Establishment of national standards of SARS-CoV-2 variants in Taiwan.

Objectives: In response to the pandemic, the Taiwan Food and Drug Administration (TFDA) established an initial SARS-CoV-2 RNA national standard based on the original Wuhan strain. However, with the depletion of the first national standard and continued mutation of the virus, the establishment of new national standards was imminent. Methods: Hence, new candidate national standards were established by heat-inactivation for 30 min for six representative strains of SARS-CoV-2, comprising the original strain and five variants with anticipated concentrations of 7.70 Log10 international units (IU)/mL each. To enhance the credibility of these national standards, the TFDA extended invitations to both domestic and international institutions to participate in a collaborative study. A total of eight participants contributed eleven datasets, incorporating two methods and targeting four distinct genes. Results: Based on these collective findings, the quantified viral RNA concentrations for each SARS-CoV-2 national standard strain are 7.69, 7.70, 7.69, 7.44, 7.52, and 7.29 Log10 IU/mL with Wuhan, alpha, beta, gamma, delta, and omicron strain, respectively. Conclusions: These newly established national standards will continue to be made available to the industry, serving as a fundamental reference for the development and quality control of nucleic acid in vitro diagnostic (IVD) reagents in Taiwan.

A collaborative study to establish the national standard for SARS-CoV-2 RNA nucleic acid amplification techniques (NAAT) in Taiwan.

The conventional PCR remains a valuable method to detect the newly emergent coronavirus rapidly and accurately. Our investigation aimed to establish the standard materials of SARS-CoV-2 for NAAT detection. We provided formalin-inactivated SARS-CoV-2 and confirmed RNA copy numbers. In addition, the virus genome was confirmed with whole-genome sequencing and identified as Wuhan/WI04/2019. Seven laboratories were invited for this collaborative study, according to the reporting data, we determined the SARS-CoV-2 with the unit of 6.35 Log10 copies/mL as the national standard. The availability of the national standard (NS) of SARS-CoV-2 will facilitate the standardization and harmonization of SARS-CoV-2 NAAT assays.

Potentially conjugative plasmids harboring Tn6636, a multidrug-resistant and composite mobile element, in Staphylococcus aureus.

OBJECTIVES: This study aimed to provide detailed genetic characterization of Tn6636, a multidrug-resistant and composite mobile element, in clinical isolates of Staphylococcus aureus. METHODS: A total of 112 ermB-positive methicillin-susceptible S. aureus (MSSA) and 224 ermB-positive methicillin-resistant S. aureus (MRSA) isolates collected from 2000 to 2015 were tested for the presence of Tn6636. Detection of the plasmids harboring Tn6636 was performed by S1 nuclease digestion pulsed-field gel electrophoresis (PFGE) analysis, conjugation test, and whole genome sequencing (WGS). RESULTS: Prevalence of Tn6636 in MSSA is higher than that in MRSA. Ten MSSA isolates and 10 MRSA isolates carried Tn6636. The 10 MSSA isolates belonged to three sequence types (ST), including ST7 (n = 6), ST5 (n = 3), and ST59 (n = 1). The 10 MRSA isolates belonged to ST188 (n = 8) and ST965 (n = 2). Analysis of plasmid sequences revealed that Tn6636 was harbored by six different mosaic plasmids. In addition to resistance genes, some plasmids also harbored toxin genes. CONCLUSION: The presence of multi-resistant Tn6636 in plasmids of both MSSA and MRSA with various STs suggests its broad dissemination. Results indicate that Tn6636 has existed for at least 16 years in Taiwan. The mosaic plasmids harboring Tn6636 can be transferred by conjugation. Ongoing surveillance of Tn6636 is essential to avoid continued spreading of resistant plasmids.

Heterogeneity of Molecular Characteristics among Staphylococcus argenteus Clinical Isolates (ST2250, ST2793, ST1223, and ST2198) in Northern Taiwan.

Staphylococcus argenteus is an emerging pathogen that is recognized as non-pigmented Staphylococcus aureus. However, the molecular characteristics of S. argenteus and its virulence factors have not been well studied. The present study analyzed 96 isolates of S. argenteus recovered from blood. Identification of S. argenteus was based on results of MALDI-TOF MS and lacking crtM gene. All 96 isolates were methicillin-susceptible. Multilocus sequence typing (MLST) revealed four sequence types: ST2250 (n = 72), ST2793 (n = 12), ST1223 (n = 10), and ST2198 (n = 2). All 72 ST2250 isolates harbored CRISPR loci with polymorphism of direct repeats and spacers, but no other STs carried CRISPR loci. To date, ST2793 isolates have rarely been reported in other countries. Collagen-binding adhesin gene (cna) and staphylococcal enterotoxin type C (sec) were detected in 12 (100%) and 8 (67%) ST2793 isolates, respectively. ST1223 has been reported as food poisoning pathogens, and enterotoxin gene clusters (egc) were detected in all 10 isolates, while seb gene was detected in three isolates. Two ST2198 isolates carried bone sialoprotein-binding protein gene (bbp), belonging to agr type IV. Our focus on the heterogeneity of molecular characterization in four ST types of S. argenteus revealed that S. argenteus had been isolated as early as 2000. Each ST type of S. argenteus harbors particular genetic markers that may contribute to their virulence.

groESL sequence determination, phylogenetic analysis, and species differentiation for viridans group streptococci.

The full-length sequences of the groESL genes (also known as cpn10/60) of Streptococcus anginosus, Streptococcus constellatus, Streptococcus gordonii, and Streptococcus sanguis and the near full-length sequence of the groESL genes of Streptococcus intermedius, Streptococcus bovis, Streptococcus mitis, Streptococcus mutans, Streptococcus oralis, and Streptococcus salivarius were determined. The lengths of the groES genes from the 10 species listed above ranged from 282 to 288 bp, and the full-length sequences of groEL determined for 4 species (S. anginosus, S. constellatus, S. gordonii, and S. sanguis) revealed that each was 1,623 bp. The intergenic region (spacer) between the groES and groEL genes varies in size (15 to 111 bp) and sequence between species. The variation of the groES sequences among the species tested was greater (62.1 to 95.1% nucleotide sequence identities) than that of the groEL sequences (77.2 to 95.2% nucleotide sequence identities). Phylogenetic analysis of the groES and groEL genes yielded evolutionary trees similar to the tree constructed by use of the 16S rRNA gene. The intraspecies variation of the spacer was minimal for clinical isolates of some species. The groESL sequence data provide an additional parameter for identification of viridans group streptococcal species.