Optimization of magnetic bead-based nucleic acid extraction for SARS-CoV-2 testing using readily available reagents.

The COVID-19 pandemic has highlighted the need for generic reagents and flexible systems in diagnostic testing. Magnetic bead-based nucleic acid extraction protocols using 96-well plates on open liquid handlers are readily amenable to meet this need. Here, one such approach is rigorously optimized to minimize cross-well contamination while maintaining sensitivity.

Influenza Classification Suite: An automated Galaxy workflow for rapid influenza sequence analysis.

Influenza viruses continually evolve to evade population immunity, and the different lineages are assigned into clades based on shared mutations. We have developed a publicly available computational workflow, the Influenza Classification Suite, for rapid clade mapping of sequenced influenza viruses. This suite provides a user-friendly workflow implemented in Galaxy to automate clade calling and antigenic site extraction. Workflow input includes clade definition and amino acid index array files, which can be customized to identify any clades of interest. The Influenza Classification Suite provides rapid, high-resolution understanding of circulating influenza strain evolution to inform influenza vaccine effectiveness and the need for potential vaccine reformulation.

Assessment of partial sequencing of the 65-kilodalton heat shock protein gene (hsp65) for routine identification of Mycobacterium species isolated from clinical sources.

We assessed the ability of an in-house database, consisting of 111 hsp65 sequences from putative and valid Mycobacterium species or described groups, to identify 689 mycobacterial clinical isolates from 35 species or groups. A preliminary assessment indicated that hsp65 sequencing confirmed the identification of 79.4% of the isolates from the 32 species examined, including all Mycobacterium tuberculosis complex isolates, all isolates from 13 other species, and 95.6% of all M. avium-M. intracellulare complex isolates. Identification discrepancies were most frequently encountered with isolates submitted as M. chelonae, M. fortuitum, M. gordonae, M. scrofulaceum, and M. terrae. Reexamination of isolates with discrepant identifications confirmed that hsp65 identifications were correct in a further 40 isolates. This brought the overall agreement between hsp65 sequencing and the other identification methods to 85.2%. The remaining 102 isolates had sequence matches below our acceptance criterion, had nondifferential sequence matches between two or more species, were identified by 16S rRNA sequencing as a putative taxonomic group not contained in our database, or were identified by hsp65 and 16S rRNA gene sequencing as a species not in our biochemical test database or had conflicting identifications. Therefore, to incorporate the unconfirmed isolates it was necessary to create 29 additional entries in our hsp65 identification database: 18 associated with valid species, 7 indicating unique sequences not associated with valid or putative species or groups, and 4 associated with unique, but currently described taxonomic groups. Confidence in the hsp65 sequence identification of a clinical isolate is best when sequence matches of 100% occur, but our data indicate that correct identifications can be confidently made when unambiguous matches exceeding 97% occur, but are dependent on the completeness of the database. Our study indicates that for hsp65 sequencing to be an effective means for identifying mycobacteria a comprehensive database must be constructed. hsp65 sequencing has the advantage of being more rapid and less expensive than biochemical test panels, uses a single set of reagents to identify both rapid- and slow-growing mycobacteria, and can provide a more definitive identification.

Use of an internal positive control in a multiplex reverse transcription-PCR to detect West Nile virus RNA in mosquito pools.

We report on the use of West Nile virus Armored RNA as an internal positive control (IPC) for the extraction and reverse transcription-PCR (RT-PCR) of RNA extracted from field-collected mosquitoes and on a multiplex real-time Taqman RT-PCR to simultaneously detect the 3' noncoding region of West Nile virus and the West Nile virus NS5-2 region comprising the IPC. Mosquito pools from the province of British Columbia, Canada (n = 635), were tested in duplicate and found to be negative for West Nile virus and positive for the IPC. Known West Nile virus-positive supernatants from mosquito pools from the provinces of Alberta and Manitoba were tested in duplicate and found to be positive for both regions of the West Nile virus genome. The mean cycle threshold (Ct) value for the IPC in batch extraction controls +/- 2 standard deviations was found to be 36.43 +/- 1.78 cycles. IPCs of 98.4% (624) of West Nile virus-negative pools fell within this range, indicating the reproducibility of RNA extraction and RT-PCR for pools varying in mosquito genus and number. A comparison of mosquito pool genera revealed no significant genus effect on the Ct value of the IPC. The incorporation of West Nile virus Armored RNA as an IPC allows monitoring of RNA extraction and RT-PCR and detection of false-negative results due to failures in these processes or to PCR inhibition, respectively.

Novel cryptosporidium genotypes in sporadic cryptosporidiosis cases: first report of human infections with a cervine genotype.

In this study, we genotyped parasites from the fecal specimens of sporadic cryptosporidiosis cases in British Columbia from 1995 to 1999. Genotyping was conducted by polymerase chain amplification of the internal transcribed spacer region, a hypervariable region in the 18S rRNA gene and the Cryptosporidium oocyst wall protein gene. Subsequent analysis was by restriction fragment length polymorphism and DNA sequencing. We identified two new Cryptosporidium genotypes in humans. One of these genotypes has been found recently in deer in New York state. The other genotype has not been identified in humans or animals. These results have important implications for drinking water quality strategies, especially for communities that obtain drinking water supplies from surface sources located in forested regions with deer populations.