Ultrafast RNA extraction-free SARS-CoV-2 detection by direct RT-PCR using a rapid thermal cycling approach.

The COVID19 pandemic has underlined the need for quick and high-throughput SARS-CoV-2 detection assays. Here we report the development of a direct RT-PCR detection method that can reliably detect SARS-CoV-2 gRNA in nasopharyngeal swab samples in under 27 minutes without needing nucleic acid extraction. Fluorescence readouts were highly linear, robust, and sensitive with a LoD95% of determined at 1.46 copies/µL as determined by RT-PCR on a surrogate sample panel containing clinical samples with varying SARS-CoV-2 viral load. We benchmarked our direct RT-PCR method against a reference qPCR method in 368 nasopharyngeal swab samples, confirming a sensitivity score of 99.4% and a specificity score of 98.5% as compared to the reference method. In summary, we here describe a novel rapid direct RT-PCR method to detect SARS-CoV-2 gRNA in clinical specimens, which can be completed in significantly less time compared to conventional PCR methods making it ideal for large-scale screening applications.

Whole-genome typing and characterization of blaVIM19-harbouring ST383 Klebsiella pneumoniae by PFGE, whole-genome mapping and WGS.

OBJECTIVES: We utilized whole-genome mapping (WGM) and WGS to characterize 12 clinical carbapenem-resistant Klebsiella pneumoniae strains (TGH1-TGH12). METHODS: All strains were screened for carbapenemase genes by PCR, and typed by MLST, PFGE (XbaI) and WGM (AflII) (OpGen, USA). WGS (Illumina) was performed on TGH8 and TGH10. Reads were de novo assembled and annotated [SPAdes, Rapid Annotation Subsystem Technology (RAST)]. Contigs were aligned directly, and after in silico AflII restriction, with corresponding WGMs (MapSolver, OpGen; BioNumerics, Applied Maths). RESULTS: All 12 strains were ST383. Of the 12 strains, 11 were carbapenem resistant, 7 harboured blaKPC-2 and 11 harboured blaVIM-19. Varying the parameters for assigning WGM clusters showed that these were comparable to STs and to the eight PFGE types or subtypes (difference of three or more bands). A 95% similarity coefficient assigned all 12 WGMs to a single cluster, whereas a 99% similarity coefficient (or ≥10 unmatched-fragment difference) assigned the 12 WGMs to eight (sub)clusters. Based on a difference of three or more bands between PFGE profiles, the Simpson's diversity indices (SDIs) of WGM (0.94, Jackknife pseudo-values CI: 0.883-0.996) and PFGE (0.93, Jackknife pseudo-values CI: 0.828-1.000) were similar (P = 0.649). However, the discriminatory power of WGM was significantly higher (SDI: 0.94, Jackknife pseudo-values CI: 0.883-0.996) than that of PFGE profiles typed on a difference of seven or more bands (SDI: 0.53, Jackknife pseudo-values CI: 0.212-0.849) (P = 0.007). CONCLUSIONS: This study demonstrates the application of WGM to understanding the epidemiology of hospital-associated K. pneumoniae. Utilizing a combination of WGM and WGS, we also present here the first longitudinal genomic characterization of the highly dynamic carbapenem-resistant ST383 K. pneumoniae clone that is rapidly gaining importance in Europe.

High Resolution Typing by Whole Genome Mapping Enables Discrimination of LA-MRSA (CC398) Strains and Identification of Transmission Events.

After its emergence in 2003, a livestock-associated (LA-)MRSA clade (CC398) has caused an impressive increase in the number of isolates submitted for the Dutch national MRSA surveillance and now comprises 40% of all isolates. The currently used molecular typing techniques have limited discriminatory power for this MRSA clade, which hampers studies on the origin and transmission routes. Recently, a new molecular analysis technique named whole genome mapping was introduced. This method creates high-resolution, ordered whole genome restriction maps that may have potential for strain typing. In this study, we assessed and validated the capability of whole genome mapping to differentiate LA-MRSA isolates. Multiple validation experiments showed that whole genome mapping produced highly reproducible results. Assessment of the technique on two well-documented MRSA outbreaks showed that whole genome mapping was able to confirm one outbreak, but revealed major differences between the maps of a second, indicating that not all isolates belonged to this outbreak. Whole genome mapping of LA-MRSA isolates that were epidemiologically unlinked provided a much higher discriminatory power than spa-typing or MLVA. In contrast, maps created from LA-MRSA isolates obtained during a proven LA-MRSA outbreak were nearly indistinguishable showing that transmission of LA-MRSA can be detected by whole genome mapping. Finally, whole genome maps of LA-MRSA isolates originating from two unrelated veterinarians and their household members showed that veterinarians may carry and transmit different LA-MRSA strains at the same time. No such conclusions could be drawn based spa-typing and MLVA. Although PFGE seems to be suitable for molecular typing of LA-MRSA, WGM provides a much higher discriminatory power. Furthermore, whole genome mapping can provide a comparison with other maps within 2 days after the bacterial culture is received, making it suitable to investigate transmission events and outbreaks caused by LA-MRSA.