay, pp65 antigenemia assay, and the Amplicor CMV test (P-AMP). The CMV loads were measured in 384 samples from 58 human immunodeficiency virus (HIV) type 1-infected, CMV-seropositive subjects, including 13 with symptomatic CMV disease. The assays were highly concordant (agreement, 0.88 to 0.97) except when the CMV load was low. Quantitative results for plasma and PBLs were significantly correlated (Spearman rho = 0.92). For PBLs, positive results were obtained 125 days before symptomatic CMV disease by CMV bDNA 2.0 and 124 days by pp65 antigenemia assay, whereas they were obtained 46 days before symptomatic CMV disease by CMM and P-AMP. At the time of CMV disease diagnosis, the sensitivity, specificity, and positive and negative predictive values of CMV bDNA 2.0 were 92.3, 97.8, 92.3, and 97.8%, respectively, whereas they were 92.3, 93.3, 80, and 97. 8%, respectively, for the pp65 antigenemia assay; 84.6, 100, 100, and 95.7%, respectively, for CMM; and 76.9, 100, 100, and 93.8%, respectively, for P-AMP. Considering the entire follow-up, the sensitivity, specificity, and positive and negative predictive values of CMV bDNA 2.0 were 92.3, 73.3, 52.1, and 97.1%, respectively, whereas they were 100, 55.5, 39.4, and 100%, respectively, for the pp65 antigenemia assay; 92.3, 86.7, 66.7, and 97.5%, respectively, for CMM; and 84.6, 91.1, 73.3, and 95.3%, respectively, for P-AMP. Detection of CMV in plasma is technically easy and, despite its later positivity (i.e., later than in PBLs), can provide enough information sufficiently early so that HIV-infected patients can be effectively treated. In addition, these standardized quantitative assays accurately monitor the efficacy of anti-CMV treatment.

Quantity of human cytomegalovirus (CMV) DNAemia as a risk factor for CMV disease in renal allograft recipients: Relationship with donor/recipient CMV serostatus, receipt of augmented methylprednisolone and antithymocyte globulin (ATG)

A prospective longitudinal study of 87 renal allograft recipients identified 31 patients with cytomegalovirus (CMV) viraemia. Previous studies have identified CMV viraemia, donor positivity, and CMV load in urine as independent risk factors for disease following renal transpl antation. We used quantitative-competitive polymerase chain reaction (QC-PCR) to quantify the CMV DNA load in blood from these patients, and report that it is a significant and independent risk factor for CMV disease. Patients with symptomatic CMV infection had significantly higher maximum CMV loads than those with no disease (P = .0003). We also found that peak loads were significantly higher in individuals experiencing primary CMV infection (P < .01), and CMV re-infection (P < .05) compared with recipients reactivating endogenous CMV. Univariate analysis revealed that CMV DNA load in blood, donor seropositivity, and receipt of antithymocyte globulin (ATG) were all significantly associated with disease (P = .005, .04, and .05, respectively). However, the association of donor/recipient serostatus, and receipt of ATG became nonsignificant in multivariate analyses whereas the significance of the quantity of CMV DNAemia was maintained, illustrating that CMV load plays a central role in the pathogenesis of CMV disease.

Evaluation of the AmpliSensor PCR and the SHARP signal detection system for the early prediction of symptomatic CMV infection in solid transplant recipients.

Cytomegalovirus (CMV) is associated with high morbidity and mortality in transplant patients. Specific antiviral treatment at an early stage of CMV infection may effectively ameliorate, but not eliminate CMV disease in these patients. Presently, the pp65 antigenemia test on peripheral leukocytes is the method most widely used for predicting and monitoring transplant patients for active CMV infection. Nucleic acid amplification methods are less well defined since they lack standardisation. A seminested fluorometric PCR assay (AmpliSensor-CMV, BAG, Germany) and a one-step PCR with a signal-amplification step (SHARP, Abbott, Germany) specific for the fragments of the CMV UL 122 and UL 123 genes, respectively, were evaluated for the early diagnosis of CMV infection. A total of 26 recipients of heterogeneous solid organs were monitored prospectively for a median of 99 days after transplantation. By testing 371 clinical samples parallel with the pp65-antigen assay and IgM and IgG EIA assays the sensitivity, specificity, correlation and quantitation potential of both PCRs was evaluated. Eight out of 26 patients developed active CMV infection. A total of 48 samples of these patients exceeded a CMV-DNA load threshold of 15 genome equivalents/10(5) leukocytes (AmpliSensor-CMV) and 41 samples exceeded the critical cut-off for the SHARP system. The AmpliSensor PCR exceeded its threshold consistently before the clinical onset of CMV disease (median 8 days). There was very good agreement between symptomatic CMV infection in patients and AmpliSensor-PCR, SHARP PCR, and pp65-antigen results (kappa-coefficient > 0.900). IgM and IgG EIA showed moderate agreement (kappa-coefficient = 0.591 and 0.552, respectively). Both PCRs and pp65 antigen assay correlated significantly better with CMV disease than serodiagnosis. The AmpliSensor PCR allowed more precisely than the SHARP system a quantitative determination of viral load and an early and reliable prediction of active CMV infection. The use of AmpliSensor PCR may improve the diagnosis and management of active CMV infection in organ transplant recipients.

Molecular pathogenesis of human cytomegalovirus infection.

Despite progress in diagnosis and treatment of human cytomegalovirus (CMV) infection, we do not understand why, in hosts with comparable levels of immunosuppression, some CMV infections result in symptomatic CMV disease while others are limited to asymptomatic virus shedding with no discernible clinical consequences. CMV viral detection and quantification are useful for identifying those at highest risk, but do not consistently predict clinical outcome. Factors such as host genotype and immune response are active areas of research. However, the importance of CMV strain variability, recognized since 1976, is now receiving attention. Advances in technology that allow the rapid sequencing of viral DNA for purposes of strain characterization have fueled the renewed interest. The focus of this review will be to summarize our evolving knowledge of CMV strain variability and to document where possible a potential relationship to strain virulence. Studies with the UL55 (gB) envelope glycoprotein will be emphasized because of the ability to clearly identify naturally occurring variants, as well as the increasing number of reports that there are differences in biological activities that may contribute to virulence.

Diagnosis of Cytomegalovirus Infection by the Detection of Early Antigen (pp65) in Leukocytes of Kidney Transplant Patients

Objective: Cytomegalovirus (CMV) infection is a frequent complication of kidney transplantation contributing substantially to morbidity and mortality. Early diagnosis of the infection is essential to implement an effective antiviral therapy. This study is aimed at establishing the CMV antigenemia assay for the diagnosis of CMV infection, and for monitoring the effectiveness of ganciclovir therapy in kidney recipients in Kuwait. Methods: Leukocyte-enriched fractions of 215 blood specimens from 92 kidney recipients were processed for the detection of CMV-specific protein (pp65) by using a commercial kit (Incstar Corp., USA). Plasma fractions were also tested for the presence of CMV IgM antibodies, and selected samples (n = 15) were processed for virus isolation in MRC-5 diploid cell culture. Results: Regarding symptomatic (n = 35) and asymptomatic patients (n = 57) the assay showed an 86% positive and a 79% negative predictive value, compared to the 55% and 70% values, respectively, for CMV IgM antibodies. The number of positive cells was parallel with the severity of symptoms. Using a cutoff level of >5 antigen-positive leukocytes/50,000 cells, the assay reached 93% positive and 96% negative predictive values. There was a very good agreement between the assay and virus isolation. Ganciclovir therapy could be monitored effectively with the assay, and in 11 symptomatic patients treated with ganciclovir, a prompt decrease in the number of antigen-positive cells was noted. Conclusion: The CMV antigenemia assay is a valuable tool for the early diagnosis of symptomatic CMV infection and monitoring antiviral therapy in kidney transplant patients.

Rethinking chronic allograft nephropathy: the concept of accelerated senescence.

Rethinking chronic allograft nephropathy: the concept of accelerated senescence.

Association of cytomegalovirus genotype with graft rejection after liver transplantation.

The envelope glycoprotein gB of human cytomegalovirus (CMV) occurs as one of four main genotypes. Some previous studies have proposed a relationship of CMV gB genotype to the frequency of symptomatic infection and to clinical outcomes in both transplant and human immunodeficiency virus-infected populations. Our aim was to define the distribution of CMV gB genotypes and the impact on acute cellular rejection and graft/patient survival after orthotopic liver transplantation (OLT). Between October 1988 and December 1996, 325 patients underwent cyclosporine-based OLT at our center. CMV infection was surveyed prospectively and defined as viral isolation from blood or urine; 53 (16%) patients had detectable CMV. Isolates were genotyped by polymerase chain reaction amplification and restriction digest analysis. The distribution of CMV genotypes was: gB1, 19 (36%) patients; gB2, 15 (28%) patients; gB3, 13 (24%) patients; and gB4, 4 (8%) patients. Two patients (4%) had mixed infection (1 + 3, 1 + 4). Age, preOLT diagnosis, use of ganciclovir prophylaxis, basal immunosuppression, mean number of HLA donor/recipient mismatches, and United Network of Organ Sharing status were comparable among patients with different genotypes. Patients with gBl had a significantly higher mean number of acute rejection episodes (1.52+0.30 vs. 0.67+0.22; P=0.027). However, there was no difference in rejection severity, including OKT3 usage or FK506 conversion, or development of chronic rejection among patients with different genotypes. The gB genotype did not affect the development of symptomatic or tissue-invasive CMV disease, detected in 15 patients. Actuarial rates of patient (odds ratio [OR] 3.0; confidence interval [CI] 1.49-6.0) and graft (OR 2.57; CI 1.25-5.22) survival were significantly diminished in the group with CMV infection versus those without CMV (P<0.0001 for both), but there was no association with CMV genotype. (1) Patients with CMV infection had significantly reduced patient and graft survival rates at 1 and 5 years after OLT as compared with OLT recipients without CMV infection. (2) CMV genotype gB1 was associated with a higher mean number of acute rejection episodes.

Effect of oral acyclovir or ganciclovir therapy after preemptive intravenous ganciclovir therapy to prevent cytomegalovirus disease in cytomegalovirus seropositive renal and liver transplant recipients receiving antilymphocyte antibody therapy.

Organ transplant recipients who are seropositive for cytomegalovirus (CMV) and who are treated with antilymphocyte antibody (ALA) therapy have a high rate of symptomatic CMV disease. The intravenous administration of ganciclovir therapy once daily during ALA therapy decreased the incidence from 24% to 10% in patients receiving ALA as an induction therapy and from 64% to 22% in those treated for rejection. The present study was undertaken to determine whether a more intensive and sustained antiviral regimen could be more effective. From April 1995 to December 1997, all CMV seropositive renal and liver transplant recipients who received ALA therapy were treated with intravenously administered ganciclovir (5 mg/kg/day with dose adjusted for renal dysfunction) for the length of ALA therapy and then with orally administered acyclovir (400 mg three times/day) or ganciclovir (1 gm twice/day) for 3 to 4 months. The incidence of CMV viremia and of CMV disease was determined during the 6 months after completion of ALA therapy. Forty-one patients (35 renal and 6 liver transplant recipients) were studied. CMV disease occurred in 2 patients (4.9%), both of whom were treated for rejection; it occurred in 1 of 21 patients (4.8%) treated with orally administered acyclovir, and in 1 of 20 patients (5%) treated with orally administered ganciclovir. The only patient who developed CMV disease in the ganciclovir group had received only 26 days of oral antiviral therapy. No CMV disease was documented in the group of patients receiving ALA therapy as induction therapy. CMV viremia occurred in three patients in the acyclovir group (14.3%) and in one patient in the ganciclovir group (5%). Among renal transplant recipients only, 1 of 35 patients developed CMV disease (2.9%) and no case of CMV disease was documented in patients treated with orally administered ganciclovir. All six patients receiving two courses of ALA therapy each were free of CMV disease. Toxicity of the regimen was minimal, and antiviral resistance did not develop. Preemptive antiviral therapy with intravenously administered ganciclovir during ALA therapy and then orally administered ganciclovir for 3 to 4 months provides virtually complete protection against the excessive rate of CMV disease that occurs in CMV seropositive allograft recipients receiving ALA therapy.

Clinical Course of Cytomegalovirus (CMV) Viremia with and without Ganciclovir Treatment in CMV-Seropositive Kidney Transplant Recipients

This study was designed to evaluate the longitudinal history of cytomegalovirus (CMV) infection and to test the capacity of ganciclovir as effective therapy in CMV-seropositive renal transplant recipients. The CMV viremia was detected with CMV pp65 antigenemia assay in 153 renal transplants. The recipients were classified as having low-grade and high-grade CMV infections according to the severity of CMV infection. The recipients with low-grade CMV infections were observed without ganciclovir treatment, and the recipients with high-grade CMV infection were randomly assigned to ganciclovir-treated and untreated groups. The clinical course between low-grade and high-grade CMV infections was evaluated. All recipients with low-grade CMV infection (n = 62) showed spontaneous remission regardless of immunosuppresants. In high-grade CMV infection (n = 31), the ciclosporin A treated group (n = 11) showed no evidence of CMV disease, and the methylprednisolone-treated group (n = 8) showed CMV disease in 1 (25%) of 4 ganciclovir-untreated recipients. In the OKT3 group (n = 12), symptomatic CMV infection was observed in 6 (100%) ganciclovir-untreated recipients contrary to no CMV disease in the ganciclovir-treated group (p < 0.05). In conclusion, the CMV antigenemia assay is effective in monitoring CMV viremia, and ganciclovir treatment should be done during early CMV viremia in OKT3-treated recipients.

Quantitative polymerase chain reaction to predict occurrence of symptomatic cytomegalovirus infection and assess response to ganciclovir therapy in renal transplant recipients.

Cytomegalovirus (CMV) DNA levels were measured by quantitative-competitive polymerase chain reaction (PCR) in weekly leukocyte samples from 50 renal transplant recipients, including 23 with symptomatic and 27 with asymptomatic CMV infection. Peak and week 4 CMV DNA levels were higher in symptomatic subjects (P = .07 and .02, respectively). In a logistic regression model, the logarithm of the week 4 level independently predicted symptomatic infection (odds ratio, 1.78 for a 1 log10 increase; 95% confidence interval, 1.14-2.78; P = .01). All subjects whose week 4 level exceeded 1000 copies/100,000 leukocytes developed symptoms. In subjects with adequate samples for analysis, CMV levels declined exponentially with ganciclovir treatment, with an average half-life of 3.3 days. Levels exceeding 10,000 copies were associated with prolonged time to clearing of CMV DNA. Potential clinical applications of quantitative CMV PCR include predicting occurrence of symptomatic first episodes after transplantation and individualizing duration of antiviral therapy.

EFFECT OF ORAL ACYCLOVIR OR GANCYCLOVIR AFTER PREEMPTIVE INTRAVENOUS GANCYCLOVIR THERAPY TO PREVENT CYTOMEGALOVIRUS DISEASE IN CYTOMEGALOVIRUS SEROPOSITIVE RENAL AND LIVER TRANSPLANT RECIPIENTS RECEIVING ANTILYMPHOCYTE THERAPY

443 Background: Cytomegalovirus (CMV) seropositive organ transplant recipients treated with antilymphocyte antibody (ALA) therapy have a high rate of symptomatic CMV disease: 10% if no ALA, 25% if induction ALA, and 64% if anti-rejection ALA.. Administrating IV gancyclovir once daily during the period of ALA therapy decreased the incidence by 2-3 fold, so that patients treated for rejection had an incidence of CMV disease of 22%. Objective: To determine if oral antiviral therapy with acyclovir (ACV) or gancyclovir (GCV) following preemptive therapy with IV GCV administered daily during ALA therapy can decrease further the incidence of CMV disease in patients with a renal or liver transplant who are seropositive for CMV. Design: Non randomized retrospective study Methods: Since April 1995, all CMV seropositive patients treated with ALA received GCV 5mg/kg IV during the ALA therapy, followed either by ACV 400 mg TID or GCV 1gm BID for 3-4 months (with doses adjusted for renal dysfunction). The incidence of CMV disease or viremia was determined during the oral therapy and for 6 months following its completion. Results: 39 patients, 34 renal and 5 liver transplants, were studied. CMV disease or viremia occurred in 3 of 20 ACV patients (15%), with 2 of the 3 developing CMV while still on ACV. CMV disease developed in 0 of 17 patients (0%) in the oral GCV group. Two patients were treated with oral GCV followed by oral ACV; among this small group, no patient developed CMV disease. Of the 3 patients who had CMV disease, 2 had received ALA therapy for induction therapy and 1 for rejection therapy. Among patients getting oral ACV (20 patients), 14 (70%) were treated with ALA for induction therapy and 6 (30%) for rejection therapy. Among patients getting oral GCV(17 patients), 6 (35%) received ALA therapy for induction therapy, 9 (53%), for rejection therapy and 2 (12%) received 2 courses of ALA therapy for induction and then rejection therapy. Conclusions: The addition of oral GCV therapy for 3-4 months to IV therapy during ALA administration to CMV seropositive individuals provided complete protection against CMV disease and viremia. Further studies will be needed to determine the minimum duration of therapy that will be necessary to sustain this effect.

CLINICAL COURSE OF CYTOMEGALOVIRUS (CMV) VIREMIA WITH AND WITHOUT GANCICLOVIR TREATMENT IN CMV-SEROPOSITIVE RENAL TRANSPLANT RECIPIENTS: LONGITUDINAL STUDY OF CMV PP65 ANTIGENEMIA ASSAY

600 This study was designed to evaluate the longitudinal history of cytomegalovirus (CMV) infection and to test the capacity of ganciclovir as effective therapy in CMV-seropositive renal transplant recipients. The CMV viremia was detected with CMV pp 65 antigenemia assay in 153 renal transplants. The recipients were classified as having low-grade (1-10 /50,000 PBMC) and high-grade CMV infections (> 10/50,000 PBMC) according to the severity of CMV infection. The recipients with low-grade CMV infections were observed without ganciclovir treatment, and the recipients with high-grade CMV infection were randomly assigned to ganciclovir-treated and untreated groups. The clinical course between low-grade and high-grade infections was evaluated. All recipients with low-grade CMV infection (n=62) showed spontaneous remission regardless of immunosuppressants. In high-grade CMV infection (n=31), the cyclosporine-treated group (n=11) showed no evidence of CMV disease, and the methylprednisolone-treated group (n=8) showed CMV disease in 1 (25%) of 4 ganciclovir-untreated recipients. In the OKT3 group(n=12), symptomatic CMV infection was observed in 6 (100%) ganciclovir-untreated group contrary to no CMV disease in ganciclovir-treated group (P<0.05). In conclusion, CMV-seropositive recipients receiving CsA and/or steroids alone do not have an important clinical problem with CMV infection. This group does not need to receive prophyaxis and can be followed by CMV antigenemia study. But OKT3-treated recipients should receive ganciclovir during early CMV viremia to prevent significant clinical consequences.

Evaluation of a new method for early detection of active cytomegalovirus infections. A study in kidney transplant recipients

Abstract Early detection of active cytomegalovirus (CMV) infection after organ transplantation is necessary to start effective antiviral treatment. In the present study, blood specimens of kidney transplant recipients (n= 38) were monitored for the expression of CMV immediate early (IE) and late (L) mRNA using nucleic acid sequence-based amplification (NASBA). Results were compared with virus isolation, pp65 antigenemia and serology. In patients developing active CMV infection, pp65 antigen and L mRNA were detected simultaneously. At the same time, positive cell culture results could be reported to the clinic. CMV was detected significantly earlier with IE NASBA than with the other assays. However, the specificity of IE NASBA is lower than that of antigenemia, late NASBA and cell culture. Early detection of IE mRNA is especially useful for patients at high risk of developing symptomatic CMV infection in order that early, adequate antiviral therapy may be started. Late NASBA can be used to monitor further development of CMV infection, comparable to antigenemia.

Colonoscopy Findings in Renal Transplant Patients With Abdominal Symptoms

THE number of chronic renal failure patients undergoing successful renal transplantation has increased substantially in recent years. Success of this procedure is highly dependent on prevention of rejection. The consequences of the necessary immunosuppression, including opportunistic infections and neoplasms in the long term, make careful follow-up of these patients obligatory. Colonic complications following transplantation occur in approximately 3% of all renal transplant cases. The most frequently encountered complications are ischemic changes, ranging from mild mucosal ulcerations to necrosis; diverticulitis; perforation; pseudomembranous colitis caused by antibiotic use; hemorrhagic proctitis; and appendicitis. Infection with cytomegalovirus (CMV) is a substantial cause of morbidity and mortality among immunocompromised patients, and symptomatic CMV can occur at all levels of the gastrointestinal tract in renal transplant patients. These individuals have a greater likelihood of colonic involvement with CMV. Tuberculosis and fungal and other opportunistic infections of the colon are also known factors in the morbidity and mortality of renal transplant recipients. In this study, we performed colonoscopy on 13 renal transplantation patients who had symptoms related to the lower gastrointestinal system. During the procedure we obtained biopsies when any apparent lesion was present. Colonoscopic findings were normal in six patients. We detected nonspecific and coincidental findings in two patients. However, the detection of specific diseases, namely intestinal tuberculosis, cytomegalovirus (CMV) colitis, and pseudomembranous colitis in three patients, reflects the importance of colonoscopy in diagnosing and directing treatment in renal transplantation patients. The aim of this study was to emphasize the importance of colonoscopy in the diagnosis of diseases that cause lower gastrointestinal symptoms in renal transplant patients.

Evaluation of Murex CMV DNA hybrid capture assay for detection and quantitation of cytomegalovirus infection in patients following allogeneic stem cell transplantation.

Murex hybrid capture DNA assay (HCS) is a solution hybridization antibody capture assay for detection and quantitation of cytomegalovirus (CMV) DNA in leukocytes. To determine whether CMV HCS is sensitive enough to initiate and monitor antiviral therapy after allogeneic stem cell transplantation (SCT), 51 consecutive SCT recipients were prospectively screened for the appearance of CMV infection by HCS, PCR, and culture assays from blood samples. Preemptive antiviral therapy was initiated after the second positive PCR result in all patients, as previously reported, and HCS was not considered for clinical decision making. A total of 417 samples were analyzed. Of these, 21 samples were found to be positive by PCR and HCS, 88 samples were PCR positive but HCS negative, and 308 were negative by both assays. Concordance of results between PCR and HCS and between HCS and blood culture was observed in 78.9 and 95.9% of the samples assayed, respectively. PCR was found to be more sensitive than HCS, and HCS was more sensitive than the blood culture assay (P < 0.0001). Four patients with symptomatic CMV infection were PCR positive prior to the onset of CMV-related symptoms, whereas HCS detected CMV DNA in three patients prior to and one at onset of CMV disease. The numbers of genomes per milliliter of blood were higher in patients with symptomatic CMV infection than in those with asymptomatic CMV infection (P = 0.06). None of the HCS-negative patients developed CMV disease. Thus, all patients with CMV disease were correctly identified by HCS; however, the lower sensitivity limit of the HCS assay may still be insufficient to allow diagnosis of CMV infection early enough to prevent CMV disease in patients following allogeneic SCT.

Evaluation of PCR Primers for Early Diagnosis of Cytomegalovirus Infection following Liver Transplantation

The availability of microbiologic methods that detect early replication of cytomegalovirus (CMV) posttransplantation will enhance the process of initiating preemptive antiviral therapy prior to the appearance of CMV disease. Using PCR techniques we sought to determine which region of the CMV genome present in peripheral blood leukocytes (PBLs) or serum provides the highest sensitivity for the detection of CMV posttransplantation. Blood samples were prospectively collected weekly for at least 8 weeks from a cohort of 21 consecutive liver transplant recipients not receiving anti-CMV prophylaxis. Results of PCR assays were correlated with recovery of CMV in cell cultures and histopathological findings from biopsy specimens of infected organs to assess clinical symptomatic infection. Of 148 specimens, primer pairs directed to the HindIII-X fragment region of CMV detected target DNA with a 94% sensitivity, compared to an 87% sensitivity with primer pairs directed to EcoRI fragment D, 32% sensitivity with primer pairs directed to the immediate-early antigen 1 gene (IEA1 gene), and 20% sensitivity with primer pairs directed to the major immediate-early (MIE) gene. The performance characteristics in terms of the sensitivity of primers for amplifying CMV DNA associated with symptomatic infection ranged from 100% (HindIII-X) to 20% (MIE gene); however, specificity was inversely related (HindIII-X, 45%; MIE gene, 91%) to primers directed to these gene targets. When HindIII-X and EcoRI-D primer sets were used, CMV DNA from PBLs was a more sensitive target than CMV DNA from serum for the early detection of symptomatic CMV infection (17 versus 12 days). Importantly, CMV DNA was not detected in five patients with no evidence of this viral infection. In conclusion, primers directed to the HindIII-X fragment region were the most optimal for the early detection of CMV DNA in PBLs and sera from symptomatic liver transplant recipients.