Use of monoclonal antibodies for the diagnosis of cytomegalovirus infection by the detection of early antigen fluorescent foci (DEAFF) in cell culture

A pool of seven monoclonal antibodies, each reactive with cytomegalovirus (CMV) early antigens, was used in an indirect immunofluorescence method for the rapid detection of CMV-infected fibroblasts following inoculation with clinical specimens. A total of 1,639 specimens were examined, and the results were compared with those of conventional isolation procedures. The detection of CMV by early antigen fluorescent foci (DEAFF) was found to be comparable, both in terms of specificity and sensitivity, to that of conventional cell culture. Its great advantage, however, is the rapidity with which results are achieved. Thus, results were available from the DEAFF test within 24 hours of receipt of the specimens as compared to a mean of 16 days for cell culture. This single rapid assay for the detection of CMV in clinical samples may be performed by any laboratory familiar with cell culture techniques and in our hands is the preferred diagnostic method for CMV.     

PCR detection of cytomegalovirus DNA in serum as a diagnostic test for congenital cytomegalovirus infection.

PCR detected cytomegalovirus (CMV) DNA in the serum of 18 of 18 infants with symptomatic congenital CMV infection, 1 of 2 infants with asymptomatic congenital CMV infection, and 0 of 32 controls. Serum CMV PCR provided a rapid, sensitive, and specific method for diagnosis of congenital CMV infection in infants who were symptomatic at birth.     

In situ hybridization for the detection of cytomegalovirus (CMV) infection

Summary Five autopsy cases of cytomegalovirus (CMV) infections were studied. Conventional light microscopy disclosed characteristic cytopathic effects in lungs, kidneys, and brain. In one case, electron microscopy was carried out and revealed typical herpesvirus particles.In situ hybridization was done with biotin-labeled CMV-DNA probes and an avidin-alkaline phosphatase detection system. 4/5 cases were observed to contain hybridizing cells in different organs. Intensity of hybridization was related to the severity of CMV infection, roughly estimated by counting cytomegalic cells. In addition to cytomegalic cells, a high number of normal-looking epithelial and mesenchymal cell types were positive. These latter cells showed nuclear hybridizations in contrast to cytomegalic cells which hybridized both within the nuclei and the cell bodies.This modified in situ hybridization procedure is a rapid and valuable tool for the detection and final demonstration of virus infection, and will be of particular help for the examination of paraffin-embedded specimens.Dedicated to Prof. Dr. G. Seifert on the occasion of his 65th birthdayThis study was supported by a grant of the Deutsche Forschungsgemeinschaft (Lo 285/2-3) and the Hamburger Stiftung zur Förderung der Krebsbekämpfung (I 208)     

Comparison of PCR and antigen detection methods for diagnosis of Entamoeba histolytica infection

AIMS: To assess different laboratory methods for the identification of Entamoeba histolytica in clinical samples. METHODS: Antigen detection enzyme linked immunosorbent assay, polymerase chain reaction solution hybridisation enzyme linked immunoassay (PCR-SHELA), and a commercial Lightcycler PCR were compared using 101 stool and pus samples. RESULTS: Fifteen of the 101 samples were positive for E histolytica by one or more method. There were discrepancies between the results in five of these 15 samples when the assays were compared. CONCLUSIONS: All three methods performed adequately, so that the choice of assay will depend on each individual laboratory's budget and projected turnaround time.     

Comparison of nested PCR for detection of DNA in plasma with pp65 leukocytic antigenemia procedure for diagnosis of human cytomegalovirus infection.

A nested PCR was used for the detection of human cytomegalovirus (HCMV) DNA in plasma. The presence of HCMV DNA and its correlation to pp65 leukocytic antigenemia were investigated with 299 blood samples from 45 organ transplant recipients and 63 AIDS patients. Of the 53 samples positive by nested PCR, 52 (98%) were also positive for leukocytic antigenemia and 23 had high levels of antigenemia (> 50 positive cells per 2 x 10(5) leukocytes). Of the 246 samples negative in PCR, only 3 (1.2%) had highly positive antigenemia. For 15 patients having a high antigenemia level in the course of their disease, consecutive blood samples were studied and also assessed for viremia in culture. The extent to which HCMV DNA, detected by PCR, was present in plasma correlated with increased levels of HCMV leukocytic antigenemia for six of the eight AIDS patients and for all the organ transplant recipients. Positivity for HCMV DNA in PCR and for viremia in cell culture was usually restricted to the highest antigenemia levels. From a total of 69 blood samples, PCR and culture gave positive results, respectively, for 17 of 32 samples (53%) and 14 of 32 samples (43%) from transplant recipients and for 15 of 37 samples (40%) and 9 of 37 samples (24%) from AIDS patients. Our findings have shown a strong correlation between high levels of leukocytic antigenemia and HCMV DNA in plasma. The detection of HCMV DNA in plasma by this nested PCR can prove HCMV dissemination in blood, but it lacks the rapidity and simplicity of the leukocytic pp65 antigenemia procedure.     

Comparison of dot-blot DNA hybridisation and immediate early nuclear antigen production in cell culture for the rapid detection of human cytomegalovirus in urine

The sensitivity and specificity of four modes of two assays, immediate early nuclear antigen detection in cell culture (IENAD) at 24 and 48 h post-infection (p.i.) by immunofluorescence using a murine monoclonal antibody, and dot-blot DNA hybridisation with overnight or prolonged autoradiography using the 32P-labelled HindIII J fragment of human cytomegalovirus (HCMV) DNA as probe, were compared for the rapid detection of HCMV in urine. The sensitivity of IENAD was enhanced by low-speed centrifugation at the time of inoculation. DNA hybridisation with overnight autoradiography was significantly less sensitive than IENAD at 24 h p.i. (P less than 0.001), and even with prolonged autoradiography the hybridisation assay was slower and significantly less sensitive than IENAD at 48 h p.i. (P less than 0.02). The specificity of the two assays was virtually 100%. The sensitivity of DNA hybridisation was thus clearly inferior to that of IENAD.     

Human cytomegalovirus (CMV) DNA in plasma reflects quantity of CMV DNA present in leukocytes.

A quantitative DNA amplification assay for human cytomegalovirus (CMV) DNA has been used to evaluate the relationship between quantities of CMV DNA in plasma and those in infected leukocytes (WBC) from human immunodeficiency virus-infected patients. The target sequence for DNA amplification was a region of the immediate-early 1 gene of CMV. The quantitation assay uses an internal control that is coamplified with each patient sample DNA and contains a sequence for detection by colorimetric hybridization with the same bases, but in different order than in the CMV immediate-early 1 region used for hybridization of amplified patient sample DNA. Results showed that patients with CMV disease had more CMV DNA in both WBC and plasma than those without disease. However, in this study, copy numbers of CMV DNA in WBC were higher than those in plasma. The gB and gH variants were the same in plasma and WBC.     

Diagnosis of cytomegalovirus infection by detection of the early antigen of cytomegalovirus in cell cultures

Conventional virus isolation and detection of cytomegalovirus (CMV) early antigen by immunofluorescence staining of cultured cells were compared in the diagnosis of CMV infection from urine specimens. By virus isolation, 33 specimens out of 333 studied were positive, and the mean length of culturing time for a positive result was 23 days (range from 6 to 45 days). By early antigen detection, 35 specimens were positive after 20 hours in culture, but the number of positive findings increased to as high as 49 after 7 days in culture. It is recommended that, in addition to the early antigen staining after one day in culture, cells should also be stained after one week in culture, because the sensitivity is essentially improved by extended culturing time.     

Early detection of active cytomegalovirus (CMV) infection after heart and kidney transplantation by testing for immediate early antigenemia and influence of cellular immunity on the occurrence of CMV infection.

To determine the incidence of active cytomegalovirus (CMV) infection after organ transplantation and its relationship with the immune system, 55 renal and 14 cardiac transplant recipients were closely monitored for active CMV infection (expression of CMV immediate early antigen in granulocytes--antigenemia--and positive cultures) and immune parameters. All 19 CMV-seronegative recipients with seronegative donors remained seronegative, showing that no CMV transmission occurred by leukocyte-depleted blood products. Primary CMV infection occurred in 4 of 11 (36%) patients with positive donors and was symptomatic in 1 (9%) patient. Active CMV infection was found in 29 of 39 (74%) seropositive patients and was symptomatic in 3 (8%) patients. CMV antigenemia was always the first indication of active CMV infection (antigenemia, on average, at day 45 +/- 15; immunoglobulin G rise at day 71 +/- 36; and positive cultures at day 70 +/- 17). Cellular immunity, as measured by lymphocyte proliferation (LPT), proved to be of importance in the prevention of active CMV infection, as 14 of 15 patients with negative LPT obtained active CMV infections with antigenemia and positive cultures, whereas 1 of 10 patients with positive LPT did so (P less than 0.0001).     

Comparison of plasma PCR and bronchoalveolar lavage fluid culture for detection of cytomegalovirus infection in adult bone marrow transplant recipients.

Plasma PCR for human cytomegalovirus (CMV) DNA was compared with bronchoalveolar lavage (BAL) fluid culture as an indicator for disseminated CMV infection. Thirteen (32.5%) of 40 consecutive bone marrow transplant (BMT) recipients were BAL fluid culture positive for CMV on day 35 post-BMT, and 9 (69%) of the 13 had positive plasma PCRs between days 28 and 49. Of the 27 with negative BAL fluid cultures, 2 (7%) had positive plasma PCRs (P < 0.001). Plasma CMV DNA in BMT recipients is a useful clinical marker for serious infection.     

Evaluation of cytomegalovirus (CMV) DNA quantification in dried blood spots: retrospective study of CMV congenital infection

We compared two protocols for extracting DNA from dried blood spots for cytomegalovirus (CMV) DNA detection and quantification by real-time PCR. Both extraction methods were reliable for the retrospective diagnosis of CMV congenital infection. Quantification of CMV DNA was valuable after normalization of viral loads with albumin gene PCR amplification results.     

Detection of cytomegalovirus (CMV) in granulocytes by polymerase chain reaction compared with the CMV antigen test.

To compare the sensitivity and suitability of detection of active cytomegalovirus (CMV) infection by using monoclonal antibodies against CMV antigen (antigen test to detect antigenemia) and the polymerase chain reaction (PCR; to detect viral DNA) in granulocytes, 19 heart and 2 lung transplant recipients were closely monitored by these tests for at least 3 months after transplantation. All patients were CMV seropositive or had a seropositive donor. In total, 201 samples were tested; 46 were positive by both tests, 9 samples showed only antigenemia, 54 samples were positive by PCR only, and 102 samples were negative by both tests. PCR was positive earlier after transplantation in eight patients, whereas antigenemia was positive earlier after transplantation in one patient. In another four patients, both tests were positive at the same time. PCR was, on average, positive for a longer period of time. Discordant results showing a positive antigen test and a negative PCR were partly due to sampling error; some were positive by PCR on retesting. Samples which were negative by the antigen test and positive by PCR were taken at the beginning or at the end of an active CMV infection. In two patients, no active CMV infection was detected by the antigen test, cultures of urine and saliva, or serology, although PCR was positive for a long period of time in the two patients.     

Quantitation of cytomegalovirus (CMV) DNA by real-time PCR for occurrence of CMV disease in HIV-infected patients receiving highly active antiretroviral therapy

In HIV-infected patients treated with highly active antiretroviral therapy (HAART) included in the Predivir cohort, we have evaluated the usefulness of CMV DNA quantitation by a TaqMan PCR assay from peripheral blood leukocytes (PBLs) to predict CMV disease occurrence. In parallel with the immune restoration after treatment by HAART, the percentage of positive samples decreased progressively from 7.3% at Day 0 to 3.5% at Month 12. Among the CMV markers, the smallest concordance with PBL CMV TaqMan PCR, as evaluated by kappa, was observed with pp65 antigenemia, whereas concordance with all other CMV markers was high. Among the 16 patients with CMV DNA copies at least once >100/150,000 cells, CMV disease occurred in six during follow-up, whereas among the 159 patients with CMV DNA copies always <10/150,000 cells, CMV disease occurred in three and among the seven patients with CMV DNA copies >10 and <100 occurred in only one. In univariate Cox models, all the CMV markers including PBL CMV TaqMan PCR >10/150,000 cells (RR: 27.6, IC95: 7.1-107.2), the CD4 cell count <75 cells/mm(3) and the HIV viral load >100,000 copies/ml were predictive for CMV disease. In a stepwise multivariate analysis, which should be interpreted with caution due to the small number of events (n = 10), three covariates were associated independently with CMV disease: pp65 antigenemia >100 nuclei/200,000, PBL CMV TaqMan PCR >10 copies/150,000 cells and HIV viral load remaining or increasing >100,000 copies/ml.     

PCR detection of viral DNA in serum as an ancillary analysis for the diagnosis of acute mononucleosis-like syndrome due to human cytomegalovirus (HCMV) in immunocompetent patients.

BACKGROUND: Serologic tests are occasionally inconclusive for the diagnosis of mononucleosis-like syndrome due to human cytomegalovirus (HCMV). OBJECTIVES: To determine the value of viral DNA detection in serum by PCR as an ancillary test for the diagnosis of HCMV mononucleosis. STUDY DESIGN: Sera from 34 previously healthy individuals with HCMV mononucleosis, obtained within 1 month after the onset of symptoms, were assayed for viral DNA by three commercial PCRs (QCA gB, QCA MIE and Amplicor CMV Monitor). Sera from 30 patients with evidence of past HCMV infection served as controls. RESULTS: Viral DNA was detected in 20% of the samples from patients with HCMV mononucleosis by both QCA procedures, but in none of the controls. All samples tested negative by the Amplicor CMV Monitor. CONCLUSION: Analysis of sera for the presence of HCMV DNA is of limited value for the diagnosis of HCMV mononucleosis.     

Comparison of enzyme immunoassay antigen detection,nucleic acid hybridization and PCR assay in the diagnosis ofChlamydia trachomatis infection

An enzyme immunoassay (EIA) antigen detection system (Micro Trak, Syva), nucleic acid hybridization (PACE 2, Gen-Probe) and polymerase chain reaction (PCR) assay (Amplicor, Hoffmann-La Roche) were evaluated for the detection ofChlamydia trachomatis in a high-risk female population. Of 234 specimens, 42 (18 %) were positive. The respective sensitivity of the EIA, RNA hybridization and the PCR was 81, 90 and 88 %. When additionally performed on diluted specimens, PCR gave positive results for three of four PCR-negative specimens from EIA- and RNA-hybridization-positive women and a sensitivity of 95 %. Thus, both techniques employing gene technology offered a clear improvement in sensitivity over the EIA. Future improvements in the PCR should be directed towards the elimination of polymerase inhibition.     

Comparison of quantitative cytomegalovirus (CMV) PCR in plasma and CMV antigenemia assay: clinical utility of the prototype AMPLICOR CMV MONITOR test in transplant recipients

The correlation between the prototype AMPLICOR CMV MONITOR test (Roche Molecular Systems), a quantitative PCR assay, and the cytomegalovirus (CMV) pp65 antigenemia assay was evaluated in transplant recipients. Sequential blood specimens were collected on 29 patients (491 specimens), the leukocyte fraction was tested by CMV antigenemia, and quantitative PCR was performed on plasma specimens. None of the 15 patients (242 specimens) who were antigenemia negative were positive for CMV DNA by PCR, and none of these patients developed active CMV disease. There were 14 antigenemia-positive patients, 8 of whom developed active CMV disease. In all patients, there was a good association between the antigenemia and PCR assays. Ganciclovir-resistant virus was isolated from three patients with active CMV disease. These three patients had persistently elevated levels of antigenemia and CMV DNA by PCR when resistance to ganciclovir developed. This standardized, quantitative CMV PCR assay on plasma has clinical utility for the diagnosis of active disease and in monitoring the response to antiviral therapy in transplant recipients.     

Comparison of different techniques for detection of cytomegalovirus infection following bone marrow transplantation

A total of 317 different clinical samples obtained from patients following bone marrow transplantation and 56 blood and urine samples from seronegative control persons were screened for the presence of human cytomegalovirus (CMV) using virus culture and slot-blot hybridization. Immunohistochemical techniques using monoclonal antibodies to various viral antigens and in situ hybridization techniques were also utilised for detection of CMV in tissue samples. In cell suspensions of blood, bone marrow and effusions, and in liver biopsies, CMV DNA could be demonstrated more often by slot-blot and in situ hybridization techniques than by virus culture or immunostaining of viral antigens. For detection of CMV in lung biopsies and other clinical samples containing mainly cell-free virus, such as urine, bronchial lavage and throat washings, virus culture was found to be at least as sensitive as the hybridization techniques. Immunostaining proved to be a fast and sensitive technique for detection of CMV in tissues and may thus provide additional information about viral replication and clinical relevance of the virus infection.     

Comparison of two nested PCR, cell culture, and antigen detection for the diagnosis of upper respiratory tract infections due to influenza viruses.

Influenza surveillance requires sensitive and rapid diagnostic methods. Different diagnostic procedures have been evaluated on a selected set of nasal swabs sample collected from patients presenting with acute respiratory infection. One hundred fifty-four samples collected during the peak of the influenza epidemic recorded during winter of 1997-1998 in the south of France were processed for influenza detection using antigen detection (ELISA-immunocapture assay), two different nested RT-PCR assays (targeting M and HA genes), and cell culture. Among 154 samples, 93 (60.4%) were positive for influenza detection. Forty specimens (26%) were positive by ELISA, 77 (50%) by culture, 88 (57.1%) using the multiplex HA-PCR and 76 (49.4%) using the M-PCR. Multiplex HA-PCR was thus the most sensitive test. The PCR assay offers an alternative to culture for influenza detection. Nevertheless, culture is efficient for influenza diagnosis and is the only technique that allows the reference centres to collect viral strains and characterise fully new variants.