Comparison of quantitative cytomegalovirus antigenemia assay with culture methods and correlation with clinical disease.

Blood samples, obtained predominantly from human immunodeficiency virus-infected patients and solid-organ and bone marrow transplant recipients, were submitted to the clinical laboratory for detection of cytomegalovirus (CMV) and were processed by three methods: conventional culture, centrifugation culture, and CMV antigenemia assay with monoclonal antibodies (Clonab CMV; Biotest Diagnostic Corporation, Denville, N.J.) to CMV antigens. Of 496 blood samples tested, 107 were positive by one or more methods: 56 were positive by conventional culture, 27 were positive by centrifugation culture, and 97 were positive for CMV antigen (Ag) by the antigenemia assay. Forty-seven samples were positive by the CMV antigenemia assay only; in these samples, a mean of 12 Ag-positive cells was detected per 200,000 polymorphonuclear leukocytes examined. In contrast, samples positive by the CMV antigenemia assay and both culture methods had a mean of 193 Ag-positive cells, and samples positive by the CMV antigenemia assay and conventional culture alone had a mean of 157 Ag-positive cells. In the antigenemia assay, paraformaldehyde fixation resulted in superior cell morphology when compared with acetone fixation. Use of immunofluorescence staining reduced sample processing time and the complexity of reagent preparation in comparison with immunoperoxidase staining. Differences in the sensitivities between the immunofluorescence and immunoperoxidase staining techniques for detection of antigenemia were minor, with discrepant samples showing only one or two Ag-positive cells. Clinical disease was generally associated with high-level antigenemia, but exceptions were noted. The CMV antigenemia test is a rapid, quantitative assay that greatly facilitated the rapid diagnosis of CMV infection. However, quantitation of antigenemia is labor-intensive, requires processing of samples soon after collection, and does not always correlate with clinical disease in the individual patient.     

Comparison of several fixation methods for cytomegalovirus antigenemia assay.

Two fixation methods based on formaldehyde or acetone for qualitative cytomegalovirus antigenemia assay were evaluated on 405 consecutive blood samples. Cytomegalovirus was detected in 40 samples by the antigenemia assay: 36 were detected by formaldehyde fixation; 22, by acetone; and 18, by both methods. Differences were statistically significant (P = 0.0043). In addition, four fixation methods (two based on formalin [with and without permeabilization] and two using acetone at different fixation times) for quantitative antigenemia assay in a different set of 32 samples from known viremic patients were evaluated. Formalin-based methods were superior to acetone-based methods, showing statistically significant differences in either the number of positive samples detected (P < 0.02; McNemar test) or the mean positive cell counts (P < 0.003; two-tailed Student's t test for paired samples). No differences between the two formalin-based methods were found. We recommend the formaldehyde fixation procedure without subsequent permeabilization because of its simplicity and sensitivity.     

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.     

Comparison of sequential cytomegalovirus isolates in a patient with lymphoma and failing antiviral therapy.

BACKGROUND: Long-term anti-cytomegalovirus (CMV) treatments in immunocompromised patients are hampered by resistance to antiviral drugs. Longitudinal changes in the resistance genotype may depend on changes in selective pressure and the complexity of CMV isolates. OBJECTIVE: To evaluate longitudinal changes in the CMV resistance genotype and phenotype along with strain-specific variability in a patient with non-Hodgkin's lymphoma in whom successive anti-CMV treatments failed. STUDY DESIGN: The resistance phenotype and genotype of seven CMV isolates collected from one patient during a 2-year follow-up period were retrospectively analysed. In parallel, we used glycoprotein B (gB) genotyping, and a- and UL10-13-sequence analysis to study CMV interstrain variability. RESULTS: The patient was infected by at least three CMV strains plus variants of the parental strains. Resistance to ganciclovir, cidofovir and foscarnet was successively detected during the follow-up period. UL97 protein kinase changes responsible for resistance to ganciclovir were initially detected at residues 591 and 592, and then at position 594. Decreased sensitivity to foscarnet coincided with the appearance of amino acid substitution N495K in DNA polymerase, whereas cross-resistance to ganciclovir and cidofovir was due to the L501I substitution. CONCLUSIONS: The CMV isolates obtained from our patient were complex mixtures of strains. Changes in resistance genotypes depended on resistance selective pressure and were not linked to interstrain variation.     

Comparison of culture and the antigenemia assay for detection of cytomegalovirus in blood specimens submitted to a reference laboratory.

We compared the antigenemia assay (AA) with tandem shell vial cultures (SVCs) and tube cultures (TCs) for detection of cytomegalovirus (CMV) in 343 blood specimens. For 249 specimens, the AA was performed in duplicate with two different commercially available monoclonal antibody reagents (Biotest Diagnostic Corporation and Argene Biosoft). Specimens considered true positives were positive in either culture system or both AAs. Only specimens which were negative in both cultures and positive in a single AA were tested retrospectively with a CMV PCR assay. CMV recovery rates were also calculated to determine if increased specimen age resulted in decreased positivity. CMV recovery rates for the AA and the combination of both cultures were 20.0 and 5.0% at 3 to 18 h, 20.2 and 14.0% at 18 to 35 h, 12.5 and 7.8% at 36 to 52 h, and 18.8 and 6.3% at 64 to 75 h, respectively. The sensitivities and specificities of the Biotest AA, the Argene AA, SVC, and TC were 84.4 and 100.0, 100.0 and 99.6, 44.4 and 100.0, and 46.0 and 100.0%, respectively. The AA was significantly more sensitive than either culture method alone and was also more sensitive than the two culture methods used in tandem (the tandem culture sensitivity was 63.5%); the Argene AA identified more positives than the Biotest AA.     

Comparison of two leukocyte extraction methods for cytomegalovirus antigenemia assay.

We carried out a prospective, parallel, and blind study on 113 blood samples from immunocompromised patients in order to compare two leukocyte extraction methods (6% dextran sedimentation and Polymorphprep separation) for cytomegalovirus (CMV) antigenemia assay. CMV was detected in 38 samples by antigenemia assay (34 by dextran sedimentation and 35 by Polymorphprep separation). No differences either in the number of positive specimens (P = 1) or in the mean CMV-positive cell counts (P = 0.41) were observed between the two leukocyte extraction methods. In conclusion, the two methods performed equally well for this assay.     

Real-time PCR assay compared to nested PCR and antigenemia assays for detecting cytomegalovirus reactivation in adult T-cell leukemia-lymphoma patients

We analyzed the efficiency of the quantitative real-time PCR assay for cytomegalovirus (CMV) reactivation in adult T-cell leukemia-lymphoma (ATL) patients and compared the results with those obtained with qualitative nested PCR and antigenemia assays. The viral load obtained by the real-time PCR assay closely paralleled the number of antigen-positive cells obtained with the antigenemia assay. Real-time PCR revealed that a large number of DNA copies could be present even in samples assessed as negative or low in antigen-positive cells (0 to 10 antigen-positive cells/50,000 cells) by antigenemia assay. CMV copy numbers did not differ between the negative and low-antigen-positive groups. When the input concentration for real-time PCR assay was 2,500 to 5,000 copies/ml, the positivity rate for the nested PCR assay was 47.3%, while the positivity rate was more than 90% at an input concentration of >/=50,000 copies/ml. Real-time PCR is more sensitive than the antigenemia and nested PCR assays. Moreover, real-time PCR was able to detect CMV reactivation earlier than the antigenemia and nested PCR assays through the use of longitudinal analysis in four ATL patients with CMV pneumonia. In longitudinal assessments, analysis of the results suggested that a cutoff level of 5,000 copies/ml might be used to initiate treatment. Real-time PCR is more suitable for monitoring CMV reactivation in ATL patients than the antigenemia and nested PCR assays. CMV viral loads of 5,000 copies/ml are proposed as the cutoff for initiating antiviral therapy in ATL patients.     

Epidemiologic analysis of reactivated cytomegalovirus antigenemia in patients with cancer

The epidemiologic features of reactivated cytomegalovirus (CMV) antigenemia were studied among 4,382 cancer patients who were cared for and tested at the University of Texas M. D. Anderson Cancer Center from 2001 to 2004. The effects of stem cell transplant (SCT) status, underlying disease, age, sex, ethnicity, and antibody status (prior to CMV exposure) on the incidence of CMV antigenemia were determined; and the CMV burdens were quantified. Antigenemia occurred in 9.3% of patients with non-SCT (n = 2511), 12.0% with autologous SCT (n = 582), and 39.1% with allogeneic SCT (n = 1289). Non-SCT patients with lymphoid tumors had a significantly higher rate of antigenemia than those with myeloid tumors (13.6% versus 3.9%) (P < 0.001); however, after allogeneic SCT, the underlying diseases had little effect, except for multiple myeloma (56.8%) (P = 0.014). Among the allogeneic SCT recipients, higher CMV antigenemia rates were also associated with female sex, older age, and positivity for pre-SCT CMV antibody. Depending on the underlying disease and its associated initial CMV risk, allogeneic SCT increased the risk by 2.6- to 29.6-fold (overall, 4.0-fold). With or without SCT, Asians had the highest CMV antigenemia rates and burdens, followed by blacks, Hispanics, and whites, and these partially correlated with antibody prevalence. Among the 808 patients with antigenemia, the circulating peak CMV burden was significantly higher among non-SCT patients (geometric mean, 18.7 positive cells per 10(6) leukocytes) than among allogeneic SCT patients (geometric mean, 7.7 positive cells per 10(6) leukocytes) or autologous SCT patients (geometric mean, 7.0 positive cells per 10(6) leukocytes) who underwent monitoring for CMV. Together, these results allow stratification of CMV risks and suggest a substantial CMV reactivation among non-SCT cancer patients and, thus, the need for better diagnosis and control.     

Prospective study of cytomegalovirus antigenemia in allograft recipients.

A recently published technique for direct detection of cytomegalovirus (CMV) antigenemia was tested prospectively in 27 transplant recipients. Eighteen patients developed active CMV infections and 10 of the 18 experienced CMV syndrome. All of the infections were detected by classical virus isolation and/or serologic techniques. No antigenemia was demonstrated.     

Factors influencing detection of quantitative cytomegalovirus antigenemia.

Of 20 blood specimens testing positive for cytomegalovirus antigen after immediate processing, 19 (95%) remained positive when kept at room temperature for 24 h before processing. Quantitative antigenemia decreased by an average of 44% after storage. Compared with acetone fixation, formaldehyde fixation showed improved readability, fewer artifacts, and a higher degree of sensitivity.     

Comparison of PCR, antigenemia assay, and rapid blood culture for detection and prevention of cytomegalovirus disease after lung transplantation

The goal of this study was to evaluate serial cytomegalovirus (CMV) blood culture, antigenemia testing, and qualitative and quantitative plasma CMV PCR for their ability to predict CMV disease and thus to direct preemptive therapy after lung transplantation. Forty-one patients provided 414 samples for blood culture, 290 samples for antigenemia testing, and 432 samples for PCR. Seven patients developed 11 episodes of CMV disease. CMV PCR had sensitivity, specificity, and positive predictive and negative predictive values of 79, 99, 84, and 99%, respectively, compared with 48, 99, 85, and 98%, respectively, for antigenemia testing, and 8, 100, 100, and 97%, respectively, for culture. Only quantitative CMV PCR correlated with disease stage: asymptomatic patients had a mean of 1,500 CMV DNA copies/ml, whereas patients who developed CMV disease had 5,087 copies/ml 12 to 4 weeks before symptoms and 32,000 copies/ml at diagnosis. Furthermore, CMV PCR-measured DNA increased 5- to 10-fold immediately preceding symptoms. PCR and antigenemia test values decreased with anti-CMV therapy. CMV DNA (as detected by PCR), but not antigenemia, persisted in patients who later developed recurrent CMV disease. The data indicate that lung transplant recipients will benefit from monitoring of CMV disease by plasma CMV PCR.     

Novel DNA assay for cytomegalovirus detection: comparison with conventional culture and pp65 antigenemia assay.

We compared conventional cytomegalovirus (CMV) isolation, rapid viral culture, a CMV pp65 antigenemia assay, and a novel CMV DNA hybrid capture system (HCS). A total of 309 blood samples from individuals in different risk groups were assessed by at least two of the methods mentioned above. Leukocytes were recovered either after centrifugation in Leucosep tubes containing 1.080 Ficoll for pp65 assay or after simple differential lysis steps for DNA detection. HCS was based on DNA hybridization with a CMV RNA probe and its capture by antibodies to DNA-RNA hybrids. The CMV pp65 lower matrix protein was detected by fluorescence with c10-c11 monoclonal antibody in formalin-fixed leukocytes. Concordant results were observed for 92.9, 78.3, and 82.7% of the patients when comparing (i) viral culture and the pp65 antigenemia assay, (ii) viral culture and HCS, and (iii) the pp65 antigenemia assay and HCS, respectively. Discordant results were observed between a positive HCS result and negative culture and/or pp65 results. These results were associated with relatively low DNA levels (< 20 pg/10(6) cells) and positive viruria. In conclusion, the pp65 antigenemia assay is a rapid and reliable method of detecting CMV and is preferable to culture, but the Murex HCS appears to be more sensitive for CMV detection.     

Comparison of PCR and pp65 antigenemia assay with quantitative shell vial culture for detection of cytomegalovirus in blood leukocytes from solid-organ transplant recipients.

This study compared PCR and an assay for cytomegalovirus (CMV) pp65 antigenemia (CMV-vue; INCSTAR Corp.) with a quantitative shell vial culture (QSVC) technique for the detection of CMV in serial blood specimens from 46 solid-organ transplant recipients. In a comparison based on 535 specimens tested by PCR and QSVC, CMV was detected by PCR in 41 and by QSVC in 37 of 43 recipients at risk of CMV infection. The mean number of days after transplantation of initial detection of CMV was 29.9 for PCR and 34.0 for QSVC (P = 0.01). The antigenemia assay was performed on 395 specimens, including 304 of those also tested by PCR. In these specimens, CMV was detected by the antigenemia assay, QSVC, and PCR in 30, 32, and 35 (respectively) of 38 patients at risk, with no statistically significant difference in the time to detection. Each of the assays detected CMV in similar proportions of patients with and without clinically significant CMV infection. PCR stayed positive longer after transplantation than the other assays but frequently returned to negative when more than 6 months had elapsed after transplantation. The antigenemia assay and PCR stayed positive longer after institution of antiviral therapy than QSVC. PCR can provide highly sensitive detection of CMV viremia, but a PCR assay for CMV is not yet available in kit form. The pp65 antigenemia assay and shell vial culture are quantifiable and comparable in sensitivity. Either is recommended for rapid detection of CMV in blood specimens from solid-organ transplant recipients.     

Effect of delayed specimen processing on cytomegalovirus antigenemia test results.

Blood samples held at either 4 degrees C or room temperature for 1 day had similar mean decreases in number of cytomegalovirus antigenemia-positive cells (52 to 55%) and similar false-negative test results (13 to 14%). After 2 days, samples held at 4 degrees C showed no further decline, whereas samples held at room temperature had a mean 81% decrease in positive cells, a 32% false-negative rate, and a more marked deterioration in cell morphology.     

Comparison of three assays for cytomegalovirus detection in AIDS patients at risk for retinitis

The purpose of this study was to determine the sensitivity and specificity of three different methods of cytomegalovirus (CMV) detection for AIDS patients at risk for CMV retinitis. Patients with CD4(+) counts of <100/microl and negative baseline screening eye examinations were tested for CMV infection by (i) pp65 antigenemia expression in leukocytes, (ii) the Digene Hybrid Capture CMV DNA System, and (iii) the Roche Amplicor Qualitative PCR Test. The incidence of CMV retinitis in our study of 296 patients at the Medical Center of Louisiana-New Orleans HIV Outpatient Clinic was 7. 2 per 100 person-years (a total of 20 episodes in 18 patients from April 1997 to February 1999). Receiver operating characteristic curves were calculated for each assay to determine optimal cutoff points which maximized the sensitivity and specificity of each assay. The sensitivities of the assays compared to the eye examinations were 80% for the pp65 antigenemia assay (cutoff, >0 cell per 1.5 x 10(5) leukocytes), 85% for the Digene assay (cutoff, 1,400 genome copies/ml of whole blood), and 60% for the Amplicor assay. The specificities of the assays were 84, 84, and 87%, respectively. The Digene assay with a cutoff of >/=1,400 genome copies/ml gave optimal sensitivity and specificity and was found to have predictive values equal to those of the more technically cumbersome antigenemia assay.     

Human recombinant myeloperoxidase antiviral activity on cytomegalovirus.

In vitro incubation of human cytomegalovirus (Towne strain) with 8 U/ml human recombinant myeloperoxidase plus sodium chloride and glucose nearly abolished viral infectivity. To assay the effect on intracellular infection, cell toxicity of the enzymes was first studied. Even the high dose of 16 U/ml of recombinant myeloperoxidase plus 10 mU/ml glucose oxidase did not decrease MRC5 cell growth. By contrast, this dose reduced proliferation of activated THP1 cells. Even half of the myeloperoxidase dose proved slightly toxic to these cells. Non-cytotoxic concentrations of the reagents were used to monitor their effect on cytomegalovirus infection. In MRC5 cells, even the low dose of 4 U/ml myeloperoxidase plus glucose oxidase inhibited synthesis of cytomegalovirus early antigens, as tested by immunofluorescence. Viral release in the supernatant was decreased by 4 logs. In THP1 cells, which produce endogenously hydrogen peroxide, myeloperoxidase alone (8 U/ml) decreased the formation of early and late antigens by 53 and 44%, respectively.