Assessment of automated DNA extraction coupled with real-time PCR for measuring Epstein-Barr virus load in whole blood, peripheral mononuclear cells and plasma.

BACKGROUND: Epstein-Barr virus (EBV) DNA load monitoring in blood has been shown to be essential for the diagnosis of EBV-associated diseases. However, the methods currently used to assess EBV DNA load are often time-consuming and require prior blood separation. OBJECTIVES: The aim of this study was to evaluate the relative diagnostic value of EBV DNA load monitoring in whole blood, peripheral blood mononuclear cells (PBMCs) and plasma after automated DNA extraction using the MagNA Pure extractor followed by LightCycler real-time quantitative PCR (LC-PCR). STUDY DESIGN: First, EBV DNA load was assessed retrospectively after automated or manual extraction on 104 PBMC specimens. Second, EBV DNA load was determined prospectively with the automated extraction procedure in the whole blood, PBMCs and plasma of 100 samples from patients with EBV-related diseases (group 1, n = 20), HIV-seropositive individuals (group 2, n = 66), and healthy EBV carriers (group 3, n = 14). RESULTS: A good correlation was observed between automated and manual extraction on 104 PBMC specimens (r = 0.956; P < 0.0001). In the prospective study, 67 samples were positive in both whole blood and PBMCs, with a good correlation between EBV DNA loads in whole blood and PBMCs (r = 0.936; P < 0.0001). Only 18/100 samples were positive in plasma. Higher viral loads were regularly observed in the three blood compartments from group 1 than from groups 2 and 3. CONCLUSION: This study demonstrated that an automated extraction of EBV DNA is easier to perform in whole blood or plasma than in PBMCs and facilitates the standardisation of EBV DNA measurement by real-time quantitative PCR. The quantitative detection of EBV DNA load in whole blood appeared more sensitive than in plasma for infectious mononucleosis in immunocompetent patients, probably because of a rapid loss of plasmatic EBV DNA. In transplant patients, EBV DNA load monitoring in whole blood and in plasma turned out to be equivalent in terms of feasibility and accuracy for the early diagnosis of post-transplant lymphoproliferative diseases (PTLDs).     

Quantitation of varicella-zoster virus DNA in whole blood, plasma, and serum by PCR and electrochemiluminescence

We describe a highly sensitive assay for quantitation of varicella-zoster virus (VZV) DNA in blood, involving PCR amplification, solution hybridization with Tris-(2, 2'-bipyridine)-ruthenium(II) chelate-labeled probes, and measurement by electrochemiluminescence (ECL). Extraction and amplification efficiencies were monitored by the inclusion of internal control (IC) DNA, mimicking the VZV target, in the DNA extraction. Viral DNA load was calculated from the ratio of VZV and IC ECL signals. The lower limit of sensitivity was 20 VZV DNA copies/ml of plasma or serum and 80 copies/ml of whole blood. In reconstruction experiments, expected and calculated VZV DNA loads were in excellent accordance. Blood specimens from 42 VZV-infected patients were tested for the presence of VZV DNA and showed detection rates of 86% in patients with varicella and 81% in patients with herpes zoster. In specimens obtained during the first week after onset of the rash, detection rates were 100 and 89%, respectively. Viral DNA was detected in all immunocompromised patients with herpes zoster, emphasizing the risk of disseminated disease in this patient group. VZV DNA load was similar in patients with varicella and multidermatomal herpes zoster and lower in patients with unidermatomal zoster. Despite the cell-associated nature of the virus, VZV DNA was detected in serum and plasma at high copy numbers, and at similar frequencies compared to whole-blood specimens. Quantitation of VZV DNA in blood is of potential importance for diagnosis and clinical management of VZV-infected patients. Plasma and serum provide convenient matrices for this purpose.     

Use of real-time PCR to measure Epstein-Barr virus genomes in whole blood

The measurement of the Epstein–Barr viral load in peripheral blood has been recognised as an important way of monitoring the response to treatment in patients with Epstein–Barr virus (EBV)-related malignancies. In particular, EBV load in transplant recipients can be used as a predictive parameter for Post-transplant Lymphoproliferative Disorder (PTLD). The aim was to develop a rapid and reliable PCR protocol for the quantification of the cell-associated EBV genome. Real-time PCR using TaqMan methodology was established. This technique was applied to determine the EBV load in various study groups including healthy controls, transplant recipients, patients on haemodialysis, and patients with infectious mononucleosis. The baseline level of EBV genomes in the immunosuppressed renal transplant recipients was significantly different from that in the healthy controls.     

Comparison of commercial extraction systems and PCR assays for quantification of Epstein-Barr virus DNA load in whole blood

The automation of DNA extraction and the use of commercial quantitative real-time PCR assays could help obtain more reliable results for the quantification of Epstein-Barr virus DNA loads (EBV VL). This study compared two automated extraction platforms and two commercial PCRs for measurement of EBV VL in 10 EBV specimens from Quality Control for Molecular Diagnostics (QCMD) and in 200 whole-blood (WB) specimens from transplant (n = 137) and nontransplant (n = 63) patients. The WB specimens were extracted using the QIAcube or MagNA Pure instrument; VL were quantified with the EBV R-gene quantification kit (Argene) or the artus EBV RG PCR kit (Qiagen) on the Rotor-Gene 6000 real-time analyzer; and the results were compared with those of a laboratory-developed PCR. DNA was extracted from the QCMD specimens by use of the QIAamp DNA minikit and was quantified by the three PCR assays. The extraction platforms and the PCR assays showed good correlation (R, >0.9; P, <0.0001), but as many as 10% discordant results were observed, mostly for low viral loads (<3 log(10) copies/ml), and standard deviations reached as high as 0.49 log(10) copy/ml. In WB but not in QCMD samples, Argene PCR tended to give higher VL values than artus PCR or the laboratory-developed PCR (mean difference for the 200 WB VL, -0.42 or -0.36, respectively). In conclusion, the two automated extraction platforms and the two PCRs provided reliable and comparable VL results, but differences greater than 0.5 log(10) copy/ml remained between the two commercial PCRs after common DNA extraction.     

Comparison of quantitative competitive PCR with LightCycler-based PCR for measuring Epstein-Barr virus DNA load in clinical specimens

The aim of this study was to develop a LightCycler-based real-time PCR assay for monitoring the Epstein-Barr virus (EBV) DNA load in unfractionated whole blood. This assay was compared with quantitative competitive PCR (Q-PCR) for EBV. The LightCycler-based assay was highly sensitive and reproducible when quantifying plasmid DNA in either the presence or absence of healthy donor blood DNA. Amplifying plasmid DNA in DNA backgrounds from different donors slightly increased the variation of quantification, indicating that clinical specimen DNA has an influence on quantification. In most transplant recipients, a good correlation was observed between EBV DNA load dynamics determined by LightCycler and Q-PCR in follow-up samples, although the correlation between absolute values of EBV DNA loads was weak and occasional samples were false negative in the LightCycler assay. In 253 cross-sectional blood samples from patients with Burkitt's lymphoma, infectious mononucleosis, or human immunodeficiency virus infection, a weak but significant correlation between the two methods was found (r(2) = 0.37, P < 0.001). Our results indicate that the clinical specimen DNA background may influence the absolute values of EBV DNA load in LightCycler analyses but that this effect is rare. LightCycler PCR is very well suited for monitoring of EBV DNA load dynamics, and its diagnostic value is comparable to that of Q-PCR. To avoid false negativity or underestimation of viral load, future internal calibration of the LightCycler is recommended. This would also enhance EBV load assay standardization and interinstitute comparisons.     

Comparison between plasma and whole blood specimens for detection of Aspergillus DNA by PCR

Ninety-six plasma and whole blood specimens from nine selected patients were analyzed for the presence of Aspergillus DNA. Nineteen specimens from three patients with proven aspergillosis were PCR positive in both materials, whereas an additional 22 were PCR positive in whole blood only. All 36 samples from six patients without signs of aspergillosis were negative in both assays. We conclude that although plasma and whole blood spiked with Aspergillus conidia showed an identical lower detection limit (10 CFU), the sensitivity of plasma PCR was lower than that of PCR performed on whole blood samples.     

Use of quantitative competitive PCR to measure Epstein-Barr virus genome load in the peripheral blood of pediatric transplant patients with lymphoproliferative disorders.

A quantitative competitive PCR (QC-PCR) assay for Epstein-Barr virus (EBV) has been developed to provide accurate measurement of EBV genome load in pediatric transplant recipients at risk for developing posttransplant lymphoproliferative disorder (PTLD). The assay quantifies between 8 and 5,000 copies of the EBV genome in 10(5) lymphocytes after a 30-cycle amplification reaction. For 14 pediatric patients diagnosed with PTLD, the median EBV genome load was 4,000, and 13 of the 14 patients had values of >500 copies per 10(5) lymphocytes. Only 3 of 12 control transplant recipients not diagnosed with PTLD had detectable viral genome loads (median value, 40). This median was calculated by using the highest value obtained by PCR testing on each of these patients posttransplantation. PCR values of >500 copies per 10(5) lymphocytes appear to correlate with a diagnosis of PTLD. By a modified protocol, the EBV genome copy number in latently infected adults was estimated to be <0.1 copy per 10(5) lymphocytes.     

Increased susceptibility to HIV-1 of peripheral blood lymphocytes in acute infection with Epstein-Barr virus

Epstein-Barr virus (EBV) is an important pathogen in human immunodeficiency virus (HIV)-infected individuals that causes lymphoma and other lymphoproliferative disorders upon disease progression; however, interaction between the two viruses during acute infection is not well known. Expression of CCR5, a major coreceptor for HIV, was enhanced on CD4+ T cells from patients with acute EBV infection. Furthermore, susceptibility of those cells to R5-HIV-1, but not X4-HIV-1, was increased. EBV effects on CCR5 expression on or susceptibility to R5-HIV-1 of CD4+ T cells did not require coinfection of the same cell with the two viruses, because CD4+ T cells from patients with acute EBV infection were not infected with EBV. Considering that both HIV and EBV are transmitted by intimate contact, such possible interaction between the two viruses may have implications for viral transmission and the pathogenesis of HIV disease.     

Enumeration of immunoglobulin-bearing lymphocytes in whole peripheral blood.

A new method is described for the detection by immunofluorescence of lymphocyte populations in whole peripheral blood. Lymphocytes are identified by their lack of intracellular peroxidase, and the absolute number of different populations can be accurately enumerated by avoiding procedures for isolating mononuclear cells before fluorescent staining. Among twenty-two normal individuals the mean +/- s.d. percentage and absolute number of lymphocytes with stable surface immunoglobulin were as follows: IgM, 8-1+/-2-3%, 142+/-60/mu1; IgD,8-9+/-2-2%, 212+/-85/mu1;IgA, 2-2+/-1-0%, 41+/-24/mu1; IgG, 2-3+/-1-6%, 60+/-47/mu1. It was confirmed that the detection of true IgG-bearing lymphocytes depends on washing the blood at 37degrees before staining or the use of F(ab)2 anti-IgG antibodies. The proportion of lymphocytes stained by whole IgG polyvalent anti-immunoglobulin antibody, which detects both true immunoglobulin-bearing lymphocytes (B cells) and Fc receptor cells was constant (20-5+/-1-0%) both in and between individuals, but the absolute numbers (380+/-117/mu1) varied with the lymphocyte count.     

Comparison of two highly automated DNA extraction systems for quantifying Epstein-Barr virus in whole blood

BACKGROUND: Optimal automated molecular methods are needed to monitor Epstein-Barr virus (EBV) infections in transplant recipients. OBJECTIVES: To compare the extraction of EBV DNA from whole blood using the COBAS Ampliprep and the MagNA Pure instruments (Roche) for quantifying EBV DNA by real-time PCR. STUDY DESIGN: EBV DNA content was determined on clinical samples extracted by both systems. RESULTS: The detection limit was 2.16log(10)copies/mL using the COBAS Ampliprep extraction system. Specificity was 100% and we saw no cross-contamination. Extraction was linear from 2.60 to 5.60log(10)copies/mL. The intra-assay variation was 1.91% for 3.60, 2% for 4.60 and 4.51% for 5.60log(10)copies/mL; inter-assay variation was 4.88%. Sixty-six samples were tested: 26 were positive and 28 were negative by both methods. One sample was MagNA Pure positive/COBAS Ampliprep negative (virus load 3.15log(10)copies/mL) and 10 samples were MagNA Pure negative/COBAS Ampliprep positive (virus loads from 1.59 to 3.51log(10)copies/mL) (P<0.0001). Both methods gave similar quantitative results (average difference 0.07log(10)copies/mL) which were well correlated (r=0.73, P<0.001). CONCLUSIONS: The COBAS Ampliprep extraction system is comparable to the MagNA Pure and offers a high reliability for extracting EBV DNA from whole blood.     

Quantification of Epstein-Barr virus load in peripheral blood of human immunodeficiency virus-infected patients using real-time PCR

Epstein-Barr virus (EBV) reactivation is more likely to occur in immunocompromised patients with subsequent higher susceptibility to EBV-associated lymphoproliferations. In contrast to transplant recipients, limited data are available concerning the EBV load in HIV-infected patients, with or without AIDS-related non-Hodgkin's lymphomas. We developed a TaqMan real-time PCR assay, allowing both the EBV genome and a cellular gene to be quantified in order to obtain a reliable normalized measurement of the EBV load in peripheral blood mononuclear cells (PBMCs). With a wide 6-log(10) quantification range and inter-assay variations of less than 24%, this quantitative PCR was sufficiently accurate and reproducible for routine follow-up. The EBV load was determined in PBMCs from 113 HIV-infected patients, 11 patients with primary HIV infection and 24 HIV-seronegative healthy controls. The rates of EBV detection were similar in the three groups. However, EBV loads were higher in the HIV-infected group (P < 0.00001) except for the patients with primary HIV infection. Unexpectedly, EBV loads were not correlated with the clinical stages of HIV infection or HIV replication, and did not depend on the degree of immunodepression, as judged by CD4+ counts. This study contributes towards the definition of the baseline EBV load during HIV infection and stresses the broad inter-individual variability of the EBV load in HIV-infected patients. Real-time PCR provides a useful tool that can be used in further longitudinal studies to assess the relevance of the EBV load to identify HIV-infected patients with a high risk of EBV-associated lymphoproliferations.     

Toward standardization of Epstein-Barr virus DNA load monitoring: unfractionated whole blood as preferred clinical specimen

Epstein-Barr virus (EBV) DNA load monitoring in peripheral blood has been shown to be a useful tool for the diagnosis of aberrant EBV infections. In the present study we compared the relative diagnostic values of EBV DNA load monitoring in unfractionated whole blood and simultaneously obtained serum or plasma samples from Burkitt's lymphoma (BL) patients, transplant recipients, human immunodeficiency virus (HIV)-infected individuals, and infectious mononucleosis (IM) patients by a quantitative competitive PCR (Q-PCR). The EBV DNA load in BL patients was mainly situated in the cellular blood compartment (up to 4.5 x 10(6) copies/ml). EBV DNA loads in unfractionated whole blood and parallel serum samples showed no correlation. In transplant recipients, IM patients, and HIV-infected patients, the EBV burden in the circulation was almost exclusively restricted to the cellular blood compartment, because serum or plasma samples from these patients yielded negative results by Q-PCR, despite high viral loads in corresponding whole-blood samples. A 10-fold more sensitive but qualitative BamHI-W-repeat PCR occasionally revealed the presence of EBV at <2,000 copies of EBV DNA per ml of serum. Spiking of 100 copies of EBV DNA in samples with negative Q-PCR results excluded the presence of inhibitory factors in serum or plasma that influenced the Q-PCR result. Serum samples from all populations were often positive for beta-globin DNA, indicating cell damage in vivo or during serum preparation. We conclude that serum is an undesirable clinical specimen for EBV DNA load monitoring because it omits the presence of cell-associated virus and uncontrolled cell lysis may give irreproducible results or overestimation of the DNA load. Unfractionated whole blood is strongly preferred since it combines all blood compartments that may harbor EBV and it best reflects the absolute viral burden in the patient's circulation.     

Human antibodies to cedar pollen secreted by human peripheral lymphocytes transformed by Epstein-Barr virus]

To establish immortalized human B-cells secreting antibodies to cedar pollen allergens, peripheral blood lymphocytes from 13 donors were transformed with Epstein-Barr virus. Of 5000 micro culture wells with transformed cell growth, supernatants from 88 wells were found to contain antibodies to pollen allergens. Fourteen supernatants reacted with a cedar allergen Cry j1 and 10 reacted with Cry j2. IgM class antibodies were predominant.     

Epidemiology of Epstein-Barr virus, cytomegalovirus, and Kaposi's sarcoma-associated herpesvirus infections in peripheral blood leukocytes revealed by a multiplex PCR assay

A multiplex polymerase chain reaction (PCR) has been developed for the simultaneous detection of Epstein-Barr virus (EBV), cytomegalovirus (CMV), and Kaposi's sarcoma-associated herpesvirus (KSHV) in a clinical sample. Primers of multiplex PCR were designed to amplify specific regions of the EBV EBNA1, CMV IE2, and KSHV LANA genes. This multiplex PCR assay was found to have detection sensitivities of 1-10 copies of purified viral DNA cloned into the plasmid. To assess diagnostic and pre-clinical applications with this method, we utilized KSHV-positive primary effusion lymphoma (PEL) cells, EBV-positive Burkitt's lymphoma cells, CMV-infected fibroblast cells, and clinically prepared peripheral blood leukocytes (PBLs) that had been infected with viruses. We found that this multiplex PCR assay has high sensitivity and specificity for simultaneous detection of EBV, CMV, and KSHV genomes in a single amplification from a clinical material. Using this multiplex PCR assay, we investigated the prevalence of EBV, CMV, and KSHV in PBL samples from normal Japanese randomly selected. KSHV, EBV, and CMV genomes were detected in samples from 2 (0.2%), 377 (39.5%), and 27 (2.8%) of the 953 blood donors, respectively. Interestingly, both EBV and CMV genomes were detected in samples from all KSHV-positive donors.     

A quantitative ultrastructural study of peripheral blood lymphocytes containing parallel tubular arrays in Epstein-Barr virus and cytomegalovirus mononucleosis.

In the normal peripheral circulation there exists a subpopulation of lymphocytes that is ultrastructurally distinct. This lymphocyte is identified with the electron microscope by the presence of cytoplasmic microtubulelike inclusions called parallel tubular arrays (PTAs) and contains Fc-receptors for cytophilic antibody. In this study, lymphocytes containing PTAs (PTA-lymphocytes) were quantitated from serial peripheral blood specimens obtained from two patients with Epstein-Barr virus (EBV) mononucleosis and two patients with cytomegalovirus (CMV) mononucleosis. These data were then correlated with the clinical state of the patient. It was determined that both the percentage and absolute number of PTA-lymphocytes were highest during the acute phase of the illness. In follow-up specimens, three of the four patients' absolute lymphocyte count fell to within normal limits before the absolute PTA-lymphocyte count. In one patient, the absolute PTA-lymphocyte count was significantly elevated 13 months after the initial clinic visit. Although the PTA-lymphocyte count was highest during the acute phase of the illness, there was no consistent correlation with the clinical state of the patient during follow-up. The estimation of absolute PTA-lymphocyte counts was determined to be valid after a morphometric analysis of the cellular areas occupied by PTAs during the acute and convalescent phases of the disease revealed no statistical differences. Electron microscopy was also performed on the peripheral blood of a patient with syphilis. Although a hematologic workup of this patient during the acute phase of his illness revealed a large number of atypical lymphocytes, electron-microscopic examination of the same specimen revealed both a normal number and a normal percentage of PTA-lymphocytes. The immunologic role of this ultrastructurally distinct third population (non-T, non-B) of lymphocytes, or "killer cells," in the course of infectious mononucleosis is discussed.     

Quantitative real-time PCR detection of adenovirus in clinical blood specimens: A comparison of plasma, whole blood and peripheral blood mononuclear cells

BACKGROUND: Detection and quantification of adenovirus (ADV) in peripheral blood specimens has become an increasingly important tool in the management of immunosuppressed patients. Investigators have described the use of whole blood (WB), peripheral blood mononuclear cells (PBMC), serum and plasma but no studies have compared the utility of these different sample types for use in a clinical diagnostic assay. OBJECTIVES: To determine the optimal blood compartment for quantitative real-time measurement of adenovirus in peripheral blood specimens. STUDY DESIGN: WB, PBMC, and plasma representing 338 samples from 148 patients were tested for ADV by quantitative real-time PCR (qrt-PCR) and the results compared for concordance of both qualitative sensitivity and viral load among positive specimens. RESULTS: There was no significant difference in qualitative sensitivity among the three tested specimen types. Quantitative values of WB and plasma were similar and tended to be greater than those found in PBMC samples. Comparison of consecutive positive samples within individual patients showed that viral loads tracked similarly over time, irrespective of the sample type tested. CONCLUSION: While WB and plasma do not offer a significant increase in sensitivity over PBMC, they may offer benefits in terms of reduced processing costs and laboratory turn around time.     

Quantitation of human immunodeficiency virus type 1 DNA by competitive nested PCR assay

A quantitative competitive nested PCR assay was developed for quantifying HIV-1 proviral DNA in clinical samples. A competitor DNA was constructed from a conserved region of the HIV-1 gag gene by deleting a sequence of 18 base pairs. We quantitated HIV-1 proviral DNA copy number in clinical samples. Peripheral blood mononuclear cells (PBMCs) from 35 HIV-infected patients with a CD4 count range of 4-728 cell/mm3 were analyzed by this method. The copy numbers of HIV-1 DNA detected ranged between 518 to 67,340 copies per 10(6) CD4+ T-cells. The copy numbers correlated inversely with the CD4 counts.     

Stress and the transformation of lymphocytes by Epstein-Barr virus

Although various stressors appear to influence herpesvirus infections, the underlying mechanisms have not been studied. A prospective design was used to examine the effects of examination stress and loneliness on the transformation of B lymphocytes in mixed cultures of T and B lymphocytes by Epstein-Barr virus (EBV). Three blood samples were drawn from 42 EBV-seropositive medical students, with the baseline sample taken 1 month before their final examinations, the stress sample drawn on the first day of final examinations, and the third sample taken the first week after their return from summer vacation. A median split on the UCLA Loneliness Scale divided the subjects into high- and low-scoring loneliness groups. There were significant effects for change over trials, with the lowest transformation levels (i.e., more virus required to transform cells) found in the stress sample. There was also a significant main effect for loneliness, in which high loneliness was associated with lower transformation levels. Possible immunological pathways for the observed changes are discussed.     

HIV-2-infected individuals with undetectable plasma viremia carry replication-competent virus in peripheral blood lymphocytes

Using an optimized HIV co-culture protocol it was possible to isolate infectious HIV-2 variants from 6 HIV-2-infected individuals who had undetectable plasma viremia and maintained high CD4 T-cell numbers for prolonged periods. This shows for the first time that HIV-2-infected individuals with no demonstrable in vivo virus production carry replication-competent virus in peripheral blood mononuclear cells (PBMCs). The frequency of PBMCs with infectious virus was low, ranging from 0.01-0.9 infectious units per million (IUPM) CD4 T cells with a median value of 0.2 IUPM. In comparison, viremic HIV-2-infected individuals had a 2-log higher median infectious load (36 IUPM, range 1-673; P = 0.003). HIV-2 infectious load correlated with CD4 counts (rs = -0.88, P < 0.0001). The low infectious load in aviremic HIV-2-infected persons is reminiscent of what has been observed for HIV-1 infection controlled by highly active antiretroviral therapy.