Minimal effect of delayed sample processing on results of quantitative PCR for cytomegalovirus DNA in leukocytes compared to results of an antigenemia assay.

Quantitative cytomegalovirus antigenemia and DNAemia were determined in peripheral leukocytes of 25 patients stored for up to 72 h at room temperature (RT) and 4 degrees C before processing. Numbers of antigen-positive cells significantly decreased with time. The decline was greater at RT than at 4 degrees C. In contrast, no significant alterations in DNAemia occurred.     

Real-time PCR quantitation of hepatitis B virus DNA using automated sample preparation and murine cytomegalovirus internal control

Quantitation of circulating hepatitis B virus (HBV) DNA is important for monitoring disease progression and for assessing the response to antiviral therapy. Several commercial and 'in house' assays for HBV DNA quantitation have been described but many of these have limitations of relatively low sensitivity and limited dynamic range. This study describes the development and evaluation of a FRET-based real-time PCR assay designed to overcome these limitations and to provide accurate quantitation of DNA from all eight genotypes of HBV (A-H). The assay employs a fully automated nucleic acid extraction system permitting high-sample throughput with minimal 'hands-on' time and incorporates a murine cytomegalovirus (mCMV) internal control to prevent false negative results and under-reporting due to unrecognised problems with viral lysis, DNA purification or PCR amplification. Sensitivity, assessed by Probit analysis at the 95% detection level, was 24.4 IU/ml, associated with an extremely wide dynamic range (approximately 9 log10). Coefficients of variation were low for both intra-assay and inter-assay variability (CV%, 7-11%) and quantitative data correlated well (R2 = 0.97) with the Digene hybrid capture assay. This assay provides an ideal system for therapeutic monitoring and for studying the relationship between HBV viral load and stage of disease.     

Comparison of quantitative and qualitative PCR assays for cytomegalovirus DNA in plasma

We analyzed the performance characteristics of the qualitative AMPLICOR CMV Test (Roche Molecular Systems, Pleasanton, Calif.) and quantitative COBAS AMPLICOR CMV MONITOR Test (Roche Molecular Systems) assays and compared the performance of the AMPLICOR quantitative assay with an in-house-developed cytomegalovirus (CMV) DNA PCR assay. The quantitative AMPLICOR assay was found to be more sensitive than the qualitative AMPLICOR assay. The quantitative AMPLICOR assay has a lower limit of sensitivity of 400 CMV DNA copies/ml of plasma and is linear to 50,000 CMV DNA copies/ml of plasma. Compared to the in-house PCR assay, the AMPLICOR quantitative assay gave lower viral load values at all concentrations tested, but the difference between the two assays was not consistent across the entire dynamic range of the AMPLICOR quantitative assay. At the lower end of the assay, the viral load values obtained with the in-house PCR assay were three- to fivefold (0.5 to 0.7 log units) higher than those measured with the AMPLICOR assay. At higher input concentrations, the differences between the two assays approached 10-fold. This direct comparison of the in-house assay and the quantitative AMPLICOR assay provides the ability to compare previously published in-house data with an assay widely available for future research and clinical monitoring of patients with CMV infections.     

Optimization of quantitative detection of cytomegalovirus DNA in plasma by real-time PCR

Previous studies have shown that detection of cytomegalovirus (CMV) DNA in plasma is less sensitive than the antigenemia assay for CMV surveillance in blood. In 1,983 blood samples, plasma PCR assays with three different primer sets (UL125 alone, UL126 alone, and UL55/UL123-exon 4) were compared to the pp65 antigenemia assay and blood cultures. Plasma PCR detected CMV more frequently in blood specimens than either the antigenemia assay or cultures, but of the three PCR assays, the double-primer assay (UL55/UL123-exon 4) performed best with regard to sensitivity, specificity, and predictive values compared to antigenemia: 122 of 151 antigenemia-positive samples were detected (sensitivity, 80.1%), and there were 122 samples that were PCR positive-antigenemia negative (specificity, 93%). Samples with discrepant results had a low viral load (median, 0.5 cells per slide; 1,150 copies per ml) and were often obtained from patients receiving antiviral therapy. CMV could be detected by other methods in 15 of 29 antigenemia positive-PCR negative samples compared to 121 of 122 PCR positive-antigenemia negative samples (P < 0.001). On a per-subject basis, 21 of 25 patients (antigenemia positive-PCR negative) and all 57 (PCR positive-antigenemia negative) could be confirmed at different time points during follow-up. The higher sensitivity of the double-primer assay resulted in earlier detection compared to antigenemia in a time-to-event analysis of 42 CMV-seropositive stem cell transplant recipients, and two of three patients with CMV disease who were antigenemia negative were detected by plasma PCR prior to the onset of disease. Interassay variability was low, and the dynamic range was >5 log(10). Automated DNA extraction resulted in high reproducibility, accurate CMV quantitation (R = 0.87, P < 0.001), improved sensitivity, and increased speed of sample processing. Thus, primer optimization and improved DNA extraction techniques resulted in a plasma-based PCR assay that is significantly more sensitive than pp65 antigenemia and blood cultures for detection of CMV in blood specimens.     

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.     

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.     

Rapid quantification of hepatitis B virus DNA by automated sample preparation and real-time PCR

Monitoring of hepatitis B virus (HBV) DNA in serum by molecular methods has become the standard for assessment of the replicative activity of HBV. Several molecular assays for the detection and quantification of HBV DNA have been described. However, they usually lack automated sample preparation. Moreover, those assays, which are based on PCR, are limited by a short dynamic range (2 to 3 log units). In the present study, the use of RealArt HBV LC PCR Reagents in conjunction with automated extraction on the COBAS AMPLIPREP analyzer was evaluated. Members of an HBV proficiency program panel were tested; linearity, interassay, and intra-assay variations were determined. The performance of the assay in a routine clinical laboratory was evaluated with a total of 117 clinical specimens. When members of the HBV proficiency program panel were tested by the new molecular assay, the results were found to be within +/-0.5 log unit of the results obtained by reference laboratories. Determination of linearity resulted in a quasilinear curve over more than 6 log units. The interassay variation of the RealArt HBV LC PCR Reagents by use of the automated sample preparation protocol ranged from 16 to 73%, and the intra-assay variation ranged from 9 to 40%. When clinical samples were tested by the new assay with the automated sample preparation protocol and the results were compared with those obtained by the COBAS AMPLICOR HBV MONITOR Test with manual sample preparation, the results for 76% of all samples with positive results by both tests were found to be within +/-0.5 log unit and the results for another 18% were found to be within between 0.5 and 1.0 log unit. In conclusion, the real-time PCR assay with automated sample preparation proved to be suitable for the routine molecular laboratory and required less hands-on time.     

Clinical evaluation of PCR method for detection of cytomegalovirus DNA

Polymerase chain reaction (PCR) to detect Cytomegalovirus (CMV)-DNA from the clinical specimens is useful to diagnose CMV infection. Eighty-one specimens of 31 patients including peripheral blood, bronchioalveolar lavage fluid, biopsy tissues, feces, urine, sputum and etc. and normal peripheral blood from 59 volunteers were used in this study. After DNA extraction each samples was amplified by the seminested PCR using primers recognizing sequences in the Immediate-early gene of CMV. This PCR method specifically detected more than 10 virus copies even in the presence of the genomic DNA. CMV-DNA was detected in only one of 59 normal peripheral bloods (1.7%). Six of 31 patients were clinically diagnosed as CMV infection by anti-CMV therapy. These 6 patients were positive in the peripheral blood by PCR for CMV, and 5 of them were positive in other samples. However, 3, 5 and 1 of 25 patients, who were clinically diagnosed as not having CMV infection, were also positive in peripheral blood, in the other samples and in both, respectively. The PCR method was able to examine any clinical samples. To examine both the peripheral blood and the samples from infected organs is helpful for the diagnosis of CMV infection.     

False-positive Chlamydiazyme results during urine sediment analysis due to bacterial urinary tract infections.

Our study examined whether urinary tract infections (UTIs) would cause false-positive results when urine sediment was tested with the Chlamydiazyme (CZ) system. Thirty-six infected urine samples and fifteen controls were studied. All controls were negative. Forty-seven percent of Escherichia coli UTIs (n = 30) and 100% of Klebsiella pneumoniae UTIs (n = 4) were positive on CZ testing of urine sediment. Nine E. coli UTIs positive by CZ were negative by direct fluorescent-antibody staining. When suspensions of the pure cultures were analyzed, 47% of E. coli and 100% of K. pneumoniae samples were CZ positive. False-positive results were not related to organism biotype or urine characteristics, including pH, specific gravity, and leukocyte count. We conclude that the presence of a UTI and also bacterial contamination must be ruled out prior to urine sediment testing.     

Rapid diagnosis of extrapulmonary tuberculosis by PCR: impact of sample preparation and DNA extraction

In cases of suspected extrapulmonary tuberculosis, rapid and accurate laboratory diagnosis is of prime importance, since traditional techniques of detecting acid-fast bacilli have limitations. The major difficulty with mycobacteria is achieving optimal cell lysis. Buffers used in commercial kits do not allow this complete lysis in a number of clinical specimens. A comparison of two sample preparation methods, pretreatment with proteinase K (PK-Roche) and complete DNA purification (cetyltrimethylammonium bromide [CTAB]-Roche), was conducted on 144 extrapulmonary specimens collected from 120 patients to evaluate the impact on the Cobas-Amplicor method. Thirty patients were diagnosed with tuberculosis, with 15 patients culture positive for Mycobacterium tuberculosis. Amplification and detection of the amplicons were impaired by a high number of inhibitory specimens (39 to 52%). CTAB-Roche allowed the detection of more culture-positive specimens by PCR than PK-Roche. Comparison with the final diagnoses of tuberculosis confirmed that CTAB-Roche produced the best sensitivity (53.8%) compared to culture (43.3%), PK-Roche (16%), and smear (13%). However, the specificity of the PCR assay with CTAB-Roche-extracted material was always lower (78.8%) than those with culture (100%) and PK-Roche (96.5%). False-positive specimens were lung biopsy material, lymph node biopsy material and aspirate, or bone marrow aspirate, mainly from immunocompromised patients. Despite the efficiency of complete DNA extraction for the rapid diagnosis by PCR of extrapulmonary tuberculosis, the false-positive results challenge our understanding of PCR results.     

Quantification of human cytomegalovirus DNA by real-time PCR

A quantitative real-time PCR assay was developed to measure human cytomegalovirus (HCMV) DNA load in peripheral blood leukocytes (PBLs). The HCMV DNA load in PBLs was normalized by means of the quantification of a cellular gene (albumin). The results of the real-time PCR assay correlated with those of the HCMV pp65-antigenemia assay (P < 0.0001).     

Rapid and cost-effective baculovirus sample preparation method as a viable alternative to conventional preparation for quantitative real-time PCR

The increasing use of the baculovirus expression vector system (BEVS) has generated significant interest into techniques for quantifying baculovirus stocks. One method involves the use of quantitative real-time polymerase chain reaction (PCR). This study investigated simplifying baculovirus sample preparation for quantitative Real Time PCR to provide an alternative to current kit-based preparation methods. To achieve this goal, combinations of freeze/thaw cycles and Triton X-100 treatment were investigated. A treatment with only Triton X-100 was found to be sufficient to provide a simple, rapid and cheap alternative to kit-based preparation methods. This study also examined other factors such as primer choice to further examine the process of baculovirus quantitation by qPCR.     

Identification of cytomegalovirus in a liquid-based gynecologic sample using morphology, immunohistochemistry, and DNA real-time PCR detection

The viral cytopathic effects of cytomegalovirus (CMV) are rarely encountered in conventional cervical vaginal smears and have never been reported in a liquid-based gynecologic sample (SurePath). We present results of a liquid-based gynecologic sample (SurePath) from an asymptomatic patient with classic CMV-associated granular or dense cyanophilic intracytoplasmic and intranuclear inclusion bodies with a clear surrounding zone. These inclusions were immunohistochemically positive for CMV. The patient also had human papilloma virus (HPV)-associated koilocytes that showed a unique perinuclear immunostaining pattern suggesting coinfection with both CMV and HPV. CMV amplification using real-time polymerase chain reaction (PCR) of DNA extracted from the liquid-based sample confirmed the morphologic and immunohistochemical findings of CMV infection. These observations suggest that a liquid-based preparation can be used to assess CMV infection morphologically, immunohistochemically, and by real-time PCR.     

PCR detection of cytomegalovirus DNA in serum as a diagnostic test for congenital cytomegalovirus infection.

PCR detected cytomegalovirus (CMV) DNA in the serum of 18 of 18 infants with symptomatic congenital CMV infection, 1 of 2 infants with asymptomatic congenital CMV infection, and 0 of 32 controls. Serum CMV PCR provided a rapid, sensitive, and specific method for diagnosis of congenital CMV infection in infants who were symptomatic at birth.     

Detection of cytomegalovirus (CMV) DNA in EDTA whole-blood samples: evaluation of the quantitative artus CMV LightCycler PCR kit in conjunction with automated sample preparation

Whole blood has been found to be a reliable matrix for the detection and quantitation of cytomegalovirus (CMV) DNA. In this study, the performance of the artus CMV LightCycler (LC) PCR kit in conjunction with automated sample preparation on a BioRobot EZ1 workstation was evaluated. The accuracy, linearity, analytical sensitivity, and inter- and intra-assay variations were determined. A total of 102 clinical EDTA whole-blood samples were investigated, and results were compared with those obtained with the in vitro diagnostics (IVD)/Conformité Européene (CE)-labeled CMV HHV6,7,8 R-gene quantification kit. When the accuracy of the new kit was tested, seven of eight results were found to be within +/-0.5 log(10) unit of the expected panel results. Determination of linearity resulted in a quasilinear curve over more than 5 log units. The lower limit of detection of the assay was determined to be 139 copies/ml in EDTA whole blood. The interassay variation ranged from 15 to 58%, and the intra-assay variation ranged from 7 to 35%. When clinical samples were tested and the results were compared with those of the routinely used IVD/CE-labeled assay, 53 samples tested positive and 13 samples tested negative by both of the assays. One sample was found to be positive with the artus CMV LC PCR kit only, and 35 samples tested positive with the routinely used assay only. The majority of discrepant results were found with low-titer samples. In conclusion, use of the artus CMV LC PCR kit in conjunction with automated sample preparation on the BioRobot EZ1 workstation may be suitable for the detection and quantitation of CMV DNA in EDTA whole blood in the routine low-throughput laboratory; however, low-positive results may be missed by this assay.     

Umbilical cord blood screening for cytomegalovirus DNA by quantitative PCR.

BACKGROUND: Cytomegalovirus (CMV) infection, which is the most common congenitally transmitted infection, affects approximately 1% of neonates worldwide. Despite its prevalence, no convenient screening test for neonatal CMV infection has been implemented. OBJECTIVE: The purpose of this pilot study was to evaluate the feasibility and yield of screening umbilical cord blood for CMV DNA emiaby quantitative PCR. STUDY DESIGN: Umbilical cord blood was tested for CMV DNAemia using a commercial quantitative PCR assay. Maternal CMV serostatus at the time of delivery was assessed by testing for CMV IgG and IgM antibodies in serum. CONCLUSIONS: Screening for congenital CMV infection with PCR is easily incorporated into routine labor and delivery care using discarded cord blood specimens to identify neonates whose infection is otherwise undiagnosed. Among 433 infants tested, two (0.5%) had DNAemia detected in cord blood, one of whom was symptomatic, and both of whose mothers were CMV IgG positive and IgM negative. Viremic neonates identified by screening with PCR may be at high risk of developing long-term neurological complications of CMV infection and cannot reliably be identified using clinical presentation or maternal serology. Because of its convenience, cord blood CMV screening with PCR should be further investigated for incorporation into neonatal screening protocols.     

False-positive culture results from patient tissue specimens due to contamination of RPMI medium with Cryptococcus albidus

Cryptococcus albidus, a rare opportunist, was isolated from biopsy specimens from three patients over 4 days. An investigation showed that the specimens had been contaminated by placement in RPMI medium. The importance of rapid communication between the microbiology laboratory, the infectious diseases/infection control division, and other involved parties in the event of unusual occurrences is highlighted.     

Detection of human cytomegalovirus DNA by real-time quantitative PCR

A real-time PCR assay was developed to quantify human cytomegalovirus (CMV) DNA. This assay was used to demonstrate a higher CMV DNA load in plasma of bone marrow transplant patients than in that of blood donors. The CMV load was higher in CMV antigen-positive patients than in antigen-negative patients.