Quantification of cytomegalovirus DNA in peripheral blood leukocytes by a branched-DNA signal amplification assay.

Quantification of cytomegalovirus (CMV) DNA in blood may aid in the identification of patients at highest risk for developing CMV disease, the evaluation of new therapeutics, and the prompt recognition of drug-resistant CMV strains. A branched-DNA (bDNA) assay was developed for the reliable quantification of CMV DNA in peripheral blood leukocytes. The bDNA assay allowed for the highly specific and reproducible quantification of CMV DNA in clinical specimens. Furthermore, the bDNA assay was at least as sensitive as culture techniques and displayed a nearly 3 log10 dynamic range in quantification. Changes in CMV DNA levels measured by the bDNA assay in a human immunodeficiency virus-positive patient undergoing therapy were consistent with CMV culture, antigen, and genotype results and correlated with disease progression and resistance markers. The bDNA assay for the quantification of CMV DNA may provide a useful tool that can be used to aid physicians in monitoring disease progression, evaluating therapeutic regimens, and recognizing viral resistance and drug failure.     

The detection of human cytomegalovirus immediate early antigen in peripheral blood leucocytes.

Recently, Van der Bij et al. (1988) reported that active human cytomegalovirus (HCMV) infection could be diagnosed by the detection of HCMV immediate early antigen (IEA) directly in the peripheral blood leucocytes of renal transplant recipients. However, the indirect peroxidase technique used resulted in high background staining due to endogenous peroxidase activity and thus the detection of HCMV-IEA positive leucocytes, which are sometimes present in extremely low numbers, was not always reliable. In an attempt to solve this problem, we have evaluated the alkaline phosphatase-anti-alkaline phosphatase (APAAP) technique, immunogold-silver staining (IGSS), and several fixatives. Fixation with acetone: methanol 1:1 in conjunction with the APAAP technique proved to be the most successful method. In 155 blood samples obtained from 44 patients following renal transplantation and from three AIDS patients, the number of positive cells ranged between 1 and 700 out of 400,000 (median 2). In 23 samples from 11 patients (one AIDS patient) at least one positive cell was found. In this series there were no problems with the evaluation since strong positive signals were obtained without any background staining. We therefore recommend the use of this protocol for the rapid and reliable detection of HCMV-IEA in peripheral blood leucocytes.     

The intracellular localization of human cytomegalovirus DNA in peripheral blood leukocytes during active infections by high-resolution fluorescence in situ hybridization

Summary Although viremia is an integral part of the pathogenesis of human cytomegalovirus (HCMV) disease, the interaction between HCMV and circulating leukocytes of actively infected patients remains an area of uncertainty. It is still a matter of dispute, whether leukocytes support viral replication with subsequent production of infectious virus. In a new approach we developed and applied a sensitive fluorescence in situ hybridization assay for the precise intracellular localization of HCMV genomes in leukocytes. It was shown that in vivo HCMV genomes were exclusively localized in the cytoplasm of leukocytes, indicating that the majority of these cells are virus carriers or abortively infected. Though this method easily detects single copy genes in metaphase chromosomes, the number of HCMV DNA positive leukocytes was significantly lower than the number of HCMV pp65 antigen positive cells. In relation to the pp65 antigen positive cells, only 1–4% of these cells were DNA positive. In addition, the much lower frequency of HCMV immediate early antigen positive leukocytes in comparison to the pp65 antigen positive cells and the impossibility of detecting other viral antigens support the hypothesis that the origin of pp65 found in leukocytes results mainly from protein uptake.     

Evaluation of CMV Brite kit for detection of cytomegalovirus pp65 antigenemia in peripheral blood leukocytes by immunofluorescence.

The CMV Brite antigenemia kit was compared with culture and an established cytomegalovirus pp65 antigenemia assay (CMV AG). Of 300 clinical specimens tested, 92 were positive by CMV Brite, 83 were positive by CMV AG, and 34 were positive by culture. Discrepancies could be attributed to anticytomegalovirus therapy or low-level antigenemia.     

Polymerase chain reaction assay for detection of human cytomegalovirus.

Direct detection of human cytomegalovirus (HCMV) from clinical specimens was examined by using the polymerase chain reaction (PCR) for amplifying HCMV DNA. The efficiency of the amplification reaction was examined by using three different buffers and concentrations of deoxynucleotide triphosphates. The PCR assay was most efficient with a reaction mixture containing 17 mM ammonium sulfate, 67 mM Tris hydrochloride (pH 8.5), 7 mM MgCl2, 10 mM 2-mercaptoethanol, 170 micrograms of bovine serum albumin per ml, and each deoxynucleotide triphosphate at a final concentration of 1.5 mM. After 35 cycles of amplification, 0.15 fg of a plasmid containing the cloned target gene (corresponding to approximately six gene copies) was detected. The PCR assay correctly identified all of 24 clinical isolates of HCMV. Virus in urine specimens could be disrupted by heating at 93 degrees C for 30 min. The viral DNA was amplified directly from 5 microliters of preheated urine, with no further treatment before amplification. We tested the PCR assay on urine specimens from patients who had undergone renal transplantation that had been screened for the presence of HCMV by enzyme-linked immunosorbent assay, hybridization assay, and direct virus isolation. Specimens that were positive by one or more of these assays were screened by PCR. HCMV was consistently detected by PCR in all specimens that were positive by at least one other test. No cross-reactivity to other herpesviruses or MRC-5 cellular DNA was observed.     

Cell culture-PCR technique for detection of infectious cytomegalovirus in peripheral blood.

We determined the efficacy of PCR for the direct detection of infectious cytomegalovirus (CMV) in specimen-inoculated MRC-5 tube cultures (cell culture-PCR [CC-PCR]). Parallel testing was performed by a shell vial assay-indirect immunofluorescence assay (SVA-IFA) and isolation (conventional MRC tube cultures [TC-CPE]. The sensitivity, specificity, and positive and negative predictive values for CC-PCR, SVA-IFA, and TC-CPE were 81, 99, 94, and 94%, 86, 100, 100, and 96%, and 77, 100, 100, and 93%, respectively. Future application of CC-PCR may be directed toward its use as a screening tool for cytomegalovirus genotypic variants.     

Monitoring of renal allograft recipients by quantisation of human cytomegalovirus genomes in peripheral blood leukocytes

The ratio of human cytomegalovirus (HCMV) genomes per cellular genomes in serial peripheral blood leukocyte (PEL) extracts of renal allograft recipients was quantitated by competitive nested polymerase chain reaction (PCR). Patients were also monitored for the development of acute HCMV infection by detection of HCMV pp65 antigenemia, HCMV IgM antibodies, and viruria. Compared to qualitative nested HCMV PCR, the frequency of positive PCR results in renal allograft recipients without further evidence of acute HCMV infection was significantly reduced by quantitative HCMV PCR. HCMV DMA levels ≥1,000 copies HCMV/10⁶ copies β-globin were found to be highly indicative for the development of a clinically symptomatic HCMV infection following renal allograft transplantation. In patients treated with ganciclovir, quantitation of HCMV target sequences allowed the assessment of the efficacy of antiviral therapy. © 1994 Wiley-Liss, Inc.     

A hybrido-immunocytochemical assay for the in situ detection of cytomegalovirus DNA using digoxigenin-labeled probes

A non-radioactive hybrido-immunocytochemical assay for the detection of cytomegalovirus (CMV) DNA in infected cells was developed. Two different DNA fragments belonging to the repeated sequences of CMV genome were used to construct the hybridization probe. The probe was constructed by incorporating deoxyuridine triphosphate labeled with digoxigenin. The in situ hybridized CMV DNA probe was immunocytochemically visualized by anti-digoxigenin. Fab fragments labeled with alkaline phosphatase. This procedure permitted the DNA detection, in the nuclei of infected cells fixed at 48 h after infection, of the Towne CMV reference strain and 21 different laboratory-isolated CMV strains. Our assay demonstrated a high specificity, sensitivity and reproducibility.     

Nonradioactive PCR-enzyme-linked immunosorbent assay method for detection of human cytomegalovirus DNA.

We developed a rapid, sensitive, and specific PCR-based assay for human cytomegalovirus (HCMV). The assay includes primer and probe sequences derived from conserved HCMV nucleotide sequences and nonradioactive hybridization-confirmation. The assay detected between 10 and 100 viral genomes. All HCMV clinical isolates tested (39 of 39) gave positive reactions.     

Ultrastructural localization of peroxidatic catalase in human peripheral blood leukocytes.

Localization of peroxidatic catalase in human peripheral blood leukocytes was accomplished by the assessment of alkaline diaminobenzidine reaction in the cytoplasmic granules of normal and acatalasemic leukocytes. A modified cytochemical procedure of Novikoff and Goldfischer (Novikoff AB, Goldfischer S: J Histochem Cytochem 17:675, 1969) and of Fahimi (Fahimi HD:J Cell Biol 43:275, 1969) was employed to improve the specificity of alkaline diaminobenzidine test for catalase. Diaminobenzidine-positive reaction for peroxidative catalase was observed in large and medium-sized granules in the cytoplasm of normal neutrophils, but a striking and notable absence of this reaction was observed in acatalasemic neutrophils. The test for myeloperoxidase, with the diaminobenzide reaction performed at neutrality, disclosed positively stained granules in both normal and acatalasemic neutrophils. Similarities in size and configuration of the positively stained granules for these enzymes suggest that catalase is sequestered in organelles which may be primary or azurophilic granules. Myeloperoxidase has been shown to be localized in the primary granules by others. It is possible that catalase and myeloperoxidase may be sequestered together or separately in these granules, but the present data do not permit us to draw this distinction. The ultrastructural localization of peroxidatic catalase and myeloperoxidase has been attempted in eosinophils, lymphocytes, and platelets, and the observations are compared with those of neutrophilic granules. The localization of peroxidatic catalase in monocytes could not be assessed satisfactorily because of the difficulties encountered in proper sampling of these cells.     

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.     

Neutral endopeptidase activity in human peripheral blood leukocytes

Neutral endopeptidase (NEP; EC 3.4.24.11) efficiently hydrolyses many neuropeptides. To determine the distribution of NEP, a possible regulatory enzyme for the neuropeptide-induced leukocyte activation, among human leukocytes, we investigated the enzymatic activity of NEP in each cell type of human peripheral blood leukocytes. The activity of NEP assessed by an NEP inhibitor phosphoramidon-sensitive Met5-enkephalin degrading activity was present in neutrophils (59.0 +/- 9.1 pmol/min 10(6) cells); however, the NEP activity was virtually absent in mononuclear cells, eosinophils and basophils. Common acute lymphoblastic leukemia antigen (CALLA) detected immunocytochemically with three anti-CALLA antibodies, whose amino acid sequence has been shown to be identical with that of NEP, was also found only in neutrophils, but not in other blood leukocytes. It is suggested that NEP might regulate the neuropeptide-induced activation of human neutrophils.     

Chemiluminescent in situ hybridization for the detection of cytomegalovirus DNA.

A chemiluminescent in situ hybridization assay that could combine the sensitivity of chemiluminescent substrates, the specificity of digoxigenin-labeled probes, and the spatial morphological resolution and localization of the signal of the in situ hybridization was developed for the detection of cytomegalovirus (CMV) DNA. CMV DNA in cultured CMV-infected cells and in different clinical samples (tissue sections and cellular smears) was detected using digoxigenin-labeled probes constructed in our laboratory that were immunoenzymatically visualized employing anti-digoxigenin Fab fragments labeled with alkaline phosphatase and the chemiluminescent adamantil-1,2-dioxetane phenyl phosphate substrate for alkaline phosphatase. The luminescent signal from the hybrid formation was detected, analyzed, and measured with a high performance, low light level imaging luminograph apparatus connected to an optical microscope and to a personal computer for quantitative image analysis. Increasing values of emitted photons per second per infected cell, corresponding to the presence of hybridized CMV DNA, could be found in infected cells fixed at various times after infection, following the CMV replication cycle. When the assay was performed on different clinical samples from patients with acute CMV infections, CMV DNA was detected in all positive samples tested, both in cellular samples and in frozen and paraffin-embedded tissue sections, proving specific and sensitive. The chemiluminescent in situ hybridization assay developed in this work can be a useful tool for a sensitive and specific diagnosis of viral infection and can be easily adapted to detect and study any specific gene sequence inside the cells. The assay may also be promising for an estimation and quantification of nucleic acids present in tissue samples or cellular smears and for imaging gene expression in cells.     

Evaluation of CMV-vue antigenemia assay for rapid detection of cytomegalovirus in mixed-leukocyte blood fractions.

The CMV-vue antigenemia assay was evaluated by using mixed-leukocyte (ML) blood fractions. Of 234 ML fractions studied, 32 (14%), 23 (10%), and 20 (8.5%) were positive in the CMV-vue assay and conventional and shell vial cultures, respectively. The CMV-vue assay was more sensitive than shell vial cultures for rapid detection of cytomegalovirus. ML fractions are appropriate specimens for the cytomegalovirus antigenemia assay.     

Performance characteristics of an automated PCR assay for the quantification of cytomegalovirus DNA in plasma

The COBAS Amplicor CMV Monitor test (Roche Diagnostics), an automated polymerase chain reaction (PCR) assay for the quantification of cytomegalovirus (CMV) DNA in plasma samples, was evaluated in a routine diagnostic laboratory. Using cell culture-derived CMV and CMV-negative human plasma, the linear detection range of the assay as well as its intra-and inter-assay variabilities were assessed. The study design allowed distinguishing variations in results related to amplification and detection from those caused by differences in the efficiency of DNA extraction. The assay was able to identify the majority of samples correctly as positive with CMV DNA concentrations above the limit of detection. However, the reported values were often twofold or more different from the (theoretical) input, which could be explained partly by inefficient DNA extraction. The following values were computed for the coefficients of determination R(2): inter-assay variability excluding DNA extraction, R(2)=0.982; including DNA extraction, R(2)=0.977; intra-assay variability excluding DNA extraction, R(2)=0.992; including DNA extraction, R(2)=0.992. On balance, the test has acceptable within-run and between-run reproducibility. It therefore allows the comparison of results obtained at different time-points as well as in different laboratories, e.g. in multi-centre studies.     

Cultivation of human peripheral blood leukocytes in agar gel

The use of a modified method of cloning hematopoietic cells in semisolid nutrient media showed that among human peripheral blood leukocytes there are committed precursor cells of the granulocytic series (CFU_c). In healthy adults and children (aged from 4 days to 10 years) the number of these precursors is 0.05–6.38 and 0.2–2.9/10⁵ nucleated cells respectively. The number of CFU_c in the blood of patients with infectious mononucleosis is within normal limits (0.5–14/10⁵ nucleated cells). In acute leukemia very low colony-forming activity of the nucleated blood cells is found (0–0.3/10⁵ cells).Laboratory of Culture and Transplantation of Bone Marrow, Central Institute of Hematology and Blood Transfusion. Laboratory of Pediatric Hematology, N. I. Pirogov Second Moscow Medical Institute. (Presented by Academician of the Academy of Medical Sciences, of the USSR N. A. Fedorov.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 81, No. 5, pp. 635–636, May, 1976.     

Flow cytometric assay for quantifying opsonophagocytosis and killing of Staphylococcus aureus by peripheral blood leukocytes.

We describe a novel flow cytometric method for quantifying opsonophagocytosis and killing of Staphylococcus aureus in cell-rich plasma obtained after dextran sedimentation of erythrocytes. To analyze opsonophagocytosis, phagocytes were labeled with a phycoerythrin-conjugated monoclonal antibody and were incubated with viable staphylococci containing carboxyfluorescein as a vital fluorescent dye. Phagocytosing cells assumed a dual, orange-green fluorescence. The relative numbers of bacteria associating with phagocytes could be determined by quantifying the decrease of free green fluorescent particles. A parallel incubation of fluorescent bacteria with unlabeled cell-rich plasma was performed to assess phagocytic killing. Blood cells were lysed with 3-[(3-cholamidopropyl)-dimethyl-ammonio]-1-propanesulfonate. This detergent spared viable bacteria, and residual green fluorescent particles were counted. The decrease in the number of these particles relative to the controls yielded the degree of killing. At bacteria-to-phagocyte ratios of 1:1 and 10:1, approximately 36 and 75% of the phagocytes participated in opsonophagocytosis, respectively. Over 90% of the staphylococci were phagocyte associated after 30 to 60 min. Killing rates were on the order of 66% +/- 12% and 80% +/- 7% after 1 and 2 h of incubation, respectively. These numbers, which were confirmed by colony countings, were significantly lower than those reported in the majority of past reports.     

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.