Rapid, high-throughput, cost-effective whole-genome sequencing of SARS-CoV-2 using a condensed library preparation of the Illumina DNA Prep kit.

The ongoing COVID-19 pandemic necessitates cost-effective, high-throughput, and timely whole-genome sequencing (WGS) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) viruses for outbreak investigations, identifying variants of concern (VoC), characterizing vaccine breakthrough infections, and public health surveillance. In addition, the enormous demand for WGS on supply chains and the resulting shortages of laboratory supplies necessitated the use of low-reagent and low-consumable methods. Here, we report an optimized library preparation method (the BCCDC cutdown method) that can be used in a high-throughput scenario, where one technologist can perform 576 library preparations (6 plates of 96 samples) over the course of one 8-hour shift. The same protocol can also be used in a rapid turnaround time scenario, from primary samples (up to 96 samples) to loading on a sequencer in an 8-hour shift. This new method uses Freed et al.'s 1,200 bp primer sets (Biol Methods Protoc 5:bpaa014, 2020, https://doi.org/10.1093/biomethods/bpaa014) and a modified and condensed Illumina DNA Prep workflow (Illumina, CA, USA). Compared to the original protocol, the application of this new method using hundreds of clinical specimens demonstrated equivalent results to the full-length DNA Prep workflow at 45% of the cost, 15% of consumables required (such as pipet tips), 25% of manual hands-on time, and 15% of on-instrument time if performing on a liquid handler, with no compromise in sequence quality. Results demonstrate that this new method is a rapid, simple, cost-effective, and high-quality SARS-CoV-2 WGS protocol. IMPORTANCE: Sequencing has played an invaluable role in the response to the COVID-19 pandemic. Ongoing work in this area, however, demands optimization of laboratory workflow to increase sequencing capacity, improve turnaround time, and reduce cost without compromising sequence quality. This report describes an optimized DNA library preparation method for improved whole-genome sequencing of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pathogen. The workflow advantages summarized here include significant time, cost, and consumable savings, which suggest that this new method is an efficient, scalable, and pragmatic alternative for SARS-CoV-2 whole-genome sequencing.

Use of genomics to design a diagnostic assay to discriminate between Streptococcus pneumoniae and Streptococcus pseudopneumoniae.

Distinuishing the species of mitis group streptococci is challenging due to ambiguous phenotypic characteristics and high degree of genetic similarity. This has been particularly true for resolving atypical Streptococcus pneumoniae and Streptococcus pseudopneumoniae. We used phylogenetic clustering to demonstrate specific and separate clades for both S. pneumoniae and S. pseudopneumoniae genomes. The genomes that clustered within these defined clades were used to extract species-specific genes from the pan-genome. The S. pneumoniae marker was detected in 8027 out of 8051 (>99.7 %) S. pneumoniae genomes. The S. pseudopneumoniae marker was specific for all genomes that clustered in the S. pseudopneumoniae clade, including unresolved species of the genus Streptococcus sequenced by the BC Centre for Disease Control Public Health Laboratory that previously could not be distinguished by other methods. Other than the presence of the S. pseudopneumoniae marker in six of 8051 (<0.08 %) S. pneumoniae genomes, both the S. pneumoniae and S. pseudopneumoniae markers showed little to no detectable cross-reactivity to the genomes of any other species of the genus Streptococcus or to a panel of over 46 000 genomes from viral, fungal, bacterial pathogens and microbiota commonly found in the respiratory tract. A real-time PCR assay was designed targeting these two markers. Genomics provides a useful technique for PCR assay design and development.

Review of 16S and ITS Direct Sequencing Results for Clinical Specimens Submitted to a Reference Laboratory.

We evaluated the performance of 16S and internal transcribed spacer (ITS) region amplification and sequencing of rDNA from clinical specimens, for the respective detection and identification of bacterial and fungal pathogens. Direct rDNA amplification of 16S and ITS targets from clinical samples was performed over a 4-year period and reviewed. All specimens were from sterile sites and submitted to a reference laboratory for evaluation. Results of 16S and ITS were compared to histopathology, Gram and/or calcofluor stain microscopy results. A total of 277 16S tests were performed, with 64 (23%) positive for the presence of bacterial DNA. Identification of an organism was more likely in microscopy positive 16S samples 14/21 (67%), compared to 35/175 (20%) of microscopy negative samples. A total of 110 ITS tests were performed, with 14 (13%) positive. The yield of microscopy positive ITS samples, 9/44 (21%), was higher than microscopy negative samples 3/50 (6%). Given these findings, 16S and ITS are valuable options for culture negative specimens from sterile sites, particularly in the setting of positive microscopy findings. Where microscopy results are negative, the limited sensitivity of 16S and ITS in detecting and identifying an infectious agent needs to be considered.

Epidemiologic and Genotypic Review of Carbapenemase-Producing Organisms in British Columbia, Canada, between 2008 and 2014.

Carbapenemase-producing organisms (CPOs) are a serious emerging problem for health care facilities worldwide. Owing to their resistance to most antimicrobial therapies, CPOs are difficult to treat and pose a challenge for infection prevention and control. Since 2010, lab-based surveillance for CPOs and PCR-based testing were implemented in British Columbia (BC), Canada. A review of CPOs in BC from 2008 to March 2014 was done to characterize the resistance mechanisms and possible clonal strain transmission and to compare pulsed-field gel electrophoresis (PFGE), multilocus sequence typing (MLST), and plasmid restriction fragment length polymorphism (RFLP) as molecular typing tools. During this study period, a total of 177 CPO cases were identified. Patient demographics and travel history were reviewed, and a descriptive analysis was carried out. PFGE profiles, MLST, and plasmid RFLP analysis for a subset of Escherichia coli, Klebsiella pneumoniae, and Enterobacter species isolates were obtained and analyzed. Our findings demonstrate that CPOs have been increasing in number in BC over time, from 1 isolate/year retrospectively identified in 2008 and 2009 to 82 isolates in 2013 and 30 isolates in the first quarter of 2014. Overall, K. pneumoniae isolates lack clonality, although some seemingly related clusters have been found. Plasmid analysis showed evidence of the spread of plasmids carrying carbapenemase-encoding genes between the examined isolates. Analysis of Enterobacter cloacae isolates revealed a more clonal nature of these CPOs in BC. The presence of related clusters provides evidence of interpatient organism transmission both within and between institutions. Although in our study, NDM-harboring E. cloacae isolates appeared to spread clonally, the spread of carbapenem resistance in K. pneumoniae seems to be plasmid mediated.

Rapid Detection of KPC, NDM, and OXA-48-Like Carbapenemases by Real-Time PCR from Rectal Swab Surveillance Samples.

We describe a multiplex real-time PCR assay for use on the ABI 7500 Fast TaqMan platform to detect all currently described Klebsiella pneumoniae carbapenemases (KPC), New Delhi metallo-ß-lactamases (NDM), and the OXA-48-like family of carbapenemases from bacterial culture lysates or sample enrichment broth lysates.

Successful approach to treatment of Helicobacter bilis infection in X-linked agammaglobulinemia.

Helicobacter bilis, an unusual cause of chronic infections in patients with X-linked agammaglobulinemia (XLA), is notoriously difficult to diagnose and eradicate. Based on the limited number of cases reported worldwide, we highlight the typical features of H. bilis infection in XLA and provide a rational and successful approach to diagnosis and treatment of this challenging infection.