Comparative Evaluation of Three Automated Systems for DNA Extraction in Conjunction with Three Commercially Available Real-Time PCR Assays for Quantitation of Plasma Cytomegalovirus DNAemia in Allogeneic Stem Cell Transplant Recipients▿

Limited data are available on the performance of different automated extraction platforms and commercially available quantitative real-time PCR (QRT-PCR) methods for the quantitation of cytomegalovirus (CMV) DNA in plasma. We compared the performance characteristics of the Abbott mSample preparation system DNA kit on the m24 SP instrument (Abbott), the High Pure viral nucleic acid kit on the COBAS AmpliPrep system (Roche), and the EZ1 Virus 2.0 kit on the BioRobot EZ1 extraction platform (Qiagen) coupled with the Abbott CMV PCR kit, the LightCycler CMV Quant kit (Roche), and the Q-CMV complete kit (Nanogen), for both plasma specimens from allogeneic stem cell transplant (Allo-SCT) recipients (n = 42) and the OptiQuant CMV DNA panel (AcroMetrix). The EZ1 system displayed the highest extraction efficiency over a wide range of CMV plasma DNA loads, followed by the m24 and the AmpliPrep methods. The Nanogen PCR assay yielded higher mean CMV plasma DNA values than the Abbott and the Roche PCR assays, regardless of the platform used for DNA extraction. Overall, the effects of the extraction method and the QRT-PCR used on CMV plasma DNA load measurements were less pronounced for specimens with high CMV DNA content (>10,000 copies/ml). The performance characteristics of the extraction methods and QRT-PCR assays evaluated herein for clinical samples were extensible at cell-based standards from AcroMetrix. In conclusion, different automated systems are not equally efficient for CMV DNA extraction from plasma specimens, and the plasma CMV DNA loads measured by commercially available QRT-PCRs can differ significantly. The above findings should be taken into consideration for the establishment of cutoff values for the initiation or cessation of preemptive antiviral therapies and for the interpretation of data from clinical studies in the Allo-SCT setting

Relationship between pp65 antigenemia levels and real-time quantitative DNA PCR for Human Cytomegalovirus (HCMV) management in immunocompromised patients

<p>Abstract</p> <p>Background</p> <p>Quantitative real-time PCR assays, which are more rapid and practical than pp65 antigenemia determination, are progressively becoming the preferred method for monitoring Human Cytomegalovirus (HCMV) reactivation. However, the relationship between HCMV DNA and antigenemia levels is still under investigation. The aim of this study was to analyse the relationship between HCMV DNA and pp65 antigenemia levels in order to identify clinically useful threshold values for the management of patients.</p> <p>Methods</p> <p>475 consecutive samples from 156 immunosuppressed patients were tested for HCMV by pp65 antigenemia and Real-time PCR assay.</p> <p>Results </p> <p>136 out of 475 consecutive samples derived from 48 patients showed evidence of HCMV infection. HCMV DNA was detected in 106 samples, pp65 antigen in 3, and both markers in 27. pp65 antigen detection was associated with higher HCMV DNA levels. The cut-off HCMV DNA level that best predicted pp65 antigenemia in this series of samples was 11,500 copies/ml, but different threshold levels could be observed for specific groups of patients. HCMV disease was observed in 5 out of 48 patients with active HCMV infection. The presence of clinical symptoms was associated with positive pp65 and with higher antigenemia levels. Higher HCMV DNA load at the onset of viral replication was correlated to the development of clinical symptoms.</p> <p>Conclusion</p> <p>Both pp65 antigenemia and HCMV DNA load can be useful for the prospective monitoring of immunocompromised subjects. Specific cut-off levels capable of triggering preemptive antiviral treatment should be determined in accordance to the type of test used and the characteristics of patients and prospectively validated.</p

A922 Sequential measurement of 1 hour creatinine clearance (1-CRCL) in critically ill patients at risk of acute kidney injury (AKI)

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The use of a multiplex real-time PCR assay for diagnosing acute respiratory viral infections in children attending an emergency unit

International audienc

Combining thermophilic aerobic reactor (TAR) with mesophilic anaerobic digestion (MAD) improves the degradation of pharmaceutical compounds

International audienceThe removal efficiency of nine pharmaceutical compounds from primary sludge was evaluated in two different operating conditions: (i) in conventional Mesophilic Anaerobic Digestion (MAD) alone and (ii) in a co-treatment process combining Mesophilic Anaerobic Digestion and a Thermophilic Aerobic Reactor (MAD-TAR). The pilot scale reactors were fed with primary sludge obtained after decantation of urban wastewater. Concerning the biodegradation of organic matter, thermophilic aeration increased solubilization and hydrolysis yields of digestion, resulting in a further 26% supplementary removal of chemical oxygen demand (COD) in MAD-TAR process compared to the conventional mesophilic anaerobic digestion. The highest removal rate of target micropollutants were observed for caffeine (CAF) and sulfamethoxazole (SMX) (>89%) with no substantial differences between both processes. Furthermore, MAD-TAR process showed a significant increase of removal efficiency for oxazepam (OXA) (73%), propranolol (PRO) (61%) and ofloxacine (OFL) (41%) and a slight increase for diclofenac (DIC) (4%) and 2 hydroxy-ibuprofen (2OH-IBP) (5%). However, ibuprofen (IBP) and carbamazepine (CBZ) were not degraded during both processes. Anaerobic digestion affected the liquid-solid partition of most target compounds. Sorbed fraction of pharmaceutical compounds on the sludge tend to decrease after digestion, this tendency being more pronounced in the case of the MAD-TAR process due to much lower concentration of solids

Human cytomegalovirus quantification in toddlers saliva from day care centers and emergency unit: A feasibility study.

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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 200,000 PBL, equivalent to the threshold value of 50 pp65-positive polymorphonuclear cells per 200,000 leukocytes, was equivalent to 3.4 log(10) genome copies in 200 μl 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