Comparison between viremia and antigenemia for detection of cytomegalovirus in blood.

In a prospective study, 139 serial blood samples from 15 transplant recipients were assessed for the presence of cytomegalovirus (CMV) by virus isolation (CMV viremia) and by direct staining of CMV antigens (CMV Ag) in blood leukocytes (CMV antigenemia). CMV was isolated from 23 samples, whereas CMV Ag was detected in 44 specimens. All positive samples were from a total of nine patients who were diagnosed as having active CMV infections. In seven patients, active CMV infections were diagnosed by virus isolation from blood and urine and by a significant rise of CMV-specific antibodies. In these patients, 21 of the 23 blood samples which were positive for CMV by cell culture were also positive by direct CMV Ag detection. Moreover, CMV Ag were detected in 23 of the 116 culture-negative samples. Twenty of these samples were from the acute phase of infection in the same seven patients. The remaining three CMV Ag-positive specimens were from the other two patients, from whom CMV was not isolated but who had serological evidence of concomitant active CMV infections. These results suggest that direct detection of CMV Ag in peripheral blood leukocytes is as specific as and more sensitive than current isolation techniques. Furthermore, by its sensitivity and inherent rapidity the antigen detection test proved to be the earliest diagnostic marker of active CMV infection in eight of the nine patients. Finally, it was shown that monoclonal antibodies to CMV immediate early antigens are a prerequisite for demonstration of CMV antigenemia.     

Monitoring levels of human cytomegalovirus DNA in blood after liver transplantation.

We evaluated a semiquantitative PCR assay prospectively in 40 liver transplant recipients as an aid in making a prompt diagnosis of cytomegalovirus (CMV) infection. For 2 months after transplantation, clinical specimens from patients were tested weekly by PCR, virus isolation from peripheral blood and urine, and CMV serology. The incidence of active CMV infection was 70%. The levels of CMV DNA determined by hybridization of PCR samples and densitometric scanning of blots were assigned a score of 1 to 4 by comparison with four external standards amplified in parallel and corresponding to a range of 80 to 80,000 genomes. The first detection of CMV in blood by PCR occurred at a mean of 15 days, and high-level PCR scores of 3 or 4 were obtained 21 days after transplantation, whereas viremia occurred 33 days after transplantation. Significantly higher levels of CMV DNA were seen in patients with CMV disease (P < 0.05) than in asymptomatic patients. The prevalence of symptomatic CMV infection was 30%. The positive predictive value of PCR was 48%, while the negative predictive value was 100%. After treatment, the clearance of CMV DNA was always observed and the disappearance of symptoms occurred concomitantly with undetectable PCR signals.     

Low predictive value of polymerase chain reaction for diagnosis of cytomegalovirus disease in liver transplant recipients.

The polymerase chain reaction (PCR) and viral culture techniques were prospectively compared for the detection of cytomegalovirus (CMV) in blood samples from 24 liver transplant recipients. Nine patients had one or more episodes of viremia, seven of which were clinically symptomatic infections. All samples in which CMV was isolated by culture were positive by the PCR. However, the PCR result was also positive for one or more samples from 11 patients who never developed CMV-related symptoms. Although the PCR is a very sensitive technique for CMV detection in blood samples from liver transplant recipients, it is not useful as a marker of symptomatic CMV disease.     

Detection of cytomegalovirus in peripheral leukocytes by different methods.

Screening leukocytes for cytomegalovirus (CMV) by shell vial assay gave unsatisfactory results. Of 10 positive specimens (114 samples), only 2 showing CMV could be detected. Disruption of leukocytes prior to their use in shell vial assays increased the sensitivity of CMV detection considerably: of 32 leukocyte specimens from transplant patients with signs of CMV disease, 13 were positive with disruption and only 3 were positive without. With this modification, 17 transplant patients with suspected CMV infection were regularly screened. Viremia could be detected in 10 cases by the shell vial assay and in 11 cases by the direct detection of immediate early antigen. On the average, viremia was detected 11 days before immunoglobulin M or typical clinical symptoms.     

Cytomegalovirus (CMV) antigenemia assay is more sensitive than shell vial cultures for rapid detection of CMV in polymorphonuclear blood leukocytes.

We compared the cytomegalovirus (CMV) antigenemia assay with shell vial cultures of polymorphonuclear leukocyte (PMNL)-enriched blood fractions for rapid diagnosis of CMV viremia. PMNL fractions of 280 blood specimens from 171 patients (170 solid-organ transplant recipients and 1 patient undergoing pretransplant evaluation) were inoculated in shell vial and conventional CMV cultures. A commercially available kit (CMV-vue kit; INCSTAR Corp.) was used for the CMV antigenemia assay, in which PMNL preparations were stained with monoclonal antibodies directed against the CMV protein pp65. Mixed-leukocyte blood fractions from the same blood specimens were inoculated in parallel shell vial and conventional cultures. CMV viremia (defined by the isolation of CMV in conventional cultures) was detected in 32 (13%) of 245 PMNL fractions included in the final analysis. Twenty-eight (87.5%) were also positive in the CMV antigenemia assay, whereas 22 (69%) were positive in shell vial cultures. Ten (4%) additional PMNL fractions positive only in the CMV antigenemia assay were from eight patients with active CMV infections (six patients), who had previous or subsequent episodes of CMV viremia (seven patients), or in whom CMV was isolated in cultures of simultaneously obtained mixed-leukocyte fractions (three patients). Overall, the CMV antigenemia assay was significantly more sensitive than shell vial cultures for detection of CMV in the PMNL fraction of blood leukocytes (P < 0.01, McNemar's test), and we recommend it as the method of choice for rapid diagnosis of CMV viremia.     

A prospective study of cytomegalovirus and herpes simplex virus disease in renal transplant recipients.

A prospective study of 84 renal graft recipients demonstrated cytomegalovirus (CMV) disease after transplantation in 37% of patients. Reactivation infection was found in 20 of 44 patients (46%) who were seropositive for CMV prior to transplant and primary CMV disease occurred in 11 of 40 (28%) initially seronegative patients. Nearly all cases of primary disease (91%) were associated with symptoms and in these cases CMV was probably acquired via the donated kidneys. Only 35% of the reactivation infections were associated with clinical symptoms. Actuarial life tables indicated that CMV disease did not reduce the length of graft survival. Herpes simplex virus (HSV) infections were diagnosed in 44 (52%) of the patients and included a fatal case of disseminated disease associated with hepatitis.     

Detection of cytomegalovirus using PCR in serum from renal transplant recipients.

AIMS--To develop a polymerase chain reaction (PCR) assay for the detection of cytomegalovirus (CMV) DNA in serum and leucocytes of renal transplant recipients and compare this assay with CMV culture and serodiagnosis. METHODS--Monthly specimens were obtained from 12 patients starting immediately before transplant. CMV infection was monitored by IgM enzyme linked immunosorbent assay, virus culture and PCR on serum and leucocytes. RESULTS--Two of four IgG positive patients had reactivation of CMV disease confirmed by culture, three of eight seronegative patients had a primary infection, one confirmed by serology and two by culture. PCR was positive earlier than conventional methods in three cases and concurrently in two. No positive PCR reactions occurred in the seven patients who remained negative by culture and serology. CONCLUSIONS--CMV DNA is detectable in serum; serum may be positive before virus is detectable by buffy coat culture; and PCR may be useful as an early indication of potential CMV disease in renal transplant recipients.     

Lack of evidence for systemic cytomegalovirus reactivation in maintenance hemodialysis patients

The risk of cytomegalovirus (CMV) reactivation among hemodialysis (HD) patients is unknown. In 52 HD patients from a single center, CMV serology and quantitative PCR were performed. The detection limit of PCR was 20 copies/ml. Here, PCR ruled out CMV viremia, despite CMV-IgM seropositivity in 15.4% patients.     

Determination of the number of blood samples needed for optimal detection of cytomegalovirus viremia in immunocompromised patients using a shell-vial assay

To establish the number of blood samples necessary for the diagnosis of viremic episodes caused by cytomegalovirus (CMV), a prospective analysis was conducted of 238 patients (38 renal transplant recipients and 200 HIV-infected patients) who developed CMV viremia. The usefulness of samples and the volume of blood required to demonstrate the presence of viremia by CMV was also studied. The first blood sample was diagnostic for CMV viremia in 53.3% of the viremic patients; the second sample documented an additional 22.2% of cases of viremia (75.5% of infected patients); and the third sample demonstrated viremia in the remaining 24.5%. Thus, a diagnosis of CMV viremia was established in every patient (100% of episodes of viremia). In this study, the use of three 3 ml blood samples collected at 24 h intervals was sufficient to detect all episodes of CMV viremia in patients clinically suspected to have disseminated disease.     

Comparison between the clinical and laboratory features of enterovirus and West Nile virus infections

The seasonality and clinical features of enterovirus (EV) infections overlap with those of West Nile virus (WNV). The purpose of this study was to determine the frequency of EV detection in patients being tested for WNV and to look for features that could be used to distinguish between infections with these two viruses. Nucleic acid amplification testing (NAT) for EV was performed on all plasma samples submitted for WNV testing in 2003 and 2004. Demographics, clinical features, and laboratory results for patients with documented EV viremia were compared with those for patients with confirmed WNV infection (as diagnosed by NAT and/or serology). NAT for EV was positive on 50 of 1,784 serum or plasma samples submitted for WNV testing (2.8%). Clinical information was compared for 45 patients with EV viremia and 214 patients with WNV infection. Patients with EV viremia were younger and less likely to have heart disease or a travel history (P<0.05). The EV viremia cases were distributed throughout the whole province while the WNV cases were predominantly in the southern part of the province. Symptoms were remarkably similar, although patients with WNV infection were more likely to have anorexia, dizziness, rash, and cranial nerve palsy (P<0.05). There are no consistent differences in the features of WNV infection and enteroviral viremia so diagnostic tests for both viruses should be performed when WNV is present in local mosquitoes.     

Reconstitution of lymphocyte populations and cytomegalovirus viremia or disease after allogeneic peripheral blood stem cell transplantation

Early reconstitution of lymphoid populations was monitored by immunophenotyping in 57 allogeneic peripheral blood stem cell (allo-PBSC) transplant patients either with or without cytomegalovirus (CMV) viremia or disease. Cell counts for total lymphocytes and CD4(+) T cells above the percentile 60th at day 14 postransplant were associated significantly with CMV viremia-free survival within 120 days after transplant. Recovery of total lymphocyte, CD3(+), and CD8(+) T-cell counts proceeded at a more rapid rate in CMV viremic patients than in nonviremic patients, irrespective of whether preemptive treatment with ganciclovir had been prescribed. Significant expansion of CD8(+) and CD8(+) CD57(+) T-cell subsets was associated with recovery from viremia and no progression to CMV disease. Immunophenotyping may provide useful information for the clinical management of CMV infection in allo-PBSC transplant recipients.     

Human cytomegalovirus-specific T-cell immune reconstitution in preemptively treated heart transplant recipients identifies subjects at critical risk for infection

Human cytomegalovirus (CMV) infection represents a major threat for heart transplant recipients (HTXs). CMV-specific T cells effectively control virus infection, and thus, assessment of antiviral immune recovery may have clinical utility in identifying HTXs at risk of infection. In this study, 10 CMV-seropositive (R(+)) pretransplant patients and 48 preemptively treated R(+) HTXs were examined before and after 100 days posttransplant. Preemptive treatment is supposed to favor the immune recovery. CMV DNAemia and gamma interferon enzyme-linked immunosorbent spot (ELISPOT) assay were employed to assess the viremia and immune reconstitution. HTXs could be categorized into three groups characterized by high (>100), medium (50 to 100), and low (<50) spot levels. Early-identified high responders efficiently controlled the infection and also maintained high immunity levels after 100 days after transplant. No episodes of grade ≥2R rejection occurred in the high responders. Midresponders were identified as a group with heterogeneous trends of immune reconstitution. Low responders were 41% and 21% of HTXs before and after 100 days posttransplant, respectively. Low responders were associated with a higher incidence of infection. The effect of viremia on immune recovery was investigated: a statistically significant inverse correlation between magnitude of viremia and immune recovery emerged; in particular, each 10-fold increase in viremia (>4 log(10) DNAemia/ml) was associated with a 36% decrease of the ELISPOT assay spot levels. All episodes of high viremia (>4 log(10) DNAemia/ml) occurred from 1 to 60 days after transplant. Thus, the concomitant evaluation of viremia and CMV immune reconstitution has clinical utility in identifying HTXs at risk of infection and may represent a helpful guide in making therapeutic choices.     

Increased IgM and IgM immune complex-like material in the circulation of renal transplant recipients with primary cytomegalovirus infections.

Twelve of 60 consecutive adult recipients of cadaver kidney transplants had increased polyethyleneglycol (PEG) precipitable IgM immune complex-like material in their circulation in the first 4 months after transplantation. All 10 recipients with primary CMV and two of four with secondary CMV infections had significant elevations in PEG precipitable IgM that coincided with rises in their CMV antibody titres. Ultracentrifuge analysis demonstrated two peaks of PEG precipitable material with sedimentation rates of about 20S and 40S. Total IgM immunoglobulin levels also were increased in transplant recipients with CMV infections, but this was less specific and occurred in patients without CMV infections. The Clq binding assay, which is more sensitive for IgG than IgM containing complexes, was positive in only three of 10 patients with primary CMV and none of four with secondary CMV. Granular deposits of IgM, but not IgG, were detected in the glomeruli of six of seven transplants biopsied during CMV infection. The PEG-IgM assay was not influenced by rejection or prednisone therapy. Thus, transplant patients, who develop primary CMV infections, produce elevated levels of circulating IgM and IgM immune complex-like material. These findings may help to differentiate CMV infection from transplant rejection as well as to increase our understanding of the special pathogenic properties of CMV in transplant recipients.     

Comparison of Cytomegalovirus (CMV) Enzyme-Linked Immunosorbent Spot and CMV Quantiferon Gamma Interferon-Releasing Assays in Assessing Risk of CMV Infection in Kidney Transplant Recipients

Assessing cytomegalovirus (CMV)-specific cell-mediated immunity (CMI) represents an appealing strategy for identifying transplant recipients at risk of infection. In this study, we compared two gamma interferon-releasing assays (IGRAs), Quantiferon-CMV and CMV enzyme-linked immunosorbent spot (ELISPOT), to determine the ability of each test to predict protective CMV-specific T-cell responses. Two hundred twenty-one Quantiferon-CMV and ELISPOT tests were conducted on 120 adult kidney transplant recipients (KTRs), including 100 CMV-seropositive transplant recipients (R⁺) and 20 CMV-seronegative transplant recipients of a CMV-positive donor (D⁺/R⁻). As a control cohort, 39 healthy adult subjects (including 33 CMV-seropositive and 6 CMV-seronegative subjects) were enrolled. CMV IgG serology was used as a reference for both tests. In the CMV-seropositive individuals, the ELISPOT and Quantiferon-CMV assays provided 46% concordance with the serology, 12% discordance, 18% disagreement between ELISPOT or Quantiferon-CMV and the serology, and 24% gray areas when one or both tests resulted in weak positives. None of the CMV-seronegative subjects showed detectable responses in the ELISPOT or the Quantiferon-CMV test. In transplant recipients, both the ELISPOT and Quantiferon-CMV assays positively correlated with each other and negatively correlated with CMV DNAemia in a significant way (P < 0.05). During the antiviral prophylaxis, all 20 D⁺/R⁻ KTRs we examined displayed undetectable Quantiferon-CMV and ELISPOT results, and there was no evidence of CMV seroconversion. The receiving operator curve (ROC) statistical analysis revealed similar specificities and sensitivities in predicting detectable viremia (areas under the curve [AUC], 0.66 and 0.62 for Quantiferon-CMV and ELISPOT, respectively). ELISPOT and Quantiferon-CMV values of >150 spots/200,000 peripheral blood mononuclear cells (PBMCs) and >1 to 6 IU gamma interferon (IFN-γ) were associated with protection from CMV infection (odds ratios [OR], 5 and 8.75, respectively). In transplant recipients, the two tests displayed similar abilities for predicting CMV infection. Both the ELISPOT and Quantiferon-CMV assays require several ameliorations to avoid false-negative results.     

Donated organ as a source of cytomegalovirus in orthotopic liver transplantation.

The importance of the donated organ as a source of CMV was assessed in 120 patients following orthotopic liver transplant and the CMV infections that developed in these patients were graded by severity. Forty-four recipients were CMV antibody negative pre-transplant. Eighteen of these received organs from CMV antibody positive donors and 15 (83%) developed primary CMV infections, 13 (87%) of which were symptomatic. Twenty-six received organs from CMV antibody negative donors and only 2 (8%) became CMV positive post transplant (P less than 0.001). These data suggest that there would be a considerable advantage in matching CMV antibody negative recipients with negative donors. Forty-five percent of secondary infections were asymptomatic compared with 12% of primary infections, and only 11% became disseminated compared with 53% of primary infections. The secondary infections that followed transplantation of an organ from a CMV antibody positive donor were more likely to be symptomatic and were more severe than those in patients who received seronegative livers.     

Seronegative invasive gastro-intestinal cytomegalovirus disease in renal allograft recipients a diagnostic dilemma! – Tissue PCR the saviour?

Seronegative Invasive Gastro-intestinal cytomegalovirus disease in renal allograft recipients Background –CMV as oppurtunistic infection affecting the gastrointerstinal tract is the most common cause for tissue invasive CMV disease occuring in 10–30% of organ transplant recepients. Gastrointerstinal CMV disease can be diagnosed in presence of clinical suspecion along with histopathological findings (CMV inclusions) and presence of mucosal lesion(s) on endoscopic examination with collaborative evidences via molecular technique. Aims-Few cases of CMV infection affecting the gastrointerstinal tract show no evidences of dissemintion despite use of highly sensitive molecular techniques. We encountered 6 cases where in despite strong clinical suspecion of Gastrointerstinal CMV disease there were seronegative and endoscopic negative evidences for CMV, blind tissue biopsy yeilded positive results for CMV disease with excellent improvement with antiviral therapy. Conclusions-Blind biopsy specimen for tissue PCR could serve as saviour in an immunocompromised individiual who has a strong clinical symptomatology for GI-CMV disease in absence of viremia, normal endoscopy and histopathology, so that the early therapeutic interventions could help in excellent patient and graft survival.     

Invasive pulmonary aspergillosis is associated with cytomegalovirus viremia in critically ill patients - A retrospective cohort study

Background/PurposeCytomegalovirus (CMV) viremia is associated with a higher mortality rate and prolonged intensive care unit (ICU) stay for critically ill patients. CMV infection causes transient but substantial immunosuppression for transplant recipients, increasing risk of fungal infection. The association between CMV viremia and invasive pulmonary aspergillosis (IPA) for critically ill patients is still unknown.MethodsWe retrospectively analyzed patients received bronchoalveolar lavage (BAL), galactomannan test, influenza survey and blood CMV viral load test in ICUs of a university hospital between April 2017 and May 2020. Independent risks for IPA were analyzed by multivariable logistic regression.ResultsA total of 136 patients were included. Twenty-one patients had IPA, 48 patients had CMV viremia and 22 patients had influenza. In a multivariable logistic regression model, patients with CMV viremia or influenza had higher IPA risk (adjusted odds ratio, 3.98 and 8.72; 95% CI, 1.26–12.60 and 2.64–28.82; p value = 0.019 and <0.001, respectively.). Patients with detectable CMV in BAL fluid did not have higher IPA risk (crude odds ratio, 0.95; 95% CI, 0.33–2.79; p value = 0.933). After stratifying patients by CMV viral load, the IPA risk is higher for patients with higher viral loads. There is an additive synergistic effect on IPA risk between CMV viremia and influenza infection.ConclusionFor critically ill patients, CMV viremia is an independent risk factor of IPA. Patients with higher blood CMV viral loads have a higher risk of IPA. CMV viremia and influenza have an additive synergistic effect for IPA risk in critically ill patients.     

Detection of IgM antibody to cytomegalovirus and rapid diagnosis of this virus infection by the shell vial assay

Multiple serum specimens from 10 patients with known cytomegalovirus (CMV) infections and 498 sera consecutively submitted to the laboratory for the diagnosis of CMV infection were tested for anti-CMV IgM after treatment with goat anti-human IgG and QAE-Sephadex A50 column chromatography. Specimens from all 10 patients were positive for IgM after treatment with anti-IgG, but only 8 after the column procedure. Anti-CMV IgM was detected in 23 of 498 (4.6%) specimens pretreated with anti-IgG but in only 12 (2.4%) of these samples after the sera was passed through QAE-Sephadex A50 columns. Anti-CMV IgM was detected exclusively in 2 sera after QAE-Sephadex A50 column treatment (sensitivity, 83%) and in 13 specimens pretreated with anti-IgG sera (specificity, 97%). Serology (IgG and IgM) was compared with the shell vial cell culture assay and histology for providing the first evidence of CMV infection in 28 liver transplant patients. CMV infection was detected initially by the rapid shell vial assay (24) or histology (3) in 27 (96%) of these patients. Detection of anti-CMV IgM in these patients had little value for rapid diagnosis of these infections, and suggests that serology should be recommended mainly for the diagnosis of primary CMV infections in localities in which the rapid shell vial assay is not available.     

Detection of CMV-DNA in peripheral blood leukocytes of liver transplant patients after ganciclovir treatment

Summary.  Cytomegalovirus (CMV) infections are common after transplantation, but usually successfully treated with antivirals. In this study, the detection of CMV-DNA in peripheral blood leukocytes was monitored and compared with CMVpp65-antigenemia in liver transplant patients receiving ganciclovir treatment. Twenty adult liver transplant recipients were frequently monitored for CMV up to 6 months after transplantation. CMV infections were diagnosed by pp65-antigenemia and the same specimens were used for CMV-DNA in situ hybridization. Altogether 202 blood specimens were analyzed. During the first 6 months, 14/20 patients developed CMV antigenemia and 11 were treated with ganciclovir. In all patients, CMV-DNA was detected before antigenemia (mean 15 days earlier). All patients responded to ganciclovir and pp65-antigenemia disappeared. However, 8/11 demonstrated persistence of CMV-DNA for up to 6 months. Recurrences appeared in 6/11 patients. In conclusion, detection of CMV-DNA preceded pp65-antigenemia. Persistence of CMV-DNA demonstrates that the virus is not eliminated by ganciclovir and recurrences can be expected. Received December 4, 2002; accepted March 14, 2003 Published online June 2, 2003     

Qualitative Plasma PCR Assay (AMPLICOR CMV Test) versus pp65 Antigenemia Assay for Monitoring Cytomegalovirus Viremia and Guiding Preemptive Ganciclovir Therapy in Allogeneic Stem Cell Transplantation

The performances of a commercially available qualitative plasma PCR assay (AMPLICOR CMV test; Roche Diagnostics) and the pp65 antigenemia assay (AG) were evaluated for the monitoring of cytomegalovirus (CMV) viremia in 43 allogeneic stem cell transplant recipients. In addition, the suitabilities of both assays for triggering the initiation of preemptive ganciclovir therapy were assessed. A total of 37 CMV viremic episodes were detected in 28 patients. Positivity of plasma PCR testing in one or more consecutive specimens was the only marker of CMV viremia in 18 of the 37 episodes (PCR positive and AG negative, n = 50 specimens). Five episodes were diagnosed on the basis of a single positive AG result (AG positive and PCR negative, n = 5 specimens); both assays were eventually positive (PCR positive and AG positive, n = 27 specimens) for 14 viremic episodes; for these episodes, conversion of the PCR assay result to a positive result occurred an average of 1 week before conversion of the AG result. Overall, the concordance between the two methods was 90%, and the sensitivities of the plasma PCR assay and AG for the detection of CMV viremic episodes were 86.5 and 51.3%, respectively. Two patients who tested positive by both assays simultaneously progressed to CMV end-stage organ disease, despite the initiation of preemptive ganciclovir therapy. Conversion of the AG result to a negative result upon administration of preemptive ganciclovir therapy occurred a median of 7.5 days earlier than conversion of the plasma PCR assay result. Nineteen of the 28 patients with CMV viremia received AG-guided preemptive ganciclovir therapy; had the positivity of the plasma PCR assay triggered the initiation of preemptive therapy, 9 additional patients would have been unnecessarily treated since none of them developed CMV end-stage organ disease. Although the AMPLICOR CMV assay is more sensitive than AG, the latter appears to be more suitable both for guiding the initiation of preemptive therapy and for monitoring a patient's response to antiviral therapy.