Monitoring Of Human Cytomegalovirus Infection Research Articles

Monitoring of Human Cytomegalovirus Infection in Bone Marrow and Liver Transplant Recipients by Antigenaemia Assay and Enzyme-Linked Immunosorbent Assay

Human cytomegalovirus (HCMV) infection is a common complication in transplant recipients. Sensitive, specific and timely diagnostic tests for the detection of HCMV infection remain essential for successful therapy. The results of three tests to detect HCMV in bone marrow and liver transplant recipients were compared: a pp65 antigenaemia assay, an immediate-early (IE) antigenaemia assay and an anti-HCMV immunoglobulin M (IgM) antibody enzyme-linked immunosorbent assay (ELISA). Of 1344 samples, 911 (67.8%) and 917 (68.2%) samples were positive for pp65 and IE, respectively. The coincidence level was 85.1%. There was no statistical difference after transplantation to the first positive detection of HCMV (mean first checkout time) between the pp65 and IE antigenaemia assays. Moreover, the levels of HCMV detected by the pp65 and IE antigenaemia assays were significantly correlated. The HCMV-positivity rate as detected by the anti-HCMV IgM ELISA was 11.1%, which was significantly different from the IE and pp65 antigenaemia assays. We suggest that the IE antigenaemia assay could replace the pp65 antigenaemia assay for monitoring active HCMV infection and early detection of HCMV infection.

Virological and Immunological Monitoring of Human Cytomegalovirus Infection in Heart and Small Bowel/Multivisceral Transplantation

Virological and Immunological Monitoring of Human Cytomegalovirus Infection in Heart and Small Bowel/Multivisceral Transplantation

Comparison of a new Light Diagnostics™ and the CMV Brite™ to an in-house developed human cytomegalovirus antigenemia assay

Background Antigenemia, i.e. detection and quantification of human cytomegalovirus (HCMV) peripheral blood pp65-positive leukocytes, is still one of the two major assays available for diagnosis and monitoring of HCMV infections. Objectives To evaluate the performance of a new commercial assay under development (Light Diagnostics™). Study design To compare the performance of the new assay with a commercial assay (CMV Brite™) already available on 300 blood samples from immunocompromised patients using as a reference the original in-house developed assay. Results Although 30 blood samples gave discrepant results among the 3 antigenemia assays, the Light Diagnostics™ detected an overall number of antigenemia-positive blood samples (sensitivity 84%) identical to that detected by CMV Brite™ (sensitivity 88%) and in-house assay (91/300, 30.3%). Problems of non-specific cytoplasmic staining were encountered with the CMV Brite™ assay in 219/300 (73%) blood samples. Conclusions The Light Diagnostics™ assay provides results comparable to those of the reference assay both in terms of specificity and sensitivity (number of pp65-positive leukocytes).

Monitoring human cytomegalovirus infection in transplant recipients

Human cytomegalovirus (HCMV) infection remains one of the most challenging infectious complications in both solid organ transplant (SOT) and hemopoietic stem cell transplant (HSCT) recipients. In the last two decades advances have been made in the diagnosis and monitoring of HCMV infection in SOT and HSCT recipients following introduction of quantitative assays such as rapid virus isolation in blood (viremia), quantitation of pp65 in peripheral blood leukocytes (antigenemia), and quantitation of viral genome in blood (DNAemia). The availability of these rapid diagnostic assays has allowed treatment administration during the presymptomatic phase of HCMV infection (preemptive treatment) and greatly reduced HCMV-related morbidity and mortality, particularly among HSCT recipients. Definition of clinically validated thresholds for initiating preemptive treatment in SOT and HSCT recipients is a major goal in the transplantation setting. With respect to universal prophylaxis of HCMV infection in transplant recipients, the preemptive treatment approach shows advantages in (i) treating a lower number of patients for shorter periods of time and (ii) avoiding the reported emergence of HCMV disease after interruption of anti-HCMV prophylaxis. To understand the mechanism behind long-term control of HCMV infection in transplant recipients, the HCMV-specific T-cell response must be evaluated.

Monitoring of human cytomegalovirus infection in immunosuppressed patients using real-time PCR on whole blood

Background Quantitative monitoring of human cytomegalovirus (HCMV) is currently used in the follow-up of immunosuppressed patients. Objective To investigate whether real-time PCR quantification (QPCR) of HCMV DNA could replace pp65 antigenemia. Study design We compared HCMV QPCR on whole blood (WB) and on plasma with a pp65-antigenemia assay on 192 samples. Afterwards, we tested 1310 samples from 308 immunosuppressed patients both by antigenemia assay and QPCR on WB. Results The first study comparison showed that QPCR results on WB and plasma were significantly correlated with antigenemia. QPCR on WB was more sensitive than QPCR on plasma or antigenemia, detecting 31 and 49 additional positive samples, respectively. During the second comparison, QPCR on WB and antigenemia were again correlated ( r = 0.70; p < 0.0001), but QPCR detected 244 additional positive samples. HCMV DNA was detected earlier than pp65 antigen (median difference: 14 days; range: 7–30). One, 5, 10, 50 and 100 pp65-positive cells/200,000 leukocytes corresponded to 439, 1531, 2623, 9150 and 15,671 HCMV DNA copies/mL of WB, respectively, but this equivalence differed according to the sub-group of patients considered. Conclusion QPCR on WB is the most sensitive method for the monitoring of HCMV infection in immunosuppressed patients.

Evaluation of the COBAS Amplicor HCMV Monitor for early detection and monitoring of human cytomegalovirus infection after allogeneic stem cell transplantation

Early diagnosis of human cytomegalovirus (HCMV) infection and the introduction of preemptive antiviral therapy have reduced HCMV-related mortality after allogeneic stem cell transplantation. A critical goal remains stratifying risk profiles and minimizing potential harm owing to antiviral overtreatment. We compared the commercially available standardized COBAS Amplicor CMV Monitor (CACM) to an in-house PCR assay, for the monitoring of HCMV infection. Seventy-two patients were surveyed by an in-house PCR of whole blood, quantitative viral load assessment by CACM and virus culture assays in a prospective and a retrospective study. A high concordance between CACM and PCR was documented. The viral load at onset correlated with the peak viral load (Spearman rank correlation R=0.634, P=0.0004). In patients developing HCMV disease, both viral loads were in trend higher (P=0.823, respectively P=0.053), and the viremic episodes longer (P=0.015), as compared to asymptomatically HCMV-infected patients. The serological pre-transplant status was the major risk factor for the development of HCMV disease, showing highest risk for seropositive patients receiving a seronegative graft, whereas donor type (related or unrelated) and graft type (bone marrow or peripheral blood mobilized stem cells) did not have an influence. HCMV infection proved to be a risk factor for the development of non-viral opportunistic infections (P=0.002).

Monitoring of human cytomegalovirus infections in heart transplant recipients by pp65 antigenemia.

Rapid diagnostic techniques offer the opportunity of early diagnosis of human cytomegalovirus (CMV) infection in immunocompromized patients at risk of developing CMV disease and syndrome. The use of CMV pp65 antigenemia as a predictor of CMV syndrome and disease in heart transplant recipient after induction therapy was studied retrospectively. One hundred and nineteen consecutive heart transplant recipients treated with induction therapy who survived more then 14 d were monitored for CMV infection. Ninety-four recipients were seropositive for CMV. Twenty-five recipients were seronegative for CMV and received grafts from seropositive donors. Pre-emptive therapy was used in seropositive patients when CMV pp65 antigenemia was greater than 50 antigen-positive cells per 2 x 10(5) peripheral blood leukocytes (PBL); prophylactic therapy was done only in seronegative recipient matched with seropositive donor. High-level CMV pp65 antigenemia (50 antigen-positive cells 2 x 10(5) PBL) occurred in 34% (41 of 119) of patients at a median of 44 d following transplantation. In seropositive recipients, 16% (15 of 94) of patients developed CMV invasive disease or syndrome, and in seronegative recipients 20% (5 of 25) of patients developed CMV disease or syndrome. Sixty-six per cent (62 of 94) of CMV seropositive patients were identified as not requiring pre-emptive therapy. In seropositive and seronegative recipients, the sensibility and negative predictive value of the cut-off level of 50 antigen positive cell for CMV disease and syndrome was 100%. The specificity was 79% and positive predictive value was 49%. Because of the excellent sensibility and negative predictive value of the cut-off level of 50 antigen positive cell per 2 x 10(5) PBL, application of pre-emptive therapy guided by high level of CMV pp65 antigenemia in the context of induction therapy allow to omit antiviral therapy in many at risk patients. In the context of pre-emptive and prophylactic therapy, the cut-off level of 50 antigen positive cell do not allow to predict with accuracy the development of CMV disease or syndrome.

Monitoring of human cytomegalovirus infection by antigenemia assay in seropositive renal transplant recipients

s of the 12th ISIIH 2325 doses up to 100 mg per day with second drug therapy of azathioprine or alternatively cyclophosphamide. After the transfer to our hospital a plethora of laboratory studies for rheumatologic, inflammatory, and infectious processes where obtained and except for a very modest decrease in C4 all where normal. A further contrast enhanced MRT of the brain showed extensive rightsided white matter disease with significant extension into the left hemisphere. An HIV ELISA, a CD4 count, and a JC virus PCR were obtained.The ELISA was negative; but the CD4 count was 52 and JCV-VL was 2888 copies/ml (normal ~80). An exhaustive literature search revealed minimal therapeutic options for the JC virus infection, and the patient was discharge to hospice in the care of her physician on a suggested regimen of a prednisone taper, and hopefully therapeutic alphainterferon and cidofovir treatment. As of February 2002 the patient remains bed bound. This devastating case of JC virus infection illustrates the confounding diagnostic dilemmas frequently encountered by practitioners. In this instance a consistent explanation is found with the realization that the patient probably suffers from advanced iatrogenic immunosuppression and the resultant possible opportunistic infections. Valacyclovir for the prevention of CMV disease after renal transplantation: A 5year follow-up *Hans-H. Neumayer, Jean-Paul Squifflet, David Lowance, Christophe M. Legendre *Department of Nephrology, University Hospital Charit&, Berlin, Germany Objective: Valacyclovir 2g four times daily for 90 days has been shown (Lowance et al., NEJM 1999: 340:146270) to reduce the incidence of cytomegalovirus (CMV) infection and disease and acute graft rejection (GR) at six months vs placebo after renal transplantation. Among the CMV seropositive patients (R+) the incidence of CMV disease was reduced from 6% to 1% and there was a reduction in biopsy confirmed acute GR from 36% to 30%. CMV disease was significantly reduced in the donor-positive recipient-negative (D+R-) group from 45% to 16% and rate of biopsy confirmed acute GR from 52% to 26% compared to placebo. This retrospective review of patients’ medical notes was designed to assess whether the benefits extend to increased graft function and survival during 5 years follow-up. Methods: Data were available for 449 of the 616 patients enrolled in the original study, P66/H73-012. The primary population for analysis was the ITT population of all patients randomised in the original study. The primary endpoint was time to allograft failure up to 5 years posttransplant. Rates of acute GR, chronic GR, CMV disease and survival were secondary endpoints. Results: No significant treatment difference was observed for time to allograft failure in either the R+ or D+Rpatients although the benefits in preventing CMV disease and acute GR are still evident at 5 years.

Diagnosis and monitoring of human cytomegalovirus infection in transplant recipients

Diagnosis and monitoring of human cytomegalovirus infection in transplant recipients

Monitoring of human cytomegalovirus infections in pediatric bone marrow transplant recipients by nucleic acid sequence-based amplification.

In the diagnosis of human cytomegalovirus (HCMV) infection, it is very important to distinguish symptomatic from asymptomatic infection. The nucleic acid sequence-based amplification (NASBA) technique was compared with single and nested polymerase chainreaction (PCR) methods. For NASBA detection, the beta2.7 transcript was chosen as a target because of its abundant active HCMV-specific expression. Of 20 pediatric bone marrow transplant (BMT) recipients, 8 developed HCMV-related clinical symptoms. The clinical sensitivities and specificities were 50% and 100% for single PCR, 100% and 67% for nested PCR, and 100% and 83% for NASBA, respectively. Follow-up of HCMV infections in pediatric BMT recipients showed that NASBA could both detect viral transcript prior to the onset of clinical symptoms and reflect clinical improvement due to antiviral therapy. These data suggest that NASBA should be useful for both predicting HCMV disease development and monitoring the effect of antiviral therapy.

Use of the human cytomegalovirus (HCMV) antigenemia assay for diagnosis and monitoring of HCMV infections and detection of antiviral drug resistance in the immunocompromised.

Quantification of viral load in blood has proven to be helpful in the follow-up of disseminated HCMV infections in immunocompromised patients. (i) To describe the antigenemia assay and its optimization and (ii) to analyze the use of the antigenemia assay for the diagnosis and monitoring of HCMV infections and for the detection of treatment failures. This article is intended to give an overview of our experience in the use of the antigenemia assay. In solid organ transplant recipients and patients with AIDS, HCMV symptomatic infections mostly appear when antigenemia values are > 300 pp65-positive PBL/2 x 10(5) examined. To avoid the appearance of overt HCMV disease antiviral treatment could be administered when antigenemia levels are > 100 pp65-positive PBL/2 x 10(5) examined. Bone marrow transplant recipients show symptomatic HCMV infections when antigenemia values are > 100 pp65-positive PBL/2 x 10(5) examined. This group of patients should be treated when antigenemia levels are < 10 pp65-positive PBL/2 x 10(5) examined due to the higher mortality rate associated with HCMV complications. A decrease in antigenemia levels during therapy indicates a good response to antiviral drug, while stable or increasing values document a treatment failure and the emergence of drug-resistant HCMV strains.

Highly sensitive single-step PCR protocol for diagnosis and monitoring of human cytomegalovirus infection in renal transplant recipients.

A multiplex, single-step PCR protocol for the detection of human cytomegalovirus (HCMV) DNA is described. The protocol amplifies regions of the viral LA and IE genes and employs elevated temperatures for both reagent mixing and primer annealing together with product detection by silver staining on polyacrylamide gels. This assay detects one to five HCMV genomes in clinical samples containing up to 100 ng of human DNA, a level of sensitivity equivalent to that of more complex assays involving either nested PCR or postamplification hybridization. As well as being of importance in clinical situations where high-sensitivity qualitative diagnosis is required, this assay is also applicable to the monitoring of HCMV infection in renal transplant recipients. Due to its multiplex format the assay provides quantitative information, in that samples from which a single target is amplified contain on average sevenfold fewer viral genomes per 10(6) leukocytes than those from which both targets are amplified. When weekly blood leukocyte DNA preparations from renal transplant patients were assayed, findings of three consecutive tests in which both HCMV targets were amplified were highly indicative of patients who had developed very high loads of HCMV (100% sensitivity, 88% specificity). We thus show that the same simple PCR assay which permits highly sensitive HCMV diagnosis can also be used for the efficient identification of transplant recipients at risk of clinically significant infection.

Early virus isolation, early structural antigen detection and DNA amplification by the polymerase chain reaction in polymorphonuclear leukocytes from AIDS patients with human cytomegalovirus viraemia.

Fifty AIDS patients were investigated for human cytomegalovirus (HCMV) viraemia when potentially HCMV-related clinical symptoms or syndromes were observed. Nine patients underwent prolonged virologic follow-up, while 41 additional patients were examined only once or sporadically. Concentrated preparations of polymorphonuclear leukocytes (PMNL) from 153 blood samples were obtained for monitoring: (1) early virus isolation in cell cultures 24 h p.i. (viraemia); (2) early structural antigen detection in cytospin preparations (antigenemia); and (3) HCMV DNA in blood (DNAemia) through DNA amplification by the polymerase chain reaction (PCR). Viraemia and antigenemia were quantitated, whereas evaluation of DNAemia was only qualitative. A good correlation between levels of viraemia and antigenemia was consistently found except during ganciclovir treatment. HCMV-related clinical symptoms were observed when the number of infected PMNL was greater than 100 per 2 x 10(5) cells examined. All 56 blood samples positive for viraemia and antigenemia were also PCR-positive, whereas 44 samples (39 of which taken from patients with ascertained HCMV infection in blood) were positive by PCR only. Viraemia and antigenemia were often unrelated to HCMV organ syndromes, such as retinitis, in which only DNAemia was often detected. Prolonged ganciclovir treatment kept viraemia, antigenemia and even DNAemia at a low or negative level, yet drug discontinuation led to rapid progression of HCMV infection in blood. In addition, prolonged antiviral treatment could induce appearance of ganciclovir-resistant HCMV strains, requiring alternative foscarnet therapy. In conclusion, determination of viraemia and antigenemia appears essential for correct clinical management and antiviral treatment of disseminated HCMV infections in AIDS patients. However, PCR is the most sensitive method for diagnosis and monitoring of HCMV infections in blood at a pre-clinical stage.

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.