In-house Real-time RT-PCR Assays Research Articles

Evaluation of Analytical and Clinical Performance and Usefulness in a Real-Life Hospital Setting of Two in-House Real-Time RT-PCR Assays to Track SARS-CoV-2 Variants of Concern.

Since the end of 2020, multiple severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern (VOCs) have emerged and spread worldwide. Tracking their evolution has been a challenge due to the huge number of positive samples and limited capacities of whole-genome sequencing. Two in-house variant-screening RT-PCR assays were successively designed in our laboratory in order to detect specific known mutations in the spike region and to rapidly detect successively emerging VOCs. The first one (RT-PCR#1) targeted the 69-70 deletion and the N501Y substitution simultaneously, whereas the second one (RT-PCR#2) targeted the E484K, E484Q, and L452R substitutions simultaneously. To evaluate the analytical performance of these two RT-PCRs, 90 negative and 30 positive thawed nasopharyngeal swabs were retrospectively analyzed, and no discordant results were observed. Concerning the sensitivity, for RT-PCR#1, serial dilutions of the WHO international standard SARS-CoV-2 RNA, corresponding to the genome of an Alpha variant, were all detected up to 500 IU/mL. For RT-PCR#2, dilutions of a sample harboring the E484K substitution and of a sample harboring the L452R and E484Q substitutions were all detected up to 1000 IU/mL and 2000 IU/mL, respectively. To evaluate the performance in a real-life hospital setting, 1308 and 915 profiles of mutations, obtained with RT-PCR#1 and RT-PCR#2, respectively, were prospectively compared to next-generation sequencing (NGS) data. The two RT-PCR assays showed an excellent concordance with the NGS data, with 99.8% for RT-PCR#1 and 99.2% for RT-PCR#2. Finally, for each mutation targeted, the clinical sensitivity, the clinical specificity and the positive and negative predictive values showed excellent clinical performance. Since the beginning of the SARS-CoV-2 pandemic, the emergence of variants-impacting the disease's severity and the efficacy of vaccines and therapies-has forced medical analysis laboratories to constantly adapt to the strong demand for screening them. Our data showed that in-house RT-PCRs are useful and adaptable tools for monitoring such rapid evolution and spread of SARS-CoV-2 VOCs.

Establishment of an in-house real-time RT-PCR assay for the detection of severe acute respiratory syndrome coronavirus 2 using the first World Health Organization international standard in a resource-limited country.

BackgroundThe COVID‐19 pandemic caused by SARS‐CoV‐2 remains public health burdens and many unresolved issues worldwide. Molecular assays based on real‐time RT‐PCR are critical for the detection of SARS‐CoV‐2 in clinical specimens from patients suspected of COVID‐19.ObjectiveWe aimed to establish and validate an in‐house real‐time RT‐PCR for the detection of SARS‐CoV‐2.MethodologyPrimers and probes sets in our in‐house real‐time RT‐PCR assay were designed in conserved regions of the N and E target genes. Optimized multiplex real‐time RT‐PCR assay was validated using the first WHO International Standard (NIBSC code: 20/146) and evaluated clinical performance.ResultsThe limit of detection validated using the first WHO International Standard was 159 IU/ml for both E and N target genes. The evaluation of clinical performance on 170 clinical samples showed a positive percent agreement of 100% and the negative percent agreement of 99.08% for both target genes. The Kappa value of 0.99 was an excellent agreement, the strong correlation of C t values observed between two tests with r 2 = 0.84 for the E gene and 0.87 for the N gene. Notably, we assessed on 60 paired saliva and nasopharyngeal samples. The overall agreement was 91.66%, and Kappa value of 0.74 showed a high agreement between two types of samples. When using nasopharyngeal swabs as the reference standard, positive percent agreement, and negative percent agreement were 91.83% and 90.90%, respectively.ConclusionIn the present study, we established and validated an in‐house real‐time RT‐PCR for molecular detection of SARS‐CoV‐2 in a resource‐limited country.

Multi-center evaluation of cepheid xpert® xpress SARS-CoV-2 point-of-care test during the SARS-CoV-2 pandemic

BackgroundWith the outbreak of SARS-CoV-2, rapid diagnostics are paramount to contain the current pandemic. The routinely used realtime RT-PCR is sensitive, specific and able to process large batches of samples. However, turnaround time is long and in cases where fast obtained results are critical, molecular point of care tests (POCT) can be an alternative. Here we report on a multicenter evaluation of the Cepheid Xpert Xpress SARS-CoV-2 point-of-care test.Study designThe Xpert Xpress SARS-CoV-2 assay was evaluated against the routine in-house real-time RT-PCR assays in three medical microbiology laboratories in The Netherlands. A sensitivity and specificity panel was tested consisting of a dilution series of SARS-CoV-2 and ten samples containing SARS-CoV-2 and a range of other seasonal respiratory viruses. Additionally, 58 samples of patients positive for SARS-CoV-2 with different viral loads and 30 tested negative samples in all three Dutch laboratories using an in-house RT-PCR, were evaluated using Cepheids Xpert Xpress SARS-CoV-2 cartridges.ResultsXpert Xpress SARS-CoV-2 point of care test showed equal performance compared to routine in-house testing with a limit of detection (LOD) of 8.26 copies/mL. Other seasonal respiratory viruses were not detected. In clinical samples Xpert Xpress SARS-CoV-2 reaches an agreement of 100 % compared to all in-house RT-PCRsConclusionCepheids GeneXpert Xpert Xpress SARS-CoV-2 is a valuable addition for laboratories in situations where rapid and accurate diagnostics are of the essence.

Establishment of an in-house one-step real-time RT-PCR assay for detection of Zaire ebolavirus

Ebola virus is a deadly causative agent with a high mortality rate of up to 90%, therefore it has been classified by the Center for Disease Control and Prevention (CDC) as a category A biological agent. The World Health Organization (WHO) recommended using RT-PCR based assays to rapidly detect the virus. In the present study, we established an in-house assay for detection of Zaire ebolavirus via real-time RT-PCR. The nucleotide sequence of the Zaire ebolavirus nucleoprotein (NP) gene was retrieved from the Genbank for designing primer pairs and probes using Primer Express 3.0 software. The RNA positive control was generated by in vitro RNA transcript synthesis. The optimal components in the 20 μl final volume of the real-time RT-PCR assay were 10 μl 2X QuantiTectProbeRT-PCR master mix, 0,6 μM of each primer, 0,1 μM of the probe, 0,2 μl RT mix and 5 μl of RNA template. The thermal cycle conditions were as follows: 50oC for 30 min, 95°C for 15 min, then 45 cycles of 15 s at 94°C, 60s at 60°C. The limit of detection of the assay was 100 copies/reaction and 1414 FFU/ml with the positive RNA panel and sample panel of RNA extracted from cell culture supernatants of cells infected with Zaire ebolavirus 2014/Gueckedou-C05, respectively. The specificity of this assay was 100% when tested with the positive RNA panel of Ebola virus and other haemorrhagic fever viruses. In conclusion, we successfully established an in-house real-time RT-PCR assay for detection of Zaire ebolavirus in Vietnam with a limit of detection of 1414 FFU/ml and specificity of 100%.

Comparative evaluation of the CerTest VIASURE flu A, B & RSV real time RT-PCR detection kit on the BD MAX system versus a routine in-house assay for detection of influenza A and B virus during the 2016/17 influenza season

BackgroundDiagnosing of influenza rapidly and accurately helps clinicians to initiate appropriate treatment options and isolation protocols. Unnecessary antimicrobial treatment and laboratory testing can also be reduced. Assess commercial alternatives to in-house assays that may not only reduce laboratory technician “hands on” time but also the laboratory turnaround time is of interest. ObjectivesWe evaluated the performance of the VIASURE Flu A, B & RSV Real Time RT-PCR Detection Kit (CerTest Biotec) for detecting Influenza A and B viruses. Study designDuring the 2016/17 influenza season 532 clinical samples were tested with the VIASURE assay on the BD MAX™ system versus an in-house real time RT-PCR assay with discrepant results resolved by a real time RT-PCR assay at a national reference laboratory. ResultsThe VIASURE assay on the BD MAX showed a sensitivity of 99.5% (95% CI: 97.3–100) and a specificity of 99.1% (95% CI: 97.4–99.8) for detection of Influenza A virus. The positive predictive and negative predictive values were 98.5% (95% CI: 95.8–99.7) and 99.7% (95% CI: 98.3–100) respectively. Influenza B virus detection could not be evaluated due to a low positivity rate. The BD MAX platform offered the flexibility of several daily runs, shorter hands-on-time and shorter turnaround time than the in-house assay. ConclusionsThe VIASURE assay on the BD MAX performed well and is now implemented in our clinical laboratory.

Hepatitis E virus serology and PCR: does the methodology matter?

Hepatitis E virus (HEV) genotype 3 is an emerging pathogen in the developed world. As the clinical manifestations and routine laboratory parameters are often nonspecific, accurate diagnostic tests are crucial. In the current study, the performance of six serological assays and three PCR assays for the detection of HEV was evaluated. In the setting of the Ghent University Hospital, patients with clinically suspected HEV infection were tested for the presence of HEV IgM and IgG as well as HEV RNA. Serology was performed using six commercial HEV ELISA assays: Biorex, Wantai and Mikrogen IgM and IgG. HEV RNA was detected using one commercial assay (Altona RealStar®), and two optimized in-house real-time RT-PCR assays (according to Jothikumar et al., 2006 and Gyarmati et al., 2007). In addition, all three PCR assays were performed on 16 external quality control (EQC) samples. In a period of 39 months (January 2011-April 2014), 70 patients were enrolled. Using different ELISA assays, the prevalence of antibodies varied from 5.7% to 14.3% for HEV IgM and from 15.7% to 20.0% for IgG. All but two of the results of the PCR assays performed on clinical samples agreed. However, 10 out of 16 EQC samples results showed major discrepancies. We observed important differences in the performance of various serological and PCR assays. For this reason, results of both serological and molecular tests for HEV should be interpreted with caution.

Hepatitis E virus serology and PCR. Does the methodology matter?

Hepatitis E virus (HEV) genotype 3 is an emerging pathogen in the developed world. As the clinical manifestations and routine laboratory parameters are often nonspecific, accurate diagnostic tests are crucial. In the current study, the performance of six serological assays and three PCR assays for the detection of HEV was evaluated. In the setting of the Ghent University Hospital, patients with clinically suspected HEV infection were tested for the presence of HEV IgM and IgG as well as HEV RNA. Serology was performed using six commercial HEV ELISA assays: Biorex, Wantai and Mikrogen IgM and IgG. HEV RNA was detected using one commercial assay (Altona RealStar®), and two optimized in-house real-time RT-PCR assays (according to Jothikumar et al., 2006 and Gyarmati et al., 2007). In addition, all three PCR assays were performed on 16 external quality control (EQC) samples. In a period of 39 months (January 2011-April 2014), 70 patients were enrolled. Using different ELISA assays, the prevalence of antibodies varied from 5.7% to 14.3% for HEV IgM and from 15.7% to 20.0% for IgG. All but two of the results of the PCR assays performed on clinical samples agreed. However, 10 out of 16 EQC samples results showed major discrepancies. We observed important differences in the performance of various serological and PCR assays. For this reason, results of both serological and molecular tests for HEV should be interpreted with caution.

Evaluation of the Xpert Flu test and comparison with in-house real-time RT-PCR assays for detection of influenza virus from 2008 to 2011 in Marseille, France

Rapid documentation of respiratory specimens can have an impact on the management of patients and their relatives in terms of preventive and curative measures. We compared the results of the Xpert® Flu assay (Cepheid) with three real-time RT-PCR assays using 127 nasopharyngeal samples, of which 75 were positive for influenza A (with 52 identified as A/HINI-2009) and 52 were positive for influenza B. The Xpert® Flu assay presented a quasi-absence of non-interpretable tests, and showed sensitivity and specificity of 100% and 100% for Flu A, 98.4% and 100% for A/HINI-2009, and 80.7% and 100% for Flu B.

Comparison of an in-house real-time RT-PCR assay with a commercial assay for detection of enterovirus RNA in clinical samples

Molecular detection of enterovirus (EV) RNA based on PCR methods is a quicker and more sensitive approach than culture methods. At present, different PCR-based methods for EV RNA detection are available, but comparisons of results obtained according to the different approaches are limited. We evaluated an in-house real-time RT-PCR assay with a commercialized TaqMan real-time RT-PCR kit for detection of EV. Consecutive clinical specimens from paediatric patients less than 6 years old with clinical suspicion of EV infection were analyzed between July and November 2010. After RNA extraction, samples were amplified both by the real-time RT-PCR commercial assay and the in-house assay. A total of 19 of 132 patients (14.4%) involving 20 samples (14 plasma samples and 6 CSF) were positive in at least one of the two assays. The sensitivity of the in-house assay when the MutaPLATE® assay was used as a reference was 90% (IC 95%; 74.35-100) and the specificity was 100% (IC 95%; 99.63-100). Cts results of two methods were statistically correlated (r = 0.774; P = 0.01). In conclusion, these two real-time RT-PCR assays are rapid and easy methods for detection of EV.

Virological evaluation of domestic water purification devices commonly used in India emphasizes inadequate quality and need for virological standards

To evaluate the performance of domestic water purification units with respect to contaminating enteric viruses. Eight domestic water purification systems widely used in India were evaluated using hepatitis E virus (HEV) as a model virus. For HEV concentration and detection, membrane filtration and real-time PCR were used respectively. Viral log reduction value (LRV) was calculated for each unit. Viral log reduction value was 0.21 for unit 6 (polyester + carbon), 1.45 for unit 4 (filter + UV), 1.52 for unit 3 (filter + chlorine), 1.70 for a carbon + exhaust indication contact disinfection unit, 2.20 for an iodine resin unit, 2.51 for a dual filter unit and 6.53 for a hollow fibre membrane unit and a gravity-fed filter unit. Thus, only the technologies employed by the latter two were efficient in complete removal of HEV. The majority of the water purifiers under use are inadequate. Virological standards in evaluating such devices need to be established urgently, in order to help manufacturers to improve the performance of such products and most importantly, to help consumers.

Clinical performance of an in-house real-time RT-PCR assay using a fluorogenic LUX™ primer for quantitation of human immunodeficiency virus type-1 (HIV-1)

The South African National Antiretroviral Treatment Guideline recommends the use of HIV viral load assays for routine monitoring of HIV-1 positive patients on Highly Active Antiretroviral Therapy (HAART). Approved commercial HIV-1 viral load assays are expensive for developing countries where a large number of patients are treated in the public sector. The evaluation of an in-house HIV-1 viral load assay (LUX assay) is described using 458 plasma specimens. Good specificity of the LUX assay was demonstrated using 50 seronegative plasma specimens. A group of 142 HIV-1 positive patients was used to assess the agreement between the LUX assay and the COBAS Amplicor assay. An intra class correlation (ICC) coefficient of 0.85 (CI 95%) indicated good agreement between the assays. The Bland–Altman model showed good agreement between the assays for ∼87% of the results (mean 0.03 [−1.26; 1.32], CI 95%). In a cohort of 55 patients followed-up longitudinally the LUX assay showed similar declines in viral load to the COBAS Amplicor assay in response to therapy. Viral rebound was detected in 5 patients out of 55 by both assays. Thus, the LUX assay compares well to the gold standard and represents an affordable alternative for high volume testing in resource limited settings.