Commercial Real-time PCR Assay Research Articles

Comparison of a Commercial Multiplex Real-Time PCR to the Cell Cytotoxicity Neutralization Assay for Diagnosis of Clostridium difficile Infections

A commercial multiplex real-time PCR assay (Cepheid Xpert C. difficile assay) for the diagnosis of Clostridium difficile infection was evaluated. The sensitivity and specificity of the Cepheid assay were 97.1% and 93.0% for fresh stools, using the cell cytotoxicity neutralization assay as the reference. Using PCR ribotyping as the reference for ribotype 027 strains, the corresponding figures were 100% and 98.1%, respectively.

Establishment of a PCR-product direct sequencing for the detection of HBV YMDD mutation

Objective To develop an assay of PCR-produet direct sequencing to detect hepatitis B virus (HBV) YMDD mutation, and compare the results gained by the sequencing and traditional real-time fluorescent PCR assays. Methods Serum samples were collected from 103 patients with chronic hepatitis B. HBV DNA were extracted from sers. YMDD mutation was detected by a commercial real-time PCR assay. Meanwhile, HBV reverse transcriptase-encoding gene was amplified by a nested PCR assay. The PCR products were directly subjected to sequencing at two directions, and the sequencing results were analyzed by NTI program. Using Kappa test, comparison was made between the results of rtM204-site mutations obtained by the direct sequencing and YMDD mutations by the real-time fluorescent PCR. Results The direct sequencing assay proved to be highly effective with bread range of detection in viral load from 500 to 1010copies/ml. And it may simultaneously avoid inhibitory effect caused by high viral load. The coincidence rates between two assays were 100% for YIDD, 97. 1% for YVDD, 76. 2% for YIDD/YVDD coexistence (Kappa = 0. 853, P < 0. 01). Conclusions The direct sequencing assay for HBV drug-resistant mutation detection is highly sensitive with broad dynamic range. It has high coincidence rate with real-time fluorescent PCR assay with advantage of detecting YMDD, YIDD and YVDD mutations simultaneously. Key words: Hepatitis B virus; Sequence analysis, DNA; Polymerase chain reaction; Mutation

Estudio comparativo entre una técnica de reacción en cadena de la polimerasa en transcripción reversa en tiempo real, un método de enzimoinmunoanálisis y el cultivo shell-vial en la detección de virus gripales A y B en pacientes adultos

The age of the patients and the type of sample are major problems in the diagnosis of influenza. Most available diagnostic techniques are highly effective in pediatric patients and in nasopharyngeal aspirates. However, in the adult population and using throat swabs, these techniques are much less reliable. We performed a prospective study comparing the efficacy of a commercial real-time reverse transcription PCR assay (RT-PCR) with that of an enzyme immunoassay (EIA) or shell vial culture (SV) in the detection of influenza A and B viruses in 125 throat swabs from adults with clinically suspected influenza during the 2007-2008 flu season. Throat swabs were subjected to rapid antigen detection for influenza viruses by means of a commercial dot-blot EIA. For the RT-PCR technique, RNA was extracted from 200 microL of each sample by the automated extraction system, EZ1 virus minikit (version 2.0). Genomic amplification of the extracted viral RNA was carried out using the OneStep RT-PCR FluA+FluB automated system with the SmartCycler amplification system. Each sample was inoculated into 2 SV of the MDCK cell line. Turnaround times were calculated from the time specimens were received in the laboratory to the time the result was reported to clinicians. The EIA system detected 27 (21.6%) positive samples, RT-PCR 62 (49.6%) positive samples, and SV 56 (44.8%) positive samples. Among the 62 positive samples, EIA detected 27 (43.5%), RT-PCR 62 (100%) and SV 56 (90.3%). With the use of RT-PCR, 38.4% of the adults studied were diagnosed on the same day samples were received. Among the total, 67.2% of diagnostic results were obtained within the first 24 hours; turnaround time was 1.1 days. The real-time RT-PCR method studied displayed high sensitivity and specificity in the detection of influenza virus in adult patients, when compared with the conventional techniques. With real-time RT-PCR, large numbers of samples can be rapidly tested and results provided the same day samples are received.

Verification of the ProPneumo-1 assay for the simultaneous detection of Mycoplasma pneumoniae and Chlamydophila pneumoniae in clinical respiratory specimens

BackgroundMycoplasma pneumoniae and Chlamydophila pneumoniae are major causes of lower and upper respiratory infections that are difficult to diagnose using conventional methods such as culture. The ProPneumo-1 (Prodesse, Waukesha, WI) assay is a commercial multiplex real-time PCR assay for the simultaneous detection of M. pneumoniae and/or C. pneumoniae DNA in clinical respiratory samples.ObjectiveThe aim of this study was to evaluate the sensitivity and specificity of the ProPneumo-1, a newly developed commercial multiplex real-time PCR assay.MethodsA total of 146 clinical respiratory specimens, collected from 1997 to 2007, suspected of C. pneumoniae or M. pneumoniae infections were tested retrospectively. Nucleic acid was extracted using an automated NucliSense easyMag (bioMerieux, Netherlands). We used a "Home-brew" monoplex real-time assay as the reference method for the analysis of C. pneumoniae and culture as the reference method for the analysis of M. pneumoniae. For discordant analysis specimens were re-tested using another commercial multiplex PCR, the PneumoBacter-1 assay (Seegene, Korea).ResultsFollowing discordant analysis, the sensitivity of the ProPneumo-1 assay for pathogens, C. pneumoniae or M. pneumoniae, was 100%. The specificity of the ProPneumo-1 assay, however, was 100% for C. pneumoniae and 98% for M. pneumoniae. The limits of detection were 1 genome equivalent (Geq) per reaction for pathogens, M. pneumoniae and C. pneumoniae. Due to the multipex format of the ProPneumo-1 assay, we identified 5 additional positive specimens, 2 C. pneumoniae in the M. pneumoniae-negative pool and 3 M. pneumoniae in the C. pneumoniae-negative pool.ConclusionThe ProPneumo-1 assay is a rapid, sensitive and effective method for the simultaneous detection of M. pneumoniae and C. pneumoniae directly in respiratory specimens.

Abbott RealTime Hepatitis C Virus (HCV) and Roche Cobas AmpliPrep/Cobas TaqMan HCV Assays for Prediction of Sustained Virological Response to Pegylated Interferon and Ribavirin in Chronic Hepatitis C Patients

Two commercial real-time PCR assays are currently available for sensitive hepatitis C virus (HCV) RNA quantification: the Abbott RealTime HCV assay (ART) and Roche Cobas AmpliPrep/Cobas TaqMan HCV assay (CAP/CTM). We assessed whether the two real-time PCR assays were more effective than Roche Cobas Amplicor HCV Monitor test, v.2.0 (CAM) for prediction of the sustained virological response (SVR) to pegylated interferon (PEG-IFN) plus ribavirin (RBV) in chronic hepatitis C. Sixty patients chronically infected with HCV genotype 1b (37 males and 23 females, 53 +/- 12 years of age) were treated with PEG-IFNalpha2b plus RBV for 48 weeks. Stored specimens at nine time points for each patient (at baseline, on treatment, and 24 weeks after treatment) were tested by the two real-time PCR assays and CAM. Twenty-six (43.3%) patients reached SVR. The positive predictive values (PPVs) for SVR of undetectable HCV RNA at week 12 by CAM, ART, and CAP/CTM were 74.3%, 88.0%, and 95.2%, respectively. An undetectable HCV RNA level by CAM, ART, and CAP/CTM correctly predicted SVR at week 4 in 100%, 100%, and 100% of patients, at weeks 5 to 8 in 91.7%, 100%, and 100% of patients, at weeks 9 to 12 in 55.6%, 75%, and 87.5% of patients, and at weeks 13 to 24 in 0%, 26.7%, and 40% of patients, respectively. Of 16 patients who relapsed after treatment, HCV RNA was detectable in 2 patients at the end of treatment by CAP/CTM but undetectable by ART and CAM. HCV RNA tests using ART and CAP/CTM are considered to be more effective at predicting SVR than CAM, and the PPV for SVR was slightly higher in CAP/CTM than in ART.

MRSA detection: comparison of two molecular methods (BD GeneOhm® PCR assay and Easy-Plex) with two selective MRSA agars (MRSA-ID and Oxoid MRSA) for nasal swabs

Screening of patients is an important component for methicillin-resistant Staphylococcus aureus (MRSA) containment. The aim of this study was to determine the utility of molecular methods compared to selective agars for MRSA detection. Consecutive high risk patients (n = 200) were screened for nasal MRSA colonization using chromogenic selective agars (MRSA ID and CMRSA) as well as the BD GeneOhm MRSA (previously known as IDI-MRSA) and the Easy-Plex commercial real time PCR assays. The sensitivities of the molecular methods were similar to the BD GeneOhm MRSA (86%) and Easy-Plex (84%). The chromogenic agars, MRSA ID and CMRSA, showed sensitivities of 62% and 84%, respectively. Sensitivity was influenced by both the MRSA antibiograms and MRSA clonal type. The specificity was >98% in all methods except for the Easy-Plex assay (90%). Sensitivity of selective agars increased with 48 hours of incubation with a corresponding decrease in specificity. In conclusion, molecular detection methods for MRSA remain sensitive and rapid but are associated with greater expense.

Testicular persistence of Parvovirus B19: Evidence for preferential infection of germ cell tumors

Human Parvovirus B19 has previously been implicated in the pathogenesis of testicular germ cell tumors, but this could not have been confirmed. This study was designed to investigate the testicular persistence of Parvovirus B19 and possible associations with germ cell tumors. Paraffin-embedded or fresh tissues from 36 germ cell tumors, 20 germ cell aplasias, 26 normal testicular tissues, 20 liver tissues, and 20 spleen tissues were evaluated by two different molecular assays: a nested PCR for Parvovirus B19 capsid genes and a commercial quantitative real-time PCR. Positive results were further confirmed by another commercial real-time PCR assay. Viral DNA was detected in 3 of 36 (8.3%) germ cell tumors, but not in other groups. Viral loads observed in all positive samples were less than 20 IU/reaction, suggesting very low levels of viral replication or latency. These results either directly or indirectly imply the involvement of Parvovirus B19 with testicular germ cell tumors. Viral persistence in normal testis, germ cell aplasia tissues, or hepatic/splenic tissues was not observed in this study.

Molecular diagnosis of sepsis in neutropenic patients with haematological malignancies

The rapid diagnosis of an infectious cause in the course of fever of unknown origin plays a pivotal role in the correct management of neutropenic patients. In this study, blood samples from febrile oncohaematological patients were tested using a novel commercial real-time PCR assay (LightCycler SeptiFast; Roche Molecular Systems) and blood culture (BacT/Alert 3D; bioMérieux). Twenty-one (20.4 %) and 34 (33 %) of the 103 samples under study tested positive by blood culture and PCR, respectively. The analysis of concordance evidenced a low correlation between the two approaches (83 %), mainly due to samples that tested negative by culture but positive using the molecular approach. Among 14 discordant cases negative by culture but positive by PCR, 12 were observed in sequential samples of patients with initial concordant results on samples drawn before the administration of a specific antimicrobial therapy. Moreover, DNA of a fastidious organism, Aspergillus fumigatus, not easily detectable by the cultural approach was rapidly detected in the two remaining discordant cases. Overall, the characteristics featured by the molecular method could be of interest in the development of new algorithms for the diagnosis of sepsis in critical patients.

Development and evaluation of real-time loop-mediated isothermal amplification for hepatitis B virus DNA quantification: A new tool for HBV management

Background Accurate quantitation of hepatitis B viral load is a critical aspect in screening and monitoring HBV infection. Objectives We used loop-mediated isothermal amplification (LAMP) to develop a real-time fluorogenic (RtF-LAMP) protocol to quantitate HBV DNA. Quantitative analysis was obtained by measuring time-to-positive (TTP), a biomarker similar to cycle threshold (Ct) in real-time PCR. Study design Sensitivity, specificity, reproducibility, and dynamic range for RtF-LAMP were evaluated using molecular and biological standards. Four hundred and two patient samples were then used to compare the performance of RtF-LAMP to a commercial real-time PCR assay (DaAn Gene Co, China). Results The lower detection limit (LDL) of RtF-LAMP by Probit analysis at the 95% detection level was 210 copies/ml, and the dynamic range was 8 orders of magnitude. The conversion factor for results obtained with the RtF-LAMP assay was 1 IU/ml equals to 4.4 copies/ml. Coefficients of variation (CV) reflected low intra-assay and inter-assay variability (4.24–12.11%). In a large number of serum samples, there was excellent an correlation between RtF-LAMP and real-time PCR ( R 2 = 0.96). There was a good agreement between the two tests except at the detection cutoff of the real-time PCR assay. Conclusion Our RtF-LAMP protocol appears to be precise, accurate and rapid. It could be a valuable tool for the detection of HBV in large clinical and epidemiological studies.

The relative test performance characteristics of two commercial assays for the detection of Mycobacterium tuberculosis complex in paraffin-fixed human biopsy specimens.

The Seeplex™ TB Detection-2 assay (Rockville, MD) is a nested endpoint PCR for the Mycobacterium tuberculosis complex (MTBC) targets IS6110 and MPB64 that utilizes dual priming oligonucleotide technology. When used to detect the presence of MTBC DNA in formalin-fixed paraffin-embedded tissue specimens, the sensitivity and specificity of this assay is equivalent to a labor-intensive traditional endpoint PCR assay and is more sensitive than a commercial real-time PCR assay.

Comparison of commercial and in-house Real-time PCR assays for quantification of Epstein-Barr virus (EBV) DNA in plasma

BackgroundEpstein-Barr virus (EBV) DNA load monitoring is known to be useful for the diagnosis and monitoring of EBV-associated diseases. The aim of this study is to compare the performance of two real-time PCR assays for EBV DNA: a commercial kit as the Q-EBV Real-Time System (Q-EBV PCR, Amplimedical, Turin, Italy) and an in-house assay (EBV RQ-PCR).ResultsThe range of linearity and the degree of precision of the two assays were similar. The clinical sensitivity of Q-EBV PCR was higher for reference samples containing less than 1,000 EBV DNA copies/ml. The absolute quantitative results of the two methods were statistically correlated (R2 = 0.7789; p < 0.0001), with the systematic overestimation by EBV RQ-PCR possibly linked to different amplification efficiency in calibration standards.ConclusionBoth the commercial and the in-house assay may be appropriate for clinical use, but common standards are advisable for comparable absolute values, as these would improve the clinical utility of EBV DNA load measurement.

Evaluation of a commercial real-time PCR assay for quantitation of Epstein-Barr virus DNA in different groups of patients

The aim of this study was to evaluate the performance of a molecular assay for quantitation of Epstein-Barr virus (EBV) DNA based on real-time PCR, and to determine EBV DNA levels in EDTA whole blood samples derived from different groups of patients. Following a manual DNA extraction protocol, real-time PCR was performed using the LightCycler EBV Quantification Kit, which demonstrated sufficient accuracy and linearity. Coefficients of variations were found to be between 6 and 42% and 5 and 34%, respectively, for inter-assay and intra-assay variations. In clinical specimens, EBV DNA was detected in all patients with acute EBV infection ( n = 34), in one of 25 adults with past EBV infection, in 16 out of 25 (64%) anti-HIV antibody positive persons, in 10 out of 25 (40%) solid organ transplant recipients, and in none of the 23 infants without history of EBV infection. When EBV DNA levels in positive specimens were compared between different groups, statistically significant differences were not found. The LightCycler EBV Quantification Kit was found to be useful for determination of EBV DNA levels in EDTA whole blood.

Evaluation of the RealArt Malaria LC real-time PCR assay for malaria diagnosis.

PCR-based methods have advantages over traditional microscopic methods for the diagnosis of malaria, especially in cases of low parasitemia and mixed infections. However, current PCR-based assays are often labor-intensive and not readily quantifiable and have the potential for contamination due to a requirement for postamplification sample handling. Real-time PCR can address these limitations. This study evaluated the performance characteristics of a commercial malaria real-time PCR assay (RealArt Malaria LC Assay; Artus GmbH, Hamburg, Germany) on the LightCycler platform for the detection of malaria parasites in 259 febrile returned travelers. Compared to nested PCR as the reference standard, the real-time assay had a sensitivity of 99.5%, specificity of 100%, positive predictive value of 100%, and negative predictive value of 99.6% for the detection of malaria. Our results indicate that the RealArt assay is a rapid (<45 min), sensitive, and specific method for the detection of malaria in returned travelers.

PL002 Regulatory networks controlling virulence, morphology and cell wall biosynthesis in Candida albicans

Q fever is a zoonotic disease, caused by the obligate intracellular bacterium Coxiella burnetii. Between 2007 and 2010, Q fever has been a major public health concern in the Netherlands, with almost 3500 human cases reported and dairy goats considered to be the most probable source. At the end of 2009, the Dutch government decided to cull all pregnant dairy sheep and dairy goats based on bulk tank milk C. burnetii positive farms, aiming to preventing shedding and to reducing environmental contamination. On bulk tank milk C. burnetii PCR positive farms, a life-time breeding ban was implemented for all remaining non-pregnant small ruminants. This study describes test results on a bulk tank milk C. burnetii PCR positive dairy goat farm on which all goats had been vaccinated against Q fever with an inactivated phase one vaccine since 2008. All pregnant dairy goats of this farm were culled in 2010, after which bulk tank milk was negative in the C. burnetii PCR. One year later, however, this farm became bulk tank milk C. burnetii PCR positive again. From all lactating animals on the farm (n = 350), individual milk samples were collected and tested using a commercial real-time PCR assay. Individual milk samples from five dairy goats appeared to be C. burnetii PCR positive. These positive goats had been born on the farm between 2002 and 2006. At postmortem examination, out of 33 mostly tissue samples per animal, only milk and mammary tissue samples were C. burnetii PCR positive. Moreover, immunohistochemical examination did not reveal the source of C. burnetii. After culling of these C. burnetii PCR milk positive animals, the bulk tank milk remained negative in C. burnetii PCR until the end of the observation period. The results indicate that vaccination of Q fever infected dairy goat farms does not completely prevent intermittent shedding of C. burnetii in probably previously infected goats. Further research is needed to investigate how and where C. burnetii multiplies in such intermittently shedding animals.