Quality Control For Molecular Diagnostics Research Articles

Evaluation of DNA extraction methods for dried blood spots in the diagnosis of congenital cytomegalovirus infection

Background Dried blood spots (DBS) may be valuable in the diagnosis of congenital cytomegalovirus (CMV) infection. However, the 2007 European Quality Control for Molecular Diagnostics (QCMD) proficiency testing programme showed that CMV DNA detection in DBS was lacking sensitivity in a considerable number of participating laboratories. Objective To compare DNA extraction methods for DBS for detecting CMV. Sensitivity and applicability of the methods for high-throughput usage were assessed. Study design Guthrie cards were spotted with CMV DNA-positive whole blood ( n = 15). DNA was extracted from the DBS using different extraction methods, followed by CMV amplification by means of real-time PCR. Results Significant differences between the extraction methods with respect to the sensitivity were found. Optimal sensitivity was achieved when samples were tested in triplicate, demonstrating that the methods in general operated around their detection limits. Triplicate testing using the protocol by Barbi et al. [Barbi M, et al. Cytomegalovirus DNA detection in Guthrie cards: a powerful tool for diagnosing congenital infection. J Clin Virol 2000;17:159–65], representing the most sensitive methods, resulted in sensitivities of 100%, 86%, and 50% for DBS with CMV DNA loads of 5–4, 4–3, and 3–2 log 10 copies/ml, respectively. This indicates that sensitivity limitations apply in the clinically relevant concentration range. Few methods appeared suitable for 96-well format high-throughput testing. Discussion When considering universal neonatal screening for congenital CMV infection, an assay which is both sensitive and applicable for high-throughput testing is required. The protocol by Barbi et al. and the BioRobot Universal System appear appropriate candidates currently available for 96-well format application in neonatal screening using DBS.

PX-24 Comparative studies of the Access® CMV IgG assay

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.

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.

Evaluation of the COBAS AmpliPrep-Total Nucleic Acid Isolation-COBAS TaqMan Hepatitis B Virus (HBV) Quantitative Test and Comparison to the VERSANT HBV DNA 3.0 Assay

Quantitative detection of hepatitis B virus (HBV) in serum or plasma has become the most direct and reliable method for monitoring chronic hepatitis B. Here, we report the performance characteristics of a real-time PCR hepatitis B DNA quantitative assay, the COBAS TaqMan (CTM) HBV test (Roche Diagnostics, Meylan, France), in combination with an automated DNA extraction on the COBAS AmpliPrep (CAP) instrument using the total nucleic acid isolation kit (TNAI kit), a generic reagent for nucleic acid isolation (both from Roche Diagnostics). The linearity, accuracy, and specificity of the CAP-TNAI-CTM HBV test were evaluated using various reference panels and standards (HBV panel 2004 from Quality Control for Molecular Diagnostics, OptiQuant HBV panel from AcroMetrix, WHO International Standard for HBV, and Teragenix hepatitis B genotype panel). Quantitative results show that the CAP-TNAI-CTM HBV test performed well with respect to linearity, accuracy, and reproducibility from at least 100 to 500,000 HBV DNA IU/ml. Based on the log(10) IU of HBV DNA/ml measured, the intra-assay variation ranged from 2.49% to 8.46% and the interassay variation ranged from 1.88% to 7.83%. The test was extremely sensitive and could detect samples containing HBV DNA below the reported quantification threshold (<30 IU/ml). All HBV genotypes were correctly amplified, and no cross-contamination occurred during the automated sample preparation. In addition, 402 human serum samples were tested comparatively to the VERSANT HBV DNA 3.0 assay (bDNA; Bayer Diagnostics, Puteaux, France). The viral load results of the CAP-TNAI-CTM test and bDNA were significantly correlated, but the agreement between the two tests was poor, with large differences between results for individual samples. The hands-on time was estimated to be reduced from 2.30 h with bDNA to 45 min with the CAP-TNAI-CTM test, and up to 84 samples were completely processed within a working day. Overall, the performance characteristics of the CAP-TNAI-CTM test demonstrated that it provides a high-throughput sensitive and reliable method for quantitation of HBV DNA levels in the routine molecular laboratory.

A real-time Taqman method for hepatitis C virus genotyping

Background: There is the need for a rapid, inexpensive method for genotyping hepatitis C virus (HCV) to support clinical practice. Objectives: To develop a real-time (Rotor-Gene™ 3000) Taqman assay for HCV genotyping in a single tube. Study design: Seven type-specific probes, two for genotypes 1–3 and one for genotype 4 were designed around genotype-specific motifs in the 5′ non-coding (NC) region to create two panels of probes. The first panel included two probes for genotype 1 detection and a single probe each for genotypes 2 and 3. The second panel had two probes for confirmation of genotypes 2 and 3 and a first line probe for genotype 4 detection. A comparative analysis of the Taqman assay against our in-house sequence-based method using 154 consecutive clinical samples, from HCV carriers in Cambridge, and four samples from the Quality Control for Molecular Diagnostics (QCMD) System was undertaken. Results: 158 samples were analysed by conventional sequencing: 49% ( n = 78) were genotype 1, 11% ( n = 18) genotype 2, 30% ( n = 47) genotype 3 and 6% ( n = 10) genotype 4. For two samples, the sequence data was heterogeneous and difficult to analyse, suggesting mixed infection and for three samples, the viral load was insufficient for sequencing. Concordant results were obtained with the novel Taqman assay for 77/78 (99%) of genotype 1 isolates (positive with both genotype 1 probes), 17/18 (94%) of genotype 2 isolates, 43/47 (91%) of genotype 3 isolates and 10/10 (100%) genotype 4 isolates. One isolate, untypeable with sequencing was genotyped with the Taqman assay. Conclusions: The Taqman assay was sensitive, specific and reliable over a wide range of viral loads and could identify mixed infections. These results highlight the potential of the Taqman assay as a fast, accurate and convenient method for routine HCV genotyping.

Comparison of Commercial Real-Time PCR Assays for Quantification of Epstein-Barr Virus DNA

Clinical research suggests a role for viral load measurement in predicting and monitoring Epstein-Barr virus (EBV)-associated diseases. The aim of this study was to assess the performance of the recently commercially available quantitative assays for EBV based on real-time PCR: the RealArt EBV LC PCR kit and the LightCycler EBV quantification kit. A total of 87 samples were analyzed: 67 samples were obtained from transplant recipients and patients with EBV-associated diseases, 8 samples were obtained from the Quality Control for Molecular Diagnostics 2002 EBV Proficiency Program, and 12 negative qualitative nested PCR samples were used as negative controls. Inter- and intra-assay variabilities were determined by running replicates of two samples. All samples were run in a LightCycler instrument. The differences between positive and negative results were not considered statistically significant (P = 0.5355). There were no false-positive results using either method for nested PCR negative-control samples. The difference in viral load values using the two different methods was considered statistically significant (P < 0.01). The logarithmic linear correlation for both assays was low (r = 0.449) but significant (P < 0.01). The LightCycler EBV quantification kit showed a wider dispersal in results but produced substantially more-accurate melting temperature profile curves. The bias towards lower measurements was considerable in comparison with higher viral load. The differences in PCR efficiency and the presence of mutations could explain the disparity between the two methods. It was concluded that confidence intervals would be required to report the results rather than plain absolute values of viral load for patient monitoring.

Highly sensitive assay for detection of enterovirus in clinical specimens by reverse transcription-PCR with an armored RNA internal control.

The objective of the present study was the development of a diagnostic reverse transcription (RT)-PCR for the specific detection of enterovirus (EV) RNA in clinical specimens controlled by an internal control (IC) RNA. The IC RNA contains the same primer binding sites as EV RNA but has a different probe region. The IC RNA was packaged into an MS2 phage core particle (armored) and was added to the clinical sample to allow monitoring of both extraction efficiency and RT-PCR efficiency. Serial dilutions of the IC RNA were made, and the detection limit of the RT-PCR was tested in a background of EV RNA-negative cerebrospinal fluid. The sensitivity and specificity of the RT-PCR assay were tested by using all 64 known EV serotypes, several non-EV serotypes, and two Quality Control for Molecular Diagnostics (QCMD) Program EV proficiency panels from 2001 and 2002. In total, 322 clinical specimens were tested by RT-PCR, and to establish the clinical utility of the RT-PCR, a comparison of the results of viral culture and RT-PCR was done with 87 clinical specimens. The lower limit of sensitivity was reached at about 150 copies of IC RNA/ml. All 64 EV serotypes were positive, while all non-EV serotypes were negative. All culture-positive samples of the 2001 QCMD proficiency panel (according to the 50% tissue culture infective doses per milliliter) were positive by RT-PCR. Invalid results, i.e., negativity for both EV RNA and IC RNA, due to inhibition of RT-PCR were observed for 33.3% of the members of the 2002 QCMD proficiency panel and 3.1% of the clinical specimens. Inhibition of RT-PCR could be relieved by the addition of 400 ng of bovine alpha-casein per microl to both the RT reaction mixture and the PCR mixture. With this optimized protocol, the results for all samples of the 2002 QCMD proficiency panel and all clinical specimens except one fecal sample (0.3%) were valid. Evaluation of the clinical samples demonstrated that EV infection could be detected in 12 of 87 samples (13.8%) by RT-PCR, while viral culture was negative. Our data show that the RT-PCR with armored IC RNA offers a very reliable and rapid diagnostic tool for the detection of EV in clinical specimens and that the addition of bovine alpha-casein relieved inhibition of the RT-PCR for 99.7% of clinical specimens.

Multicentre quality control study for detection of Mycobacterium tuberculosis in clinical samples by nucleic amplification methods

The aim of this study was to evaluate the laboratory performance of nucleic acid amplification tests (NATs) for detection of the Mycobacterium tuberculosis complex. A proficiency panel consisting of eight sputum specimens and four specimens diluted in phosphate-buffered saline (PBS) was sent to 82 laboratories in 23 countries by the Quality Control for Molecular Diagnostics (QCMD) TB programme. The performance of different NATs was analysed in combination with a questionnaire on the applied methods. Seventy-eight participants (95.2%) contributed a total of 85 evaluable data sets. The percentage of correct results on the eight sputum samples was 86.3% (586/679). Of the sputum specimens considered as 'smear-negatives' (650 CFU/250 micro L), only 61.2% (104/170) were reported positive. The percentage of correct results for the three scored PBS samples was 75.7% (193/255). The total number of false-positive results was 11 (4.3%); these were reported for seven (8.2%) of the 85 data sets. In 32 (37.6%) data sets an 'in-house' NAT method was used, and in 53 (62.4%) sets a commercial assay was tested. The percentage of data sets achieving correct results on all sputum samples was 35.3% and 37.8%, respectively. For the PBS samples this was 45.8% and 41.5%. Overall, the results of this study demonstrated that the performance of NATs for the detection of M. tuberculosis has improved since previous studies. The percentage of false-positives has decreased considerably. However, a large number of procedures still lack sufficient sensitivity for application to smear-negative samples.