Comparison of FTD SARS-CoV-2 Assay and RealStar RT-PCR kit 1.0 for the detection of SARS-CoV-2

The aim of the study was to evaluate the clinical performance of FTD SARS-CoV-2 compared to the RealStar RT-PCR kit 1.0. The analysis of 100 nasopharyngeal swabs showed an overall agreement of 88%. The positive percentage agreement was 85.6% and the negative percentage agreement was 91%.
In conclusion we observed a substantial agreement among the two methods, with discrepancies mainly observed in Bio Med Frontiers specimens  with a relatively low amount of viral RNA.

Comparison of three commercial DNA extraction kits for the enhancement of PCR assay sensitivity for Xanthomonas euvesicatoria pv. Allii

The study was carried out to evaluate three commercial DNA extraction kits, Probe GS, FitoSorb and Sorb GMO, thus identifying the most suitable for isolating the phytopathogenic bacteria Xanthomonas euvesicatoria pv. allii.
 Onion seed samples were prepared which were inoculated with bacterial concentrations ranging from 101 to 107 CFU ml-1 .
Real-time PCR was performed to determine the efficacy of the isolated DNA in enhancing the sensitivity of the assay.
The DNA extracted by Probe GS had the best detectability, having been detected at the lowest concentration used in the study 101 x 3 CFU ml-1 .
FitoSorb and Sorb GMO yielded DNA with a higher and similar limit of detection 103 x 3 CFU ml-1 .
Furthermore, Probe GS had the lowest cycle at every concentration tested as compared to the other methods.
Therefore Probe GS proved to be the most optimised kit for the extraction of X. euvesicatoria pv. allii, hence enhanced degree of sensitivity for the assay.
 The findings generated in this study can be used by phytosanitary laboratories to develop highly rapid and accurate diagnostic protocols for X. euvesicatoria pv. allii.

Compatibility of a novel filter paper-based bio-safe sputum transport kit with line probe assay for diagnosing drug-resistant tuberculosis: a single-site evaluation study

  • Near-patient access to appropriate tests is a major obstacle for the efficient diagnosis of tuberculosis (TB) and associated drug resistance.
  • We recently developed the “TB Concentration & Transport” kit for bio-safe, ambient-temperature transportation of dried sputum on Trans-Filter, and the “TB DNA Extraction” kit for DNA extraction from Trans-Filter for determining drug resistance by DNA sequencing.
  • In the present study, we evaluated the compatibility of Kit-extracted DNA with Hain’s line probe assays (LPAs), which are endorsed by National TB programmes for the detection of drug resistance in sputum collected from presumptive multidrug-resistant TB patients (n=207).
  •  Trans-Filter-extracted DNA was seamlessly integrated with the LPA protocol (Kit-LPA). The sensitivity of Kit-LPA for determining drug resistance was 83.3% for rifampicin (95% CI 52-98%), 77.7% for isoniazid (95% CI 52-94%), 85.7% for fluoroquinolones (95% CI 42-100%) and 66.6% for aminoglycosides (95% CI 9-99%), with a specificity range of 93.7% (95% CI 87-97) to 99.1% (95% CI 95-100) using phenotypic drug susceptibility testing (DST) as a reference standard.
  • A high degree of concordance was noted between results obtained from Kit-LPA and LPA (99% to 100% (κ value: 0.83-1.0)).
  •  This study demonstrates successful integration of our developed kits with LPA. The adoption of these kits across Designated Microscopy Centres in India can potentially overcome the existing challenge of transporting infectious sputum at controlled temperature to centralised testing laboratories and can provide rapid near-patient cost-effective “Universal DST” services to TB subjects residing in remote areas.

Comparative evaluation of the Thermo fisher TaqPath™ COVID-19 combo kit with the Cepheid Xpert® Xpress SARS-CoV-2 assay for detecting SARS-CoV-2 in nasopharyngeal specimens

With over 50 SARS-CoV-2 gene amplification assays that have been EUA cleared with minimal experimental validation performed, it is likely that not all of these assays are comparable in their ability to detect SARS-CoV-2 in clinical specimens. Thermo Fisher Scientific is a relatively new company in the molecular diagnostics field and the purpose of this study was to compare the performance of the Thermo Fisher TaqPath™ Combo Kit with an established test, the Cepheid Xpert® Xpress SARS-CoV-2 assay, for its ability to detect SARS-CoV-2 in nasopharyngeal specimens.
 A total of 300 randomly selected nasopharyngeal specimens were evaluated and tested by the TaqPath and GeneXpert assays. Discordant test specimens were arbitrated by performing an alternative PCR assay and Sanger sequencing.
The TaqPath assay had a 96.7 and 99.6% positive and negative agreement respectively when compared to the Xpert Xpress test.
However, after test arbitration, the three discordant specimens were arbitrated in favor of the TaqPath assay producing a positive and negative percent agreement of 100% for the TaqPath Combo Kit while the Xpress SARS-CoV-2 assay had a positive and negative percent agreement of 98.3 and 99.2% respectively.
The TaqPath Combo Kit is a high complexity assay that compares favorably with the Xpert Xpress test and can be reliably used for the detection of SARS-CoV-2 in nasopharyngeal specimens.

Evaluation of a Commercial Culture-Free Neutralization Antibody Detection Kit for Severe Acute Respiratory Syndrome-Related Coronavirus-2 and Comparison With an Antireceptor-Binding Domain Enzyme-Linked Immunosorbent Assay

  •  Severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) surrogate neutralization assays that obviate the need for viral culture offer substantial advantages regarding throughput and cost. The cPass SARS-CoV-2 Neutralization Antibody Detection Kit (GenScript) is the first such commercially available assay that detects antibodies that block receptor-binding domain (RBD)/angiotensin-converting enzyme (ACE)-2 interaction. We aimed to evaluate cPass to inform its use and assess its added value compared with anti-RBD enzyme-linked immunosorbent assays (ELISAs)
  • Serum reference panels comprising 205 specimens were used to compare cPass to plaque-reduction neutralization test (PRNT) and a pseudotyped lentiviral neutralization (PLV) assay for detection of neutralizing antibodies. We assessed the correlation of cPass with an ELISA detecting anti-RBD immunoglobulin (Ig)G, IgM, and IgA antibodies at a single timepoint and across intervals from onset of symptoms of SARS-CoV-2 infection.
  •  Compared with PRNT-50, cPass sensitivity ranged from 77% to 100% and specificity was 95% to 100%. Sensitivity was also high compared with the pseudotyped lentiviral neutralization assay (93%; 95% confidence interval [CI], 85-97), but specificity was lower (58%; 95% CI, 48-67).
  • Highest agreement between cPass and ELISA was for anti-RBD IgG (r = 0.823). Against the pseudotyped lentiviral neutralization assay, anti-RBD IgG sensitivity (99%; 95% CI, 94-100) was very similar to that of cPass, but overall specificity was lower (37%; 95% CI, 28-47). Against PRNT-50, results of cPass and anti-RBD IgG were nearly identical.
  • The added value of cPass compared with an IgG anti-RBD ELISA was modest.

Rapid and simultaneous identification of three mutations by the Novaplex™ SARS-CoV-2 variants I assay kit

The emergence of SARS-CoV-2 variants has caused an unexpected rebound globally. The World Health Organization has listed three variants (B.1.1.7, B.1.351, and P.1) as variants of concern.
To understand the epidemiology and thereby plan appropriate safety measures, differential identification of the variants is indeed critical.
 Although whole-genome sequencing is the gold standard for variant identification, it is time-consuming and relatively expensive. Therefore, a rapid, easy, and cost-effective platform targeting multiple regions of the genome is required. Here, we assessed the usefulness of the Novaplex™ SARS-CoV-2 Variants I Assay kit in identifying mutations in the variants.
We retrospectively examined 30 stored nasal swabs from COVID-19-positive patients tested between November 2020 and March 2021. RNA extracted from these swabs was subjected to the commercial kit and real-time reverse transcription-PCR was performed.
To determine the genome sequences of SARS-CoV-2 in the collected samples and deduce the consensus sequences among the identified variants, genome sequencing libraries were prepared and mapped to the reference genome.

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Four of the tested samples were determined as variants. Of them, two harbored both H69/V70 deletion and N501Y substitution, whereas two harbored E484K substitution alone.
The variant with E484K substitution alone (“R.1”) has been now categorized as a variant of interest in Japan. Additionally, the kit-based assay was found to be feasible, convenient, and user-friendly in identifying the abovementioned mutations with a turnaround time of only 2 h.

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