Quantitative real-time PCR with automated sample preparation for diagnosis and monitoring of cytomegalovirus infection in bone marrow transplant patients

BACKGROUND: In bone marrow and stem cell transplant patients, the widespread use of preemptive cytomegalovirus (CMV) antiviral therapy necessitates faster, more precise, and more sensitive quantitative laboratory methods for serial viral load monitoring. METHODS: We developed a novel CMV viral load assay using real-time PCR of plasma DNA prepared by an automated robotic workstation. Fluorescent hybridization probes directed at the glycoprotein B (gB) gene (or EcoRI D region) of CMV were used to detect and quantify PCR products. The beta-globin gene was amplified in parallel to control for the efficiency of the extraction and PCR steps. RESULTS: The assay was linear (R = 0.999) from a lower detection limit of 125 copies/mL to 5 x 10(9) copies/mL with a PCR efficiency of 1.975 (gB) or 2.02 (EcoRI D). The viral loads determined by PCRs directed at these two different viral targets were no different (n = 53; R = 0.928). The interassay CV was 3.5%, and the intraassay CV was 1-4%. Compared with a commercially available quantitative competitive PCR assay (Roche MONITOR; R = 0.59), the mean CMV viral load by real-time PCR was 3.1 times higher (mean ratio; P = 0.002). The diagnostic sensitivity and specificity of the real-time assay were 96% and 100%, respectively (n = 147), compared with 74% and 98% for a qualitative PCR assay (Roche AMPLICOR). On a subset of samples, the diagnostic sensitivity of viral culture was no greater than 50% (n = 44). Of 1115 clinical referral samples from 252 patients, 10% of the samples and 18% of the patients had low-level CMV viremia (median, 500 copies/mL). In this predominantly (85%) bone marrow transplant testing cohort, serial CMV viral load results were the predominant clinical trigger for the initiation, monitoring, and cessation of preemptive antiviral therapy. CONCLUSIONS: The combination of automated DNA preparation and semiautomated real-time fluorescent PCR detection allows for a sensitive, precise, and accurate high-throughput assay of CMV viral load that can be used as the laboratory trigger for preemptive antiviral therapy.     

Evaluation of the Epstein-Barr virus R-gene quantification kit in whole blood with different extraction methods and PCR platforms

Reliable real-time quantitative PCR assays to measure Epstein-Barr virus (EBV) DNA load (EBV) are useful for monitoring EBV-associated diseases. We evaluated a new commercial kit, EBV R-gene Quantification kit (Argene, Varilhes, France) to quantify EBV DNA load in whole blood. Assay performance was assessed with two PCR platforms (LightCycler 2.0 and SmartCycler 2.0) and three commercial DNA extraction methods. The assay was compared with our in-house real-time EBV PCR using samples from the Quality Control for Molecular Diagnostics 2006 EBV proficiency program and using 167 whole-blood specimens from individuals with infectious mononucleosis, from transplanted or HIV-infected patients, and from EBV-seropositive healthy carriers. The EBV R-gene assay was sensitive to 500 copies of EBV DNA per milliliter of whole blood with the two PCR platforms and the three extraction methods and was linear across 4 orders of magnitude. Intra- and interassay coefficients of variations were less than 20%. Nine of 10 samples tested with the EBV R-gene were in agreement with the expected qualitative results of the Quality Control for Molecular Diagnostics 2006 EBV proficiency program, and 7 of 10 samples were within +/-0.5 log units of the expected quantitative values, with discrepant results mostly observed for low viral load (ie, <1000 copies/ml). In the clinical specimens, the correlation between the R-gene assay and the in-house PCR was high (r=0.92). In conclusion, the EBV R-gene assay accurately assesses the EBV DNA load in whole blood of patients with various forms of EBV infections.     

实时定量PCR检测外周血中的巨细胞病毒

目的建立定量检测外周血中巨细胞病毒的实时定量PCR方法,并对骨髓移植病人进行巨细胞感染监测。方法构建基于LightCycler-TaqMan探针的实时定量PCR方法,对其进行方法学评价,并抽提外周血DNA,检测正常人与骨髓移植病人巨细胞病毒感染状况。结果经反应条件的优化,该方法的敏感性在10个拷贝数以上,批内与批间差异分别控制在较小范围,对骨髓移植病人巨细胞病毒感染的监测发现病毒拷贝数与病程进展和恢复呈较好的相关关系。结论实时定量PCR方法检测巨细胞病毒感染可为临床对移植患者的感染提供一种有效的监测手段。     

Absence of activation of CMV by blood transfusion to HIV-infected, CMV-seropositive patients

BACKGROUND: The Viral Activation Transfusion Study (VATS) afforded an opportunity to determine whether blood transfusions, and in particular exogenous WBCs, "activate" CMV replication in HIV-infected, CMV-seropositive patients, and whether such patients can be superinfected by additional strains of CMV transmitted via blood transfusions. STUDY DESIGN AND METHODS: A total of 531 patients were randomized to receive either WBC-reduced (WBCR) or non-WBC-reduced (NWBCR) RBC units. Plasma CMV PCR assays were performed before transfusion and weekly after transfusion for 4 weeks. NWBCR cases with evidence of possible reactivation and/or superinfection were further studied for donor viremia by DNA PCR of frozen retention segments and new genotype acquisition using gB envelope sequence analysis of pre- and posttransfusion recipient specimens. RESULTS: VATS patients received a median of two RBC units during their initial transfusion. Whether positive or negative for CMV DNA at baseline, there were no significant treatment-arm differences in the percentage of patients who had positive qualitative CMV PCR or increases in CMV viral load at follow-up. Of 50 recipients randomized to NWBCR RBC and meeting criteria for possible CMV superinfection, 25 had sufficient CMV DNA load in a baseline and one or more viremic follow-up sample to permit comparison of gB genotypes. Only two recipients showed genotype shifts. Of 125 WBC pellets prepared from the seropositive units transfused into these 50 cases, only 1 tested weakly PCR positive for CMV DNA (insufficient copy number for genotyping). CONCLUSION: There was no evidence of "activation" of CMV by blood transfusion. Among the NWBCR RBC recipients, there was little evidence of possible transmission of new CMV strains. Hence, the current policy for transfusion support of HIV-infected patients, which allows transfusion of CMV-antibody-positive blood to CMV-seropositive patients, is appropriate.     

Comparison of quantitative competitive polymerase chain reaction-enzyme-linked immunosorbent assay with LightCycler-based polymerase chain reaction for measuring cytomegalovirus DNA in patients after hematopoietic stem cell transplantation

Development of highly sensitive quantitative assays for cytomegalovirus (CMV) DNA detection is crucial for identification of immunodeficient patients at high risk of CMV disease. We designed 2 internally controlled competitive quantitative assays, enzyme-linked immunosorbent assay (ELISA)-based and real-time polymerase chain reaction (PCR) tests, using amplification of the same segment of the CMV genome. The aim of this study was to compare sensitivity, specificity, and laboratory performance characteristics of these assays. In both assays, a 159-bp segment of UL83 gene was amplified. External and internal controls were constructed by cloning the amplification product and heterogenous DNA segment flanked by target sequences for CMV-derived primers into bacterial plasmids, respectively. Real-time PCR was performed on LightCycler (Roche Diagnostics, Mannheim, Germany), and amplicons were detected using fluorescence resonance energy transfer probes. Alternatively, PCR products were labeled by digoxigenin, hybridized to immobilized probes, and detected by ELISA. The assays were tested on genomic DNA isolated from laboratory strains of CMV, QCMD control panel, and CMV DNA-positive peripheral blood DNA samples from hematopoietic stem cell transplant recipients, previously characterized by pp65 antigenemia and qualitative nested PCR. Real-time and ELISA-based PCR assays showed a linear course of 1-10(8) and 10-10(5) copies of CMV DNA per reaction, respectively. When compared with ELISA-based PCR, real-time PCR showed superiority in inter- and intra-assay reproducibility. Both assays were highly specific in detecting CMV DNA. No difference in amplification efficiency of internal or external standards and wild-type CMV DNA was found. The assays exhibited 83% concordance in CMV DNA detection from clinical samples, all discrepant samples having low CMV DNA copy numbers. There was a good correlation between viral DNA loads measured by the 2 assays. Statistically significant correlation was observed between the numbers of CMV DNA copies and pp65-positive leukocytes in the samples tested. Both variants of competitive PCR are adequately sensitive to be used for CMV DNA quantitation in clinical samples. LightCycler PCR, having superior performance characteristics and being less time-consuming, seems to be more suitable for routine diagnosis.     

Efficacy of Epstein-Barr virus removal by leukoreduction of red blood cells

BACKGROUND: Epstein-Barr virus (EBV) infection results in life-long carriage of latent virus in B lymphocytes in the majority of the adult population, including blood donors. The removal of EBV from red blood cell (RBC) components by leukoreduction was assessed. STUDY DESIGN AND METHODS: Sixteen randomly selected fresh AS-5 units were leukoreduced by filtration. B lymphocytes from preleukoreduction specimens and mononuclear cells (MNCs) from postleukoreduction specimens were assayed for EBV DNA with sensitive real-time polymerase chain reaction (PCR). RESULTS: EBV genomes were detected in CD19+ B cells in 14 of 16 preleukoreduced RBC units. EBV genomic copy number in the units ranged from 0.18 to 96.84 per 10(5) B lymphocytes representing approximately 135 to 72,630 total EBV genomes per bag. Leukoreduction rendered all but one unit EBV-negative by PCR. The lone PCR-positive unit after leukoreduction amplified 1.2 EBV genome copies from MNCs recovered from the entire unit of leukoreduced RBCs; this unit had the highest EBV viral load before leukoreduction (72,630 EBV genomes). CONCLUSIONS: These results indicate that a 4-log reduction of EBV genomic copy number can be achieved with leukoreduction of RBC units and renders most RBC units EBV-negative by sensitive PCR.     

Quantitative PCR determination of human cytomegalovirus in blood cells

We evaluated a rapid and sensitive method to determine human cytomegalovirus (CMV) DNA levels in blood cells using a quantitative polymerase chain reaction (PCR) technique. This method is based on real-time detection of PCR using a dual fluorescence-labeled probe and a sequence detector. Ten copies of CMV DNA were detected, when 1 microg of DNA from blood samples was used with this method, and a good correlation was obtained between increased concentrations of copy numbers calculated and measured copy numbers of CMV DNA (r = 0.999). Forty normal subjects exhibited no copies of CMV DNA. On the other hand, a 6-month-old girl tested positive for increased levels 4 weeks after liver transplant. This method is simple, accurate, and sensitive for the quantitative detection of CMV DNA in vivo, indicating possible applications for the diagnosis and monitoring of CMV infection.     

Prevalence of Cytomegalovirus DNA in Cord Blood and Voided Urine Obtained from Pregnant Women at the End of Pregnancy

Objective

This study was undertaken to determine the prevalence of congenital cytomegalovirus (CMV) infection in pregnant women at the end of pregnancy in Kuwait using cord blood and maternal urine.

Subjects and Methods

Urine samples were collected prior to childbirth, and cord blood was collected immediately after delivery from 983 women. Anti-CMV IgG and IgM antibodies were determined using ELISA; CMV DNA was detected using nested PCR, and viral load was calculated using real-time PCR. CMV concentration in samples was categorized as low when the viral load ≤10³ copies/µl, intermediate when the viral load = 10³−10⁴ copies/µl, and high when the viral load >10⁴ copies/µl. The cord blood serology outcome was compared to cord blood PCR, cord blood viral load, maternal urine PCR and viral load analyses.

Results

Serology showed that of the 983 cord blood samples, 89 (9%) were positive for anti-CMV IgM antibodies; PCR test showed 44 (4.5%) contained CMV DNA, and there was a high viral load in all. Maternal urine PCR showed that 9 (10.11%) women had CMV DNA, and there was a high viral load in 7 (78%). The kappa test for measures of agreement showed a reasonable agreement (0.45) between cord blood PCR and urine PCR.

Conclusion

This study showed that CMV infection in the cord blood sera of pregnant women is common in Kuwait and highlights the need for more clinically based studies to follow up newborns with congenital CMV infection.Key Words: Active cytomegalovirus infection, Cord blood, Maternal urine, Polymerase chain reaction, Viral load     

Multiplex PCR for detection of Enterobacteriaceae in blood

BACKGROUND: Rapid and sensitive methods are needed to detect the small numbers of bacteria that may sometimes contaminate units of blood during collection. A multiplex 5'-nuclease TaqMan PCR assay (PE Applied Biosystems) was used to detect several bacterial species that may contaminate blood. STUDY DESIGN AND METHODS: Oligonucleotide primers were made for regions of the 16S rRNA gene conserved in four different bacterial species: Yersinia enterocolitica and Serratia, Klebsiella, and Enterobacter species. Two probes were designed: SL-1 detected Serratia, Klebsiella, and Enterobacter species, and YE-3 detected Y. enterocolitica. RESULTS: When TaqMan PCR was performed with chromosomal DNA isolated from pure cultures of Serratia liquefaciens, Klebsiella oxytoca, Klebsiella pneumoniae, Enterobacter cloacae, and Enterobacter agglomerans, the limit of detection with probe SL-1 was 1 to 2 CFUs. For S. marcescens, the sensitivity was 8 CFUs. The limit of detection for Y. enterocolitica with probe YE-3 was 2 CFUs. When total chromosomal DNA was extracted from whole-blood samples spiked with different numbers of Y. enterocolitica, S. liquefaciens, E. cloacae, or K. pneumoniae bacteria, the TaqMan PCR detected 12 to 16 organisms in 1 mL of blood. CONCLUSION: The 5'-nuclease TaqMan PCR assay takes only 3 hours to perform and has the potential to detect very small numbers of bacteria.     

Evaluation of HTLV-I removal by filtration of blood cell components in a routine setting

BACKGROUND: WBC depletion by filtration may prevent the transmission of HTLV-I, which requires cell-to-cell contact. The removal of HTLV-I-infected cells in routinely filtered blood cell components was measured. STUDY DESIGN AND METHODS: The study was conducted in Martinique where systematic screening for HTLV-I and -II and universal leukoreduction are mandatory. HTLV-I was quantified by use of real-time PCR in 8 RBC units and 4 PLT concentrates before and after filtration. HTLV-I proviral load in PBMNCs was determined in five of the eight HTLV-I-infected blood donors. RESULTS: The amount of MNC-associated HTLV-I DNA in RBC units before filtration was 21 x 10(6)+/- 29 x 10(6) copies (mean +/- SD). HTLV-I was detected in 4 of 8 RBC units after filtration, with a number of copies in the MNC fraction ranging from 20 to 140, following a 4.9 to 5.8 log reduction. Flow cytometry analysis performed in 2 of the filtered RBC units containing detectable HTLV-I showed suboptimal and out-of-range leukoreduction (0.56 x 10(6) and 1.22 x 10(6) residual WBCs). HTLV was not detected in filtered RBCs from the blood donor with the highest percentage of HTLV-I-infected PBMCs (9%). CONCLUSION: This study confirms that HTLV-I-infected cells can be detected in filtered blood cell components and shows that optimal leukoreduction is critical for HTLV-I removal.     

Frequency and duration of plasma CMV viremia in seroconverting blood donors and recipients

BACKGROUND: Both CMV-seronegative blood and unscreened, filtered blood carry a low but definite risk of transmitting CMV infection. To explain this residual risk, evidence of cell-free viremia was sought in seroconverting and seroprevalent blood donors and seroconverting transfusion recipients by means of a plasma-based assay for CMV DNA. STUDY DESIGN AND METHODS: A CMV DNA PCR assay (COBAS Amplicor CMV Monitor, Roche) was used to detect CMV DNA in 384 paired plasma samples from 192 donors who seroconverted to anti-CMV, 488 anti-CMV EIA-positive samples from 60 seroprevalent donors, and 113 serial samples from 11 seroconverting recipients with posttransfusion CMV hepatitis. RESULTS: Three of 384 samples from 192 seroconverting donors had low levels of plasma CMV DNA (400-1600 copies/mL); one donor was positive before seroconversion, and the other two, after seroconversion. None of the 488 serial samples from 60 anti-CMV- positive donors contained CMV DNA in plasma. Three of 11 recipients demonstrated transient plasma viremia that temporally coincided with seroconversion. CONCLUSIONS: Plasma CMV DNA was detected in a small percentage of seroconverting blood donors and a larger percentage of recipients but was undetectable in seroprevalent donors. Plasma viremia in seroconverting donors may partially explain the low residual risk of CMV transmission by both CMV-seronegative and WBC-reduced seropositive blood.     

Multicenter evaluation of PCR methods for detecting CMV DNA in blood donors

BACKGROUND: CMV DNA screening may be a useful adjunct to serologic tests in distinguishing potentially infectious blood donations from those that are "CMV-safe." However, there is currently no consensus on the optimal assay method for accurate detection of CMV DNA in donors. STUDY DESIGN AND METHODS: A blinded multicenter evaluation of seven CMV PCR assays was performed by five laboratories by using coded sets of analytical controls and donor blood samples. RESULTS: Five assays displayed sufficient sensitivity for donor screening, as judged by consistent detection of a minimum of 25 CMV genome equivalents (geq) in analytical controls constructed to contain from 1 to 100 CMV geq in background DNA from 250,000 cells, while the other two assays displayed inadequate sensitivity. Three sensitive assays, two based on nested PCR directed at the UL93 and UL32 regions of the CMV genome and another test (Monitor Assay, Roche), did not detect CMV DNA in samples from any of 20 pedigreed CMV-seronegative, Western blot-negative (S-/WB-) donors. Two other assays based on nested PCR occasionally detected CMV DNA in S-WB- samples, and one sensitive nested PCR assay directed at UL123 detected CMV DNA in a large proportion (85%) of S-WB- samples. CONCLUSION: Seven CMV PCR assays currently used for research and/or diagnostic applications displayed marked variations in sensitivity, specificity, and reproducibility when applied to coded analytical and clinical control samples containing cellular DNA from the equivalent of 250,000 WBCs. These results will be useful in the selection of assays with performance characteristics appropriate to donor screening objectives. They may also help explain discrepant findings from previous studies that used PCR to determine CMV DNA prevalence in seronegative and seropositive blood donors.     

荧光定量PCR检测传染性单核细胞增多症不同血样本EBV的研究

[目的]寻找荧光定量PCR检测传染性单核细胞增多症（IM）EBV DNA拷贝量的最佳样本。[方法]采用荧光定量PCR分别检测了IM病人的外周血单个核细胞、全血基因组DNA及血浆的EBV DNA拷贝量,并比较各种方法的阳性率及拷贝量。[结果]三种不同来源的样品荧光定量PCR方法的阳性率分别为76％、69％、23％。全基因组及单个核细胞组阳性率比较经x^2检验,差异无显著性,且两组拷贝量比较差异亦无显著性。血浆组阳性率及拷贝量与前两种方法比较差异均有显著性。[结论]外周血单个核细胞是荧光定量PCR检测IM的EBV DNA量的最佳样本。     

Early detection of plasma cytomegalovirus DNA by real-time PCR after allogeneic hematopoietic stem cell transplantation

Cytomegalovirus (CMV) infection is an important cause of morbidity and mortality after allogeneic hematopoietic stem cell transplantation. Therefore, preemptive ganciclovir therapy based on early detection of CMV reactivation is widely used to prevent CMV disease. Real-time polymerase chain reaction (PCR) has been widely used for monitoring CMV reactivation as well as the antigenemia assay that detects CMV structural phosphoprotein with a molecular weight of 65,000 (pp65). We developed a real-time PCR assay system for CMV based on a double-stranded DNA-specific dye, SYBR Green I, and quantified DNA, which was extracted automatically from plasma. This real-time PCR assay and the pp65 antigenemia assay were compared in parallel with 357 blood samples obtained from 64 patients who underwent allogeneic hematopoietic stem cell transplantation (allo-HSCT). Real-time PCR assay results correlated with those of the pp65 antigenemia assay (p < 0.0001). It is noteworthy that the detection of CMV DNA by PCR preceded the first positive antigenemia by 14 days. In this study, 10 of 64 patients developed CMV disease. The antigenemia assay detected CMV reactivation earlier than the development of CMV disease only in four of 10 patients. In contrast, our real-time PCR detected CMV-DNA before the development of CMV diseases in eight of 10 patients. The real-time PCR with SYBR Green I as a detection signal is simple and readily performed, and may be a useful system for early detection of CMV reactivation after allo-HSCT.     

造血干细胞移植患者人巨细胞病毒监测的临床意义

目的探讨荧光定量聚合酶链反应对造血干细胞移植患者人巨细胞病毒活动性感染的早期诊断价值。方法收集40例造血干细胞移植患者共416份血标本,应用实时荧光定量PCR方法动态监测血浆HCMV—DNA载量,并与60例健康体检者血浆HCMV—DNA载量对比分析。结果416份血标本中96份阳性,阳性率23．1％,病毒载量介于5．0x10^2-1．0x10,拷贝／ml之间。结论实时荧光定量PCR方法检测HCMV—DNA适用于造血干细胞移植后巨细胞病毒活动性感染的早期诊断。     

Evaluation of a real-time fluorescent PCR assay for rapid detection of Group B Streptococci in neonatal blood

Streptococcus agalactiae (Group B Streptococcus: GBS) is the major causative agent of neonatal sepsis. Neonates at risk for GBS infections are empirically administered broad-spectrum antibiotics for at least 48 h pending blood culture results. A rapid assay to expedite detection of GBS would facilitate initiation of specific antibiotic therapy. Conversely, expeditious proof of absence of infection will avoid unnecessary antibiotic use. Using the LightCycler, we evaluated a hybridization probe polymerase chain reaction (PCR) assay to detect GBS-specific cfb gene target DNA sequence in blood specimens. Both sensitivity and specificity of the real-time PCR assay was 100%. The assay demonstrated 100% specificity when tested against 26 non-GBS bacteria. This method is capable of detecting as few as approximately 100 copies or 10 pg of GBS genomic DNA. This real-time PCR method is rapid, sensitive, and specific for the detection of GBS in neonatal blood samples and holds great promise in its utility in the diagnostic laboratory.     

Failure to detect human cytomegalovirus DNA in peripheral blood leukocytes of healthy blood donors by the polymerase chain reaction

The transmission of cytomegalovirus (CMV) by blood transfusion may have a major effect on certain immunocompromised patients. To protect susceptible blood recipients from infection, it is advisable to use blood components from CMV-seronegative donors. However, serologic tests are not capable of indicating which blood component actually harbors infectious virus and can transfer it to the recipient. Therefore, a sensitive method is needed for the detection of the virus itself. There have been three reports on the detection of CMV in healthy volunteer blood donors by the polymerase chain reaction (PCR). CMV DNA was found in all seropositive and most seronegative blood donors. However, many other authors have failed to confirm these data. A highly sensitive and specific PCR assay was developed for the detection of CMV DNA in peripheral blood leukocytes. With this protocol, blood samples from 116 volunteer blood donors were investigated. None of these samples proved to be positive for CMV DNA. In contrast, CMV DNA was detected in 10 of 10 renal transplant patients early in the course of active CMV infection. It can be concluded that the CMV genome copy number in the peripheral blood leukocytes of healthy individuals is beyond the detection limit of current PCR technology.     

实时荧光定量PCR检测血中曲霉菌基因的实验研究

目的：建立并评价从血标本中检测曲霉菌基因的real-time PCR方法。方法：用自行设计的高效率引物探针对标准菌株及临床疑似病人血标本进行检测,对方法的敏感性、特异性进行评价。结果检测出曲霉菌基因敏感性达10拷贝ITS I基因,相当于5～10CFU／ml,与人类基因组、其他真菌及细菌无交叉反应。29例临床疑似标本阳性率为69％,与临床诊断符合率为86％。结论：real-time PCR检测血中曲霉菌基因有较高的敏感性、特异性,有一定的临床应用前景。     

CMV DNA is rarely detected in healthy blood donors using validated PCR assays

BACKGROUND: Although serologic screening or WBC reduction of blood components can reduce the incidence of transfusion-transmitted CMV (TT-CMV) infection, 'breakthrough' cases of TT-CMV still occur and may produce serious sequelae. NAT of blood components for CMV DNA has been proposed to further reduce the risks of TT-CMV. However, large-scale studies to determine the utility of validated CMV NAT assays for donor screening have not been reported. STUDY DESIGN AND METHODS: Coded whole-blood samples (n=1000) were tested for the presence of CMV DNA using two CMV PCR assays previously validated in a multicenter trial (a nested PCR assay directed at the CMV UL93 open-reading frame and the Roche Monitor assay). Corresponding plasma samples were tested in parallel for the presence of anti-CMV using other assays (Abbott CMV EIA and Fujirebio/Olympus CMV particle agglutination assays). RESULTS: In total 416 and 514 of the samples tested as CMV-seropositive and -seronegative, respectively, by both antibody assays. The remaining 70 samples had discrepant serology results. Only 2 of the 1000 samples (both seropositive) had reproducibly detectable CMV DNA (positive in at least three of four replicates). CMV DNA was not reproducibly detected in seronegative samples or in samples with discrepant serology results. CONCLUSIONS: Although previous investigations showed frequent detection of CMV DNA in healthy CMV-seropositive (and some seronegative) blood donors, these studies were relatively small and the performance characteristics of their assays were difficult to evaluate. In contrast, the present large cross-sectional study of US donors utilized two previously validated PCR assays and demonstrated that CMV DNA is only rarely detectable in seropositive donors. Thus, the use of CMV PCR assays with optimal performance characteristics did not increase the detection of potentially infectious blood components beyond that provided by current serologic screening assays.     

Comparison of QIAsymphony automated and QIAamp manual DNA extraction systems for measuring Epstein-Barr virus DNA load in whole blood using real-time PCR

Automated and manual extraction systems have been used with real-time PCR for quantification of Epstein-Barr virus [human herpesvirus 4 (HHV-4)] DNA in whole blood, but few studies have evaluated relative performances. In the present study, the automated QIAsymphony and manual QIAamp extraction systems (Qiagen, Valencia, CA) were assessed using paired aliquots derived from clinical whole-blood specimens and an in-house, real-time PCR assay. The detection limits using the QIAsymphony and QIAamp systems were similar (270 and 560 copies/mL, respectively). For samples estimated as having ≥10,000 copies/mL, the intrarun and interrun variations were significantly lower using QIAsymphony (10.0% and 6.8%, respectively), compared with QIAamp (18.6% and 15.2%, respectively); for samples having ≤1000 copies/mL, the two variations ranged from 27.9% to 43.9% and were not significantly different between the two systems. Among 68 paired clinical samples, 48 pairs yielded viral loads ≥1000 copies/mL under both extraction systems. Although the logarithmic linear correlation from these positive samples was high (r(2) = 0.957), the values obtained using QIAsymphony were on average 0.2 log copies/mL higher than those obtained using QIAamp. Thus, the QIAsymphony and QIAamp systems provide similar EBV DNA load values in whole blood.