ABSTRACT
The ARTIC protocol uses a multiplexed PCR approach with two primer pools tiling the entire SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) genome. Primer pool updates are necessary for accurate amplicon sequencing of evolving SARS-CoV-2 variants with novel mutations. The suitability of the ARTIC V4 and updated V4.1 primer scheme was assessed using whole genome sequencing of Omicron from clinical samples using Oxford Nanopore Technology. Analysis of Omicron BA.1 genomes revealed that 93.22â% of clinical samples generated improved genome coverage at 50× read depth with V4.1 primers when compared to V4 primers. Additionally, the V4.1 primers improved coverage of BA.1 across amplicons 76 and 88, which resulted in the detection of the variant-defining mutations G22898A, A26530G and C26577G. The Omicron BA.2 sub-variant (VUI-22JAN-01) replaced BA.1 as the dominant variant by March 2022, and analysis of 168 clinical samples showed reduced coverage across amplicons 15 and 75. Upon further interrogation of primer binding sites, a mutation at C4321T [present in 163/168 (97â%) of samples] was identified as a possible cause of complete dropout of amplicon 15. Furthermore, two mutations were identified within the primer binding regions for amplicon 75: A22786C (present in 90â% of samples) and C22792T (present in 12.5â% of samples). Together, these mutations may result in reduced coverage of amplicon 75, and further primer updates would allow the identification of the two BA.2-defining mutations present in amplicon 75: A22688G and T22679C. This work highlights the need for ongoing surveillance of primer matches as circulating variants evolve and change.
Subject(s)
COVID-19 , Humans , SARS-CoV-2/genetics , Mutation , Binding SitesABSTRACT
BACKGROUND: Norovirus is a leading cause of infectious gastroenteritis, characterized by outbreaks of diarrhoea and vomiting in closed settings. Nucleic acid amplification tests allow rapid and sensitive laboratory diagnosis of norovirus, with a number of commercial platforms now available. OBJECTIVES: Evaluate the performance of the Becton Dickinson BD-MAX™System, Cepheid Xpert® Norovirus Assay, and Luminex xTAG® Gastrointestinal Pathogen Panel (GPP) for norovirus detection in stool. Assess the performance of the Xpert® Norovirus Assay and BD-MAX™ in vomit samples. STUDY DESIGN: 163 diarrhoeal stool samples were tested on four diagnostic systems (laboratory-defined real time RT-PCR (assigned as gold standard), BD MAX™, Xpert® Norovirus Assay, and xTAG® GPP). A further 70 vomit samples were tested on the Xpert and BD MAX platforms. RESULTS: In stool, sensitivity and specificity of the BD-MAX™ was 96.8% and 100%, for Xpert® Norovirus Assay was 91.9% and 100%, and for xTAG® GPP was 79.0% and 87.1%. In vomit samples positive and negative percent agreement was 95.6% and 92.0%, between the BD-MAX™ and Xpert® Norovirus. CONCLUSIONS: The BD-MAX™ System with user defined settings and the Xpert® Norovirus Assay showed acceptable sensitivity and specificity for detection of norovirus from stool and vomit. The xTAG GPP assay was less reliable for norovirus detection but can detect a number of other clinically useful enteropathogens. Clinical laboratories must consider skill mix, budget, and sample throughput to determine the best fit for their service.
Subject(s)
Feces/virology , Gastroenteritis/virology , Molecular Diagnostic Techniques/methods , Norovirus/isolation & purification , Vomiting/virology , Aged , Aged, 80 and over , Female , Humans , Male , Middle Aged , Molecular Diagnostic Techniques/instrumentation , Multiplex Polymerase Chain Reaction/instrumentation , Multiplex Polymerase Chain Reaction/methods , Norovirus/genetics , Reagent Kits, Diagnostic , Sensitivity and SpecificityABSTRACT
BACKGROUND: Taxanes such as paclitaxel and docetaxel are used successfully to treat breast cancer, usually in combination with other agents. They interfere with microtubules causing cell cycle arrest; however, the mechanisms underlying the clinical effects of taxanes are yet to be fully elucidated. METHODS: Isogenic paclitaxel resistant (PACR) MDAâMBâ231, paclitaxel resistant ZR75â1 and docetaxel resistant (DOCR) ZR75â1 cell lines were generated by incrementally increasing taxane dose in native cell lines in vitro. We used aCGH analysis to identify mechanisms driving taxane resistance. RESULTS: Taxane resistant cell lines exhibited an 18-170 fold increased resistance to taxanes, with the ZR75-1 resistant cell lines also demonstrating cross resistance to anthracyclines. Paclitaxel treatment of native cells resulted in a G2/M block and a decrease in the G1 phase of the cell cycle. However, in the resistant cell lines, minimal changes were present. Functional network analysis revealed that the mitotic prometaphase was lost in the resistant cell lines. CONCLUSION: This study established a model system for examining taxane resistance and demonstrated that both MDR and mitosis represent common mechanism of taxane resistance.
Subject(s)
Antineoplastic Agents/pharmacology , Drug Resistance, Neoplasm/genetics , Paclitaxel/pharmacology , Taxoids/pharmacology , ATP Binding Cassette Transporter, Subfamily B/genetics , ATP Binding Cassette Transporter, Subfamily B/metabolism , Cell Line, Tumor , Comparative Genomic Hybridization , Docetaxel , Drug Resistance, Multiple , Gene Expression , Humans , Inhibitory Concentration 50ABSTRACT
This study looked at 128 nasopharyngeal aspirates (NPA) and 162 throat swabs (TS) tested with the Diagenode multiplex assay on the BD Max system versus our in-house Bordetella pertussis PCR. Sensitivity and specificity were 97.3% and 100% for NPA and 88.3% and 98% for TS, respectively. Of positive NPA, 42.1% were coinfected with respiratory viruses.
