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.     

Monitoring of human cytomegalovirus infection in immunosuppressed patients using real-time PCR on whole blood.

BACKGROUND: Quantitative monitoring of human cytomegalovirus (HCMV) is currently used in the follow-up of immunosuppressed patients. OBJECTIVE: To investigate whether real-time PCR quantification (QPCR) of HCMV DNA could replace pp65 antigenemia. STUDY DESIGN: We compared HCMV QPCR on whole blood (WB) and on plasma with a pp65-antigenemia assay on 192 samples. Afterwards, we tested 1310 samples from 308 immunosuppressed patients both by antigenemia assay and QPCR on WB. RESULTS: The first study comparison showed that QPCR results on WB and plasma were significantly correlated with antigenemia. QPCR on WB was more sensitive than QPCR on plasma or antigenemia, detecting 31 and 49 additional positive samples, respectively. During the second comparison, QPCR on WB and antigenemia were again correlated (r=0.70; p<0.0001), but QPCR detected 244 additional positive samples. HCMV DNA was detected earlier than pp65 antigen (median difference: 14 days; range: 7-30). One, 5, 10, 50 and 100 pp65-positive cells/200,000 leukocytes corresponded to 439, 1531, 2623, 9150 and 15,671 HCMV DNA copies/mL of WB, respectively, but this equivalence differed according to the sub-group of patients considered. CONCLUSION: QPCR on WB is the most sensitive method for the monitoring of HCMV infection in immunosuppressed patients.     

Prospective evaluation of a new automated nucleic acid extraction system using routine clinical respiratory specimens

The aim of the study was to evaluate the MagNA Pure 96? nucleic acid extraction system using clinical respiratory specimens for identifying viruses by qualitative real‐time PCR assays. Three extraction methods were tested, that is, the MagNA Pure LC?, the COBAS Ampliprep?, and the MagNA Pure 96? with 10‐fold dilutions of an influenza A(H1N1)pdm09 sample. Two hundred thirty‐nine respiratory specimens, 35 throat swabs, 164 nasopharyngeal specimens, and 40 broncho‐alveolar fluids, were extracted with the MagNA Pure 96? and the COBAS Ampliprep? instruments. Forty COBAS Ampliprep? positive samples were also tested. Real‐time PCRs were used to identify influenza A and influenza A(H1N1)pdm09, rhinovirus, enterovirus, adenovirus, varicella zoster virus, cytomegalovirus, and herpes simplex virus. Similar results were obtained on RNA extracted from dilutions of influenza A(H1N1)pdm09 with the three systems: the MagNA Pure LC?, the COBAS Ampliprep?, and the MagNA Pure 96?. Data from clinical respiratory specimens extracted with the MagNA Pure 96? and COBAS Ampliprep? instruments were in 98.5% in agreement (P < 0.0001) for influenza A and influenza A(H1N1)pdm09. Data for rhinovirus were in 97.3% agreement (P < 0.0001) and in 96.8% agreement for enterovirus. They were in 100% agreement for adenovirus. Data for cytomegalovirus and HSV1‐2 were in 95.2% agreement (P < 0.0001). The MagNA Pure 96? instrument is easy‐to‐use, reliable, and has a high throughput for extracting total nucleic acid from respiratory specimens. These extracts are suitable for molecular diagnosis with any type of real‐time PCR assay. J. Med. Virol. 84:906–911, 2012. © 2012 Wiley Periodicals, Inc.     

Typing of human adenoviruses in specimens from immunosuppressed patients by PCR-fragment length analysis and real-time quantitative PCR

Currently, 51 human adenovirus (AdV) serotypes, which are divided into six species (A to F), are known. AdV infections are a major cause of morbidity and mortality in immunosuppressed individuals, particularly in allogeneic stem cell transplant (SCT) recipients. Any AdV species may cause life-threatening disease, but little information is available on the clinical relevance of individual serotypes. The use of serological testing for serotype identification is limited due to the impaired immune response during the posttransplant period. A new molecular approach to serotype identification is presented here that exploits variable regions within the hexon gene. All serotypes belonging to the species A, B, C, E, and F can be determined by fragment length analysis of a single PCR product. For species C, which is the most prevalent in many geographic regions, an alternative technique based on serotype-specific real-time quantitative PCR was established. Of 135 consecutive pediatric patients screened for AdV infections after allogeneic SCT, 40 tested positive. Detailed analysis revealed the presence of 10 different serotypes; serotypes 1 and 2 from species C (C01 and C02) showed the highest prevalence, accounting for 77% of the AdV-positive cases. Representatives of other species were observed less commonly: serotype A12 in 6.5%; serotype A31 in 4.5%; and B03, B16, C05, C06, D19, and F41 in 2%. The approach to rapid molecular serotype analysis presented here provides a basis for detailed studies on adenovirus epidemiology and on the transmission of nosocomial infections. Moreover, in view of the increasing importance of tailored therapy approaches, serotype identification may in the future have implications for the selection of the most appropriate antiviral treatment.     

A multiplex real-time PCR assay for rapid detection and differentiation of 25 bacterial and fungal pathogens from whole blood samples

Early detection of bloodstream infections (BSI) is crucial in the clinical setting. Blood culture remains the gold standard for diagnosing BSI. Molecular diagnostic tools can contribute to a more rapid diagnosis in septic patients. Here, a multiplex real-time PCR-based assay for rapid detection of 25 clinically important pathogens directly from whole blood in <6 h is presented. Minimal analytical sensitivity was determined by hit rate analysis from 20 independent experiments. At a concentration of 3 CFU/ml a hit rate of 50% was obtained for E. aerogenes and 100% for S. marcescens, E. coli, P. mirabilis, P. aeruginosa, and A. fumigatus. The hit rate for C. glabrata was 75% at 30 CFU/ml. Comparing PCR identification results with conventional microbiology for 1,548 clinical isolates yielded an overall specificity of 98.8%. The analytical specificity in 102 healthy blood donors was 100%. Although further evaluation is warranted, our assay holds promise for more rapid pathogen identification in clinical sepsis.     

A novel set of real-time PCRs for rapid differentiation between human cytomegalovirus wild-type and highly passaged laboratory strains

Restricting amplification of human cytomegalovirus (HCMV) DNA to wild-type (WT) HCMV is useful to exclude PCR contaminations by laboratory strains from viral cultures. A set of UL141-specific TaqMan PCRs was developed to amplify (1) WT HCMV and laboratory strain Towne(long), but not strain AD169, (2) only WT HCMV. The performance was compared to a PCR targeting the conserved sequence of HCMV glycoprotein B using 46 serum and urine samples from blood donors with primary CMV infection. Amplification was restricted to the targeted strains with the exception of Towne(long) being amplified also by PCR (2), but at a distinctly lower efficiency than WT HCMV. The coefficient of regression for linear dilutions of two clinical samples with a high concentration of HCMV DNA was 0.999 and 0.997, respectively. The correlation between both WT PCRs and the generic HCMV PCR was good, with coefficients of regression of 0.891 and 0.871 for PCR (1) and (2), respectively. The limit of detection was calculated to be 1.5 genome equivalents per PCR. The set of HCMV TaqMan PCRs enables rapid differentiation between WT and laboratory strains, which can be especially useful as even virus lysate can contaminate sensitive PCRs without prior DNA isolation. A standardized WT HCMV control would be useful to evaluate WT-specific PCR methods.     

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).     

Real-time quantitative PCR assays for detection and monitoring of pathogenic human viruses in immunosuppressed pediatric patients

A panel of 23 real-time PCR assays based on TaqMan technology has been developed for the detection and monitoring of 16 different viruses and virus families including human polyomaviruses BK virus and JC virus, human herpesviruses 6, 7, and 8, human adenoviruses, herpes simplex viruses 1 and 2, varicella-zoster virus, cytomegalovirus, Epstein-Barr virus, parvovirus B19, influenza A and B viruses, parainfluenza viruses 1 to 3, enteroviruses, and respiratory syncytial virus. The test systems presented have a broad dynamic range and display high sensitivity, reproducibility, and specificity. Moreover, the assays allow precise quantification of viral load in a variety of clinical specimens. The ability to use uniform PCR conditions for all assays permits simultaneous processing and detection of many different viruses, thus economizing the diagnostic work. Our observations based on more than 50,000 assays reveal the potential of the real-time PCR tests to facilitate early diagnosis of infection and to monitor the kinetics of viral proliferation and the response to treatment. We demonstrate that, in immunosuppressed patients with invasive virus infections, surveillance by the assays described may permit detection of increasing viral load several days to weeks prior to the onset of clinical symptoms. In virus infections for which specific treatment is available, the quantitative PCR assays presented provide reliable diagnostic tools for timely initiation of appropriate therapy and for rapid assessment of the efficacy of antiviral treatment strategies.     

Rapid quantitation of cytomegalovirus DNA in whole blood by a new molecular assay based on automated sample preparation and real-time PCR

Quantitation of cytomegalovirus (CMV) DNA in whole blood samples has gained significance in the routine diagnostic laboratory. In this study, the analytical performance of the artus™ CMV RG PCR kit in conjunction with automated sample preparation on the new QIAsymphony™ SP instrument was evaluated. Clinically referred samples were tested and results compared to those obtained with the routinely used molecular test system. Accuracy testing showed results to be within ±0.2 log₁₀ unit of the expected panel results. The assay was linear (R = 0.9972) from a lower quantification limit of 148 copies/ml to 1.3 × 10⁷ copies/ml. The between-day imprecision CV was 8–63%, and the within-run imprecision CV was 16–61%. When 100 clinically referred samples were tested, results obtained with the new test system showed an acceptable concordance with those obtained with the routinely used easyMAG™ sample preparation and CMV HHV6,7,8 R-gene™ test system. Discrepant results were found with low-titer samples containing CMV DNA concentrations under the lower limit of quantification or within half a log unit above. The time to results was comparable for both test systems. The QIAsymphony™ sample preparation and artus™ CMV RG PCR test system allows for a rapid, sensitive, precise, and accurate high-throughput quantitation of CMV DNA in whole blood in the routine diagnostic laboratory.     

Strongyloides stercoralis: detection of parasite-derived DNA in serum samples obtained from immunosuppressed patients

Parasitology Research - Strongyloidiasis is an important neglected disease, which is life threatening in immunocompromised patients. This study aimed to evaluate the frequency of Strongyloides...     

A novel real-time PCR system for simultaneous detection of human viruses in clinical samples from patients with uncertain diagnoses

A novel simultaneous detection system for human viruses was developed using a real-time polymerase chain reaction (PCR) system to identify causes of infection in clinical samples from patients with uncertain diagnoses. This system, designated as the "multivirus real-time PCR," has the potential to detect 163 human viruses (47 DNA viruses and 116 RNA viruses) in a 96-well plate simultaneously. The specificity and sensitivity of each probe-primer set were confirmed with cells or tissues infected with specific viruses. The multivirus real-time PCR system showed profiles of virus infection in 20 autopsies of acquired immunodeficiency syndrome patients, and detected frequently TT virus, cytomegalovirus, human herpesvirus 6, and Epstein-Barr virus in various organs; however, RNA viruses were detected rarely except for human immunodeficiency virus-1. Pathology samples from 40 patients with uncertain diagnoses were examined, including cases of encephalitis, hepatitis, and myocarditis. Herpes simplex virus 1, human herpesvirus 6, and parechovirus 3 were identified as causes of diseases in four cases of encephalitis, while no viruses were identified in other cases as causing disease. This multivirus real-time PCR system can be useful for detecting virus in specimens from patients with uncertain diagnoses.     

A new technique for taking blood samples from ducks and geese

A new technique for taking blood samples from ducks and geese is described. The method could possibly be used in other bird species. It presents many advantages for the operator as well as for the birds and it allows repeated blood samples to be taken, if necessary, in large quantities.     

Automated extraction and quantification of human cytomegalovirus DNA in whole blood by real-time PCR assay

The measurement of human cytomegalovirus (HCMV) DNA in blood is becoming the standard method for monitoring HCMV infection in immune-suppressed and unsuppressed patients. As various blood compartments can be used, we have compared the HCMV DNA measured in whole blood (WB), peripheral blood leukocytes (PBL), and plasma by real-time PCR. We tested 286 samples: HCMV DNA was extracted automatically from WB and PBL with the MagNA Pure instrument (Roche Molecular Biochemicals) and manually from plasma samples. The HCMV DNA from WB, PBL, and plasma was measured by real-time Light Cycler PCR. Primers and probe were located in the UL 83 region. HCMV DNA was detected more frequently in WB (88.5%) than in the PBL (65.7%) (P < 0.0001) or the plasma (55.2%) (P < 0.0001). There was a good correlation between the positive results in WB and in PBL (r = 0.68; P < 0.0001), and 3.15 log(10) genome copies in 200000 PBL, equivalent to the threshold value of 50 pp65-positive polymorphonuclear cells per 200000 leukocytes, was equivalent to 3.4 log(10) genome copies in 200 microl of WB. WB was shown to be suitable for automated extraction and the quantitation of HCMV DNA by real-time Light Cycler PCR by analysis of serial samples from representative patients of various populations. This system may be very useful for monitoring of immune-suppressed and unsuppressed patients.     

Clinical evaluation of a new fully automated enzyme immunoassay for basophil histamine release in whole blood

Inflammation Research -     

Automated extraction and quantitation of oncogenic HPV genotypes from cervical samples by a real-time PCR-based system

Accurate laboratory assays for the diagnosis of persistent oncogenic HPV infection are being recognized increasingly as essential for clinical management of women with cervical precancerous lesions. HPV viral load has been suggested to be a surrogate marker of persistent infection. Four independent real-time quantitative TaqMan PCR assays were developed for: HPV-16, -31, -18 and/or -45 and -33 and/or -52, -58, -67. The assays had a wide dynamic range of detection and a high degree of accuracy, repeatability and reproducibility. In order to minimize material and hands-on time, automated nucleic acid extraction was performed using a 96-well plate format integrated into a robotic liquid handler workstation. The performance of the TaqMan assays for HPV identification was assessed by comparing results with those obtained by means of PCR using consensus primers (GP5+/GP6+) and sequencing (296 samples) and INNO-LiPA analysis (31 samples). Good agreement was found generally between results obtained by real-time PCR assays and GP(+)-PCR system (kappa statistic=0.91). In conclusion, this study describes four newly developed real-time PCR assays that provide a reliable and high-throughput method for detection of not only HPV DNA but also HPV activity of the most common oncogenic HPV types in cervical specimens.     

Rapid detection of Staphylococcus aureus bacteremia and methicillin resistance by real-time PCR in whole blood samples

We prospectively evaluated a real-time polymerase chain reaction (PCR) approach for the rapid diagnosis of Staphylococcus aureus bacteremia and presence of the mecA gene in 902 blood samples from 468 infectious episodes of 384 patients. Eight of 12 blood culture (BC)-confirmed samples were positive by the S. aureus-specific PCR. In addition, the mecA gene PCR correctly detected all cases of BC-confirmed methicillin-resistant Staphylococcus aureus (MRSA) infection. A positive PCR result was also obtained in ten of 462 BC-negative infectious episodes, including three patients with culture-confirmed S. aureus infection at other body sites. Juliane Winter and Susanne Gebert contributed equally to this work.