Evaluation of 5 Polymerase Chain Reaction Assays for the Detection of Mpox Virus.

BACKGROUND: In 2022, the global dissemination of mpox virus (MPXV) outside endemic regions prompted the expansion of diagnostic testing worldwide. This study assesses the performance characteristics of 5 real-time polymerase chain reaction (PCR) assays in detecting MPXV during the 2022 outbreak. METHODS: Clinical specimens collected from patients across Ontario, Canada, were tested on the following assays: RealStar Orthopoxyvirus PCR and FlexStar Monkeypox virus PCR (Altona Diagnostics), Novaplex MPXV (Seegene), VIASURE Monkeypox virus Real Time PCR Reagents (CerTest Biotec), and a laboratory-developed test. Positive percent agreement (PPA), negative percent agreement (NPA), relative limit of detection (LOD), and precision were evaluated and MPXV lineages were determined using an amplicon-based whole-genome sequencing (WGS) assay. RESULTS: Swabs were collected from various anatomic sites (65 positive and 30 negative). All assays demonstrated 100% NPA (95% confidence interval, 88.4%/88.1%-100.0%), with PPA ranging from 92.2% (82.7%-97.4%) to 96.9% (89.3%-99.6%). LOD and precision were comparable across assays, with coefficient of variations <3%. WGS analysis identified 6 lineages, all belonging to subclade IIb. CONCLUSIONS: The assays exhibited excellent PPA, NPA, LOD, and precision. Ongoing performance monitoring is essential to detect assay escape mutants and ensure universal detection of evolving MPXV strains.

Coronavirus disease 2019 (COVID-19) outbreak on an in-patient medical unit associated with unrecognized exposures in common areas-Epidemiological and whole-genome sequencing investigation.

OBJECTIVE: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) hospital outbreaks have been common and devastating during the coronavirus disease 2019 (COVID-19) pandemic. Understanding SARS-CoV-2 transmission in these environments is critical for preventing and managing outbreaks. DESIGN: Outbreak investigation through epidemiological mapping and whole-genome sequencing phylogeny. SETTING: Hospital in-patient medical unit outbreak in Toronto, Canada, from November 2020 to January 2021. PARTICIPANTS: The outbreak involved 8 patients and 10 staff and was associated with 3 patient deaths. RESULTS: Patients being cared for in geriatric chairs at the nursing station were at high risk for both acquiring and transmitting SARS-CoV-2 to other patients and staff. Furthermore, given the informal nature of these transmissions, they were not initially recognized, which led to further transmission and missing the opportunity for preventative COVID-19 therapies. CONCLUSIONS: During outbreak prevention and management, the risk of informal patient care settings, such as geriatric chairs, should be considered. During high-risk periods or during outbreaks, efforts should be made to care for patients in their rooms when possible.

A Novel Approach to Detect IDH Point Mutations in Gliomas Using Nanopore Sequencing: Test Validation for the Clinical Laboratory.

The use of standard next-generation sequencing technologies to detect key mutations in IDH genes for glioma diagnosis imposes several challenges, including high capital cost and turnaround delays associated with the need for batch testing. For both glioma testing and testing in other tumor types where highly specific mutation identification is required, the high-throughput nature of next-generation sequencing limits the feasibility of using it as a primary approach in clinical laboratories. We hypothesized that third-generation nanopore sequencing by Oxford Nanopore Technologies has the capability to overcome these limitations. This study aimed to develop and validate a nanopore-based IDH mutation detection assay for clinical practice using glioma formalin-fixed, paraffin-embedded (FFPE) tissue. Glioma FFPE (n = 66) samples with confirmed IDH gene mutational status were sequenced on the MinION device using an amplicon-based approach. All cases were concordant when compared with the reference results. Limit of blank and limit of detection for the variant allele fraction were 1.5% and 3.3%, respectively, at 500× read depth per gene. Total sequencing cost per sample was CAD$50 to CAD$134 with results being available in 9 to 15 hours. These findings demonstrate that nanopore-sequencing technology can be leveraged to develop low-cost, high-performance clinical sequencing-based assays with quick turnaround times to support the detection of targeted mutations in FFPE tumor tissue.

Emergence of a mutation in the nucleocapsid gene of SARS-CoV-2 interferes with PCR detection in Canada.

The emergence of Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV-2) was met with rapid development of robust molecular-based detection assays. Many SARS-CoV-2 molecular tests target multiple genetic regions of the virus to maximize detection and protect against diagnostic escape. Despite the relatively moderate mutational rate of SARS-CoV-2, numerous mutations with known negative impact on diagnostic assays have been identified. In early 2021, we identified four samples positive for SARS-CoV-2 with a nucleocapsid (N) gene drop out on Cepheid Xpert® Xpress SARS-CoV-2 assay. Sequencing revealed a single common mutation in the N gene C29200T. Spatiotemporal analysis showed that the mutation was found in at least six different Canadian provinces from May 2020 until May 2021. Phylogenetic analysis showed that this mutation arose multiple times in Canadian samples and is present in six different variants of interest and of concern. The Cepheid testing platform is commonly used in Canada including in remote regions. As such, the existence of N gene mutation dropouts required further investigation. While commercial SARS-CoV-2 molecular detection assays have contributed immensely to the response effort, many vendors are reluctant to make primer/probe sequences publicly available. Proprietary primer/probe sequences create diagnostic 'blind spots' for global SARS-CoV-2 sequence monitoring and limits the ability to detect and track the presence and prevalence of diagnostic escape mutations. We hope that our industry partners will seriously consider making primer/probe sequences available, so that diagnostic escape mutants can be identified promptly and responded to appropriately to maintain diagnostic accuracy.

Real-time monitoring of bead-based DNA hybridization in a microfluidic system: study of amplicon hybridization behavior on solid supports.

DNA hybridization phenomena occurring on solid supports are not understood as clearly as aqueous phase hybridizations and mathematical models cannot predict some empirically obtained results. Ongoing research has identified important parameters but remains incomplete to accurately account for all interactions. It has previously been shown that the length of the overhanging (dangling) end of the target DNA strand following hybridization to the capture probe is correlated to interactions with the complementary strand in solution which can result in unbinding of the target and its release from the surface. We have developed an instrument for real-time monitoring of DNA hybridization on spherical particles functionalized with oligonucleotide capture probes and arranged in the form of a tightly packed monolayer bead bed inside a microfluidic cartridge. The instrument is equipped with a pneumatic module to mediate displacement of fluid on the cartridge. We compared this system to both conventional (passive) and centrifugally-driven (active) microfluidic microarray hybridization on glass slides to establish performance levels for the detection of single nucleotide polymorphisms. The system was also used to study the effect of the dangling end's length in real-time when the immobilized target DNA is exposed to the complementary strand in solution. Our findings indicate that increasing the length of the dangling end leads to desorption of target amplicons from bead-bound capture probes at a rate approaching that of the initial hybridization process. Finally, bead bed hybridization was performed with Streptococcus agalactiae cfb gene amplicons obtained from randomized clinical samples, which allowed for identification of group B streptococci within 5-15 min. The methodology presented here is useful for investigating competitive hybridization mechanisms on solid supports and to rapidly validate the suitability of microarray capture probes.

A concordance study of the Altona RealStar Varicella-Zoster virus real-time quantitative PCR and in-house conventional qualitative PCR.

This study compared the Altona RealStar™ VZV Kit 1.0 real time quantitative VZV PCR with in-house qualitative conventional VZV PCR on cerebrospinal fluid, mucocutaneous, and other uncommon clinical specimens. Overall, positive and negative agreement percentages were respectively 97.9% (95%CI: 93.8-99.6), 100.0% (95%CI: 93.1-100.0), and 96.3% (95%CI: 89.4-99.2) while Cohen's kappa statistic value was 0.96 (95%CI: 0.91-1.00). RealStar™ VZV quantitative PCR assay reported average quantitative viral loads of 4.4 × 105 and 1.1 × 107 copies/mL in cerebrospinal fluid and cutaneous specimens, respectively (P < 0.01). RealStar™ VZV PCR assay showed excellent agreement with in house conventional assay for various clinical specimens.

Structured oligonucleotides for target indexing to allow single-vessel PCR amplification and solid support microarray hybridization.

The combination of molecular diagnostic technologies is increasingly used to overcome limitations on sensitivity, specificity or multiplexing capabilities, and provide efficient lab-on-chip devices. Two such techniques, PCR amplification and microarray hybridization are used serially to take advantage of the high sensitivity and specificity of the former combined with high multiplexing capacities of the latter. These methods are usually performed in different buffers and reaction chambers. However, these elaborate methods have high complexity and cost related to reagent requirements, liquid storage and the number of reaction chambers to integrate into automated devices. Furthermore, microarray hybridizations have a sequence dependent efficiency not always predictable. In this work, we have developed the concept of a structured oligonucleotide probe which is activated by cleavage from polymerase exonuclease activity. This technology is called SCISSOHR for Structured Cleavage Induced Single-Stranded Oligonucleotide Hybridization Reaction. The SCISSOHR probes enable indexing the target sequence to a tag sequence. The SCISSOHR technology also allows the combination of nucleic acid amplification and microarray hybridization in a single vessel in presence of the PCR buffer only. The SCISSOHR technology uses an amplification probe that is irreversibly modified in presence of the target, releasing a single-stranded DNA tag for microarray hybridization. Each tag is composed of a 3-nucleotide sequence-dependent segment and a unique "target sequence-independent" 14-nucleotide segment allowing for optimal hybridization with minimal cross-hybridization. We evaluated the performance of five (5) PCR buffers to support microarray hybridization, compared to a conventional hybridization buffer. Finally, as a proof of concept, we developed a multiplexed assay for the amplification, detection, and identification of three (3) DNA targets. This new technology will facilitate the design of lab-on-chip microfluidic devices, while also reducing consumable costs. At term, it will allow the cost-effective automation of highly multiplexed assays for detection and identification of genetic targets.

Microfluidic device for rapid (<15 min) automated microarray hybridization.

BACKGROUND: Current hybridization protocols on microarrays are slow and need skilled personnel. Microfluidics is an emerging science that enables the processing of minute volumes of liquids to perform chemical, biochemical, or enzymatic analyzes. The merging of microfluidics and microarray technologies constitutes an elegant solution that will automate and speed up microarray hybridization. METHODS: We developed a microfluidic flow cell consisting of a network of chambers and channels molded into a polydimethylsiloxane substrate. The substrate was aligned and reversibly bound to the microarray printed on a standard glass slide to form a functional microfluidic unit. The microfluidic units were placed on an engraved, disc-shaped support fixed on a rotational device. Centrifugal forces drove the sample and buffers directly onto the microarray surface. RESULTS: This microfluidic system increased the hybridization signal by approximately 10fold compared with a passive system that made use of 10 times more sample. By means of a 15-min automated hybridization process, performed at room temperature, we demonstrated the discrimination of 4 clinically relevant Staphylococcus species that differ by as little as a single-nucleotide polymorphism. This process included hybridization, washing, rinsing, and drying steps and did not require any purification of target nucleic acids. This platform was sensitive enough to detect 10 PCR-amplified bacterial genomes. CONCLUSION: This removable microfluidic system for performing microarray hybridization on glass slides is promising for molecular diagnostics and gene profiling.

Correlation between microarray DNA hybridization efficiency and the position of short capture probe on the target nucleic acid.

The hybridization behavior of small oligonucleotides arrayed on glass slides is currently unpredictable. In order to examine the hybridization efficiency of capture probes along target nucleic acid, 20-mer oligonucleotide probes were designed to hybridize at different distances from the 5' end of two overlapping 402- and 432-bp ermB products amplified from the target DNA. These probes were immobilized via their 5' end onto glass slides and hybridized with the two labeled products. Evaluation of the hybridization signal for each probe revealed an inverse correlation with the length of the 5' overhanging end of the captured strand and the hybridization signal intensity. Further experiments demonstrated that this phenomenon is dependent on the reassociation kinetics of the free overhanging tail of the captured DNA strand with its complementary strand. This study delineates key predictable parameters that govern the hybridization efficiency of short capture probes arrayed on glass slides. This should be most useful for designing arrays for detection of PCR products and nucleotide polymorphisms.