Field-based detection of bacteria using nanopore sequencing: Method evaluation for biothreat detection in complex samples.

From pathogen detection to genome or plasmid closure, the utility of the Oxford Nanopore Technologies (ONT) MinION for microbiological analysis has been well documented. The MinION's small footprint, portability, and real-time analytic capability situates it well to address challenges in the field of unbiased pathogen detection, as a component of a security investigation. To this end, a multicenter evaluation of the effect of alternative analytical approaches on the outcome of MinION-based sequencing, using a set of well-characterized samples, was explored in a field-based scenario. Three expert scientific response groups evaluated known bacterial DNA extracts as part of an international first responder (Chemical, Biological, Radiological) training exercise. Samples were prepared independently for analysis using the Rapid and/or Rapid PCR sequencing kits as per the best practices of each of the participating groups. Analyses of sequence data were in turn conducted using varied approaches including ONTs What's in my pot (WIMP) architecture and in-house computational pipelines. Microbial community composition and the ability of each approach to detect pathogens was compared. Each group demonstrated the ability to detect all species present in samples, although several organisms were detected at levels much lower than expected with some organisms even falling below 1% abundance. Several 'contaminant' near neighbor species were also detected, at low abundance. Regardless of the sequencing approach chosen, the observed composition of the bacterial communities diverged from the input composition in each of the analyses, although sequencing conducted using the rapid kit produced the least distortion when compared to PCR-based library preparation methods. One of the participating groups generated drastically lower sequencing output than the other groups, likely attributed to the limited computer hard drive capacity, and occasional disruption of the internet connection. These results provide further consideration for conducting unbiased pathogen identification within a field setting using MinION sequencing. However, the benefits of this approach in providing rapid results and unbiased detection must be considered along with the complexity of sample preparation and data analytics, when compared to more traditional methods. When utilized by trained scientific experts, with appropriate computational resources, the MinION sequencing device is a useful tool for field-based pathogen detection in mixed samples.

Comparison of fifteen SARS-CoV-2 nucleic acid amplification test assays used during the Canadian Laboratory Response Network's National SARS-CoV-2 Proficiency Program, May 2020 to June 2021.

Background: On March 11, 2020, the World Health Organization declared a pandemic caused by the recently emerged severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). This led to increased clinical testing and decentralizing of this testing from provincial health laboratories to regional and private facilities. Leveraging the results from the Canadian Laboratory Response Network's National SARS-CoV-2 Proficiency Test (PT) Program, this study compares multiple commercial and laboratory-developed nucleic acid amplification tests, assessing both sensitivity and specificity across multiple users. Methods: Each panel consisted of six blinded, contrived-clinical samples. Panels were distributed to international, provincial and territorial laboratories and subsequently to partner facilities. Participating laboratories were asked to run these sample through their respective extraction/PCR workflows and submit results to the National Microbiology Laboratory, outlining the nucleic acid extraction platform and nucleic acid amplification test employed, as well as the viral gene target and Ct values or equivalent obtained. Data were compiled for each molecular platform and gene target used. Results: The PT schemes were deployed in May 2020, November 2020 and June 2021, resulting in 683 data sets using 37 different nucleic acid amplification tests. Over the course of three PT schemes, the average score obtained was 99.3% by participants demonstrating consistent testing between laboratories and testing platforms. Conclusion: This study confirmed the rapid and successful implementation of a Canadian PT Program and provided comparative analysis of the various emergency use authorized and laboratory developed tests employed for the detection of SARS-CoV-2 and demonstrated an overall 99.3% test concordance nationwide.

Evolution of nucleic acid amplification testing across Canada as observed through the Canadian Laboratory Response Network's SARS-CoV-2 Proficiency Test Program, May 2020 to June 2021.

To help accommodate the surge in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) clinical testing due to the coronavirus disease 2019 pandemic, the decentralization of testing from provincial public health laboratories to regional laboratories and private facilities was necessary. To further support the growing number of test sites in Canada, the National Microbiology Laboratory developed a proficiency test program for the detection of SARS-CoV-2 using nucleic acid amplification tests and administered it under an arm of the Canadian Laboratory Response Network (CLRN). Since its conception in May 2020, CLRN has conducted three proficiency test schemes, from May 2020 to June 2021, and has observed an increase in participation of more than 400%. This article will explore the evolution of CLRN's SARS-CoV-2 Proficiency Test Program and its support of the Canadian pandemic response.

A four specimen-pooling scheme reliably detects SARS-CoV-2 and influenza viruses using the BioFire FilmArray Respiratory Panel 2.1.

The COVID-19 pandemic required increased testing capacity, enabling rapid case identification and effective contract tracing to reduce transmission of disease. The BioFire FilmArray is a fully automated nucleic acid amplification test system providing specificity and sensitivity associated with gold standard molecular methods. The FilmArray Respiratory Panel 2.1 targets 22 viral and bacterial pathogens, including SARS-CoV-2 and influenza virus. While each panel provides a robust output of information regarding pathogen detection, the specimen throughput is low. This study evaluates the FilmArray Respiratory Panel 2.1 using 33 pools of contrived nasal samples and 22 pools of clinical nasopharyngeal specimens to determine the feasibility of increasing testing capacity, while maintaining detection of both SARS-CoV-2 and influenza virus. We observed 100% detection and 90% positive agreement for SARS-CoV-2 and 98% detection and 95% positive agreement for influenza viruses with pools of contrived or clinical specimens, respectively. While discordant results were mainly attributed to loss in sensitivity, the sensitivity of the pooling assay was well within accepted limits of detection for a nucleic acid amplification test. Overall, this study provides evidence supporting the use of pooling patient specimens, one in four with the FilmArray Respiratory Panel 2.1 for the detection of SARS-CoV-2 and influenza virus.

Salivary testing of COVID-19: evaluation of serological testing following positive salivary results.

BACKGROUND: Salivary detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been proposed as an alternative to nasopharyngeal or oropharyngeal swab testing. Our group previously published a study demonstrating that both testing methods identified SARS-CoV-2 using polymerase chain reaction (PCR)-based detection methodology. We therefore conducted a follow-up study using antibody testing to evaluate the accuracy of saliva versus swabs for COVID-19 detection and the durability of antibody response. METHODS: Venous blood samples were collected from consenting participants and the presence of serum antibodies for SARS-CoV-2 was evaluated on a large, automated immunoassay platform by the Roche anti-SARS-CoV-2 qualitative assay (Roche Diagnostics, Laval Quebec). Individuals with a serum antibody cut-off index (COI) ≥ 1.0 were considered positive. RESULTS: In asymptomatic and mildly symptomatic patients with a previously positive standard swab and/or saliva SARS-CoV-2 PCR-test, 42 demonstrated antibodies with 13 patients positive by swab alone, and 8 patients positive by saliva alone. CONCLUSIONS: Despite their status as 'current standard' for COVID-19 testing, these findings highlight limitations of PCR-based tests.

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry-based identification of security-sensitive bacteria: Considerations for Canadian Bruker users.

BACKGROUND: The use of matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) systems for bacterial identification has rapidly become a front line tool for diagnostic laboratories, superseding classical microbiological methods that previously triggered the identification of higher risk pathogens. Unknown Risk Group 3 isolates have been misidentified as less pathogenic species due to spectral library availability, content and quality. Consequently, exposure to higher risk pathogens has been reported within Canadian laboratory staff following the implementation of MALDI-TOF MS. This overview aims to communicate the potential risk to laboratory staff of inaccurate identification of security-sensitive biological agents (SSBA) bacteria and to provide suggestions to mitigate. METHODS: Cultures were manipulated in a Biosafety Level 3 laboratory, prepared for MALDI-TOF MS analysis via full chemical extraction and analysed on a Bruker Microflex LT instrument. Data were analyzed with Biotyper software; comparing raw spectra against MS profiles in three libraries: Bruker Taxonomy; Bruker Security-Restricted; and National Microbiology Laboratory (NML) SSBA libraries. Four years of Bruker MALDI-TOF MS data acquired in-house were reviewed. RESULTS: In general, the Bruker MS spectral libraries were less successful in identifying the SSBA bacteria. More successful was the NML library. For example, using a high score cut-off (greater than 2.0), the Bruker SR library was unable to identify 52.8% of our Risk Group 3 agents and near neighbours to the species-level with confidence, whereas the custom NML library was unable to identify only 20.3% of the samples. CONCLUSION: The last four years of data demonstrated both the importance of library selection and the limitations of the various spectral libraries. Enhanced standard operating procedures are advised to reduce laboratory exposure to SSBAs when using MALDI-TOF MS as a front line identification tool.

Dual RNA-Seq characterization of host and pathogen gene expression in liver cells infected with Crimean-Congo Hemorrhagic Fever Virus.

Crimean-Congo hemorrhagic fever virus (CCHFV) is a tick-borne virus that can cause a hemorrhagic fever in humans, with a case fatality rate of up to 40%. Cases of CCHFV have been reported in Africa, Asia, and southern Europe; and recently, due to the expanding range of its vector, autochthonous cases have been reported in Spain. Although it was discovered over 70 years ago, our understanding of the pathogenesis of this virus remains limited. We used RNA-Seq in two human liver cell lines (HepG2 and Huh7) infected with CCHFV (strain IbAr10200), to examine kinetic changes in host expression and viral replication simultaneously at 1 and 3 days post infection. Through this, numerous host pathways were identified that were modulated by the virus including: antiviral response and endothelial cell leakage. Notably, the genes encoding DDX60, a cytosolic component of the RIG-I signalling pathway and OAS2 were both shown to be dysregulated. Interestingly, PTPRR was induced in Huh7 cells but not HepG2 cells. This has been associated with the TLR9 signalling cascade, and polymorphisms in TLR9 have been associated with poor outcomes in patients. Additionally, we performed whole-genome sequencing on CCHFV to assess viral diversity over time, and its relationship to the host response. As a result, we have demonstrated that through next-generation mRNA deep-sequencing it is possible to not only examine mRNA gene expression, but also to examine viral quasispecies and typing of the infecting strain. This demonstrates a proof-of-principle that CCHFV specimens can be analyzed to identify both the virus and host biomarkers that may have implications for prognosis.

Mycobacterium chimaera Encephalitis Following Cardiac Surgery: A New Syndrome.

We report the cases of 3 patients with fatal, disseminated Mycobacterium chimaera infections following cardiac surgeries. Progressive neurocognitive decline and death were explained by active granulomatous encephalitis, with widespread involvement of other organs. This syndrome is clinically elusive and, thus, may have caused deaths in prior reported series.

Incidental post-partum isolation of Brucella melitensis in an asymptomatic woman.

A 25-year-old Somali-born female was admitted to the hospital in active labour. Following post-partum hemorrhage, Brucella melitensis grew from a blood culture and the placenta. Identification and relatedness were determined through reverse transcriptase polymerase chain reaction (RT-PCR), single nucleotide polymorphism (SNP), and whole genome sequencing. The patient and her child were completely asymptomatic at their initial assessment.

Une femme d'origine somalienne de 25 ans a été hospitalisée en travail actif. Après une hémorragie postpartum, le Brucella melitensis s'est développé dans une hémoculture et dans le placenta. La réaction de transcriptase inverse et d'amplification en chaîne de la polymérase (RT-PCR), le polymorphisme du nucléotide simple (SNP) et le séquençage du génome entier ont permis d'identifier la bactérie et d'établir sa parenté génétique. La patiente et son enfant étaient complètement asymptomatiques à l'évaluation initiale.

Evaluation of Oxford Nanopore's MinION Sequencing Device for Microbial Whole Genome Sequencing Applications.

The MinION sequencer (Oxford Nanopore Technologies) is a paradigm shifting device allowing rapid, real time long read sequencing of nucleic acids. Yet external benchmarking of this technologies' capabilities has not been extensively reported, nor has thorough evaluation of its utility for field-based analysis with sub-optimal sample types been described. The aim of this study was to evaluate the capability of the MinION sequencer for bacterial genomic and metagenomic applications, with specific emphasis placed on the quality, yield, and accuracy of generated sequence data. Two independent laboratories at the National Microbiology Laboratory (Public Health Agency of Canada), sequenced a set of microbes in replicate, using the currently available flowcells, sequencing chemistries, and software available at the time of the experiment. Overall sequencing yield and quality improved through the course of this set of experiments. Sequencing alignment accuracy was high reaching 97% for all 2D experiments, though was slightly lower for 1D sequencing (94%). 1D sequencing provided much longer sequences than 2D. Both sequencing chemistries performed equally well in constructing genomic assemblies. There was evidence of barcode cross-over using both the native and PCR barcoding methods. Despite the sub-optimal nature of samples sequenced in the field, sequences attributable to B. anthracis the target organism used in this scenario, could none-the-less be detected. Together, this report showcases the rapid advancement in this technology and its utility in the context of genomic sequencing of microbial isolates of importance to public health.

MALDI-TOF mass spectrometry and high-consequence bacteria: safety and stability of biothreat bacterial sample testing in clinical diagnostic laboratories.

We considered the application of MALDI-TOF mass spectrometry for BSL-3 bacterial diagnostics, with a focus on the biosafety of live-culture direct-colony testing and the stability of stored extracts. Biosafety level 2 (BSL-2) bacterial species were used as surrogates for BSL-3 high-consequence pathogens in all live-culture MALDI-TOF experiments. Viable BSL-2 bacteria were isolated from MALDI-TOF mass spectrometry target plates after 'direct-colony' and 'on-plate' extraction testing, suggesting that the matrix chemicals alone cannot be considered sufficient to inactivate bacterial culture and spores in all samples. Sampling of the instrument interior after direct-colony analysis did not recover viable organisms, suggesting that any potential risks to the laboratory technician are associated with preparation of the MALDI-TOF target plate before or after testing. Secondly, a long-term stability study (3 years) of stored MALDI-TOF extracts showed that match scores can decrease below the threshold for reliable species identification (<1.7), which has implications for proficiency test panel item storage and distribution.

Application of whole genome sequence analysis to the study of Mycobacterium tuberculosis in Nunavut, Canada.

Canada has one of the lowest rates of tuberculosis (TB) in the world, however, among certain sub-populations, disease incidence rates approach those observed in sub-Saharan Africa, and other high incidence regions. In this study, we applied mycobacterial interspersed repetitive unit (MIRU) variable number of tandem repeat (VNTR) and whole genome sequencing (WGS) to the analysis of Mycobacterium tuberculosis isolates obtained from Northern communities in the territory of Nunavut. WGS was carried out using the Illumina MiSeq, with identified variants used to infer phylogenetic relationships and annotated to infer functional implications. Additionally, the sequencing data from these isolates were augmented with publically available WGS to evaluate data from the Nunavut outbreak in the broader Canadian context. In this study, isolates could be classified into four major clusters by MIRU-VNTR analysis. These could be further resolved into sub-clusters using WGS. No evidence for antimicrobial resistance, either genetic or phenotypic, was observed in this cohort. Among most subjects with multiple samples, reactivation/incomplete treatment likely contributed to recurrence. However, isolates from two subjects appeared more likely to have occurred via reinfection, based on the large number of genomic single nucleotide variants detected. Finally, although quite distinct from previously reported Canadian MTB strains, isolates obtained from Nunavut clustered most closely with a cohort of samples originating in the Nunavik region of Northern Quebec. This study demonstrates the benefit of using WGS for discriminatory analysis of MTB in Canada, especially in high incidence regions. It further emphasizes the importance of focusing epidemiological intervention efforts on interrupting transmission chains of endemic TB throughout Northern communities, rather than relying on strategies applied in regions where the majority of TB cases result from importation of foreign strains.

Custom database development and biomarker discovery methods for MALDI-TOF mass spectrometry-based identification of high-consequence bacterial pathogens.

A high-quality custom database of MALDI-TOF mass spectral profiles was developed with the goal of improving clinical diagnostic identification of high-consequence bacterial pathogens. A biomarker discovery method is presented for identifying and evaluating MALDI-TOF MS spectra to potentially differentiate biothreat bacteria from less-pathogenic near-neighbour species.

Bacillus cereus Biovar Anthracis Causing Anthrax in Sub-Saharan Africa-Chromosomal Monophyly and Broad Geographic Distribution.

Through full genome analyses of four atypical Bacillus cereus isolates, designated B. cereus biovar anthracis, we describe a distinct clade within the B. cereus group that presents with anthrax-like disease, carrying virulence plasmids similar to those of classic Bacillus anthracis. We have isolated members of this clade from different mammals (wild chimpanzees, gorillas, an elephant and goats) in West and Central Africa (Côte d'Ivoire, Cameroon, Central African Republic and Democratic Republic of Congo). The isolates shared several phenotypic features of both B. anthracis and B. cereus, but differed amongst each other in motility and their resistance or sensitivity to penicillin. They all possessed the same mutation in the regulator gene plcR, different from the one found in B. anthracis, and in addition, carry genes which enable them to produce a second capsule composed of hyaluronic acid. Our findings show the existence of a discrete clade of the B. cereus group capable of causing anthrax-like disease, found in areas of high biodiversity, which are possibly also the origin of the worldwide distributed B. anthracis. Establishing the impact of these pathogenic bacteria on threatened wildlife species will require systematic investigation. Furthermore, the consumption of wildlife found dead by the local population and presence in a domestic animal reveal potential sources of exposure to humans.

Ferrets Infected with Bundibugyo Virus or Ebola Virus Recapitulate Important Aspects of Human Filovirus Disease.

UNLABELLED: Bundibugyo virus (BDBV) is the etiological agent of a severe hemorrhagic fever in humans with a case-fatality rate ranging from 25 to 36%. Despite having been known to the scientific and medical communities for almost 1 decade, there is a dearth of studies on this pathogen due to the lack of a small animal model. Domestic ferrets are commonly used to study other RNA viruses, including members of the order Mononegavirales To investigate whether ferrets were susceptible to filovirus infections, ferrets were challenged with a clinical isolate of BDBV. Animals became viremic within 4 days and succumbed to infection between 8 and 9 days, and a petechial rash was observed with moribund ferrets. Furthermore, several hallmarks of human filoviral disease were recapitulated in the ferret model, including substantial decreases in lymphocyte and platelet counts and dysregulation of key biochemical markers related to hepatic/renal function, as well as coagulation abnormalities. Virological, histopathological, and immunohistochemical analyses confirmed uncontrolled BDBV replication in the major organs. Ferrets were also infected with Ebola virus (EBOV) to confirm their susceptibility to another filovirus species and to potentially establish a virus transmission model. Similar to what was seen with BDBV, important hallmarks of human filoviral disease were observed in EBOV-infected ferrets. This study demonstrates the potential of this small animal model for studying BDBV and EBOV using wild-type isolates and will accelerate efforts to understand filovirus pathogenesis and transmission as well as the development of specific vaccines and antivirals. IMPORTANCE: The 2013-2016 outbreak of Ebola virus in West Africa has highlighted the threat posed by filoviruses to global public health. Bundibugyo virus (BDBV) is a member of the genus Ebolavirus and has caused outbreaks in the past but is relatively understudied, likely due to the lack of a suitable small animal model. Such a model for BDBV is crucial to evaluating vaccines and therapies and potentially understanding transmission. To address this, we demonstrated that ferrets are susceptible models to BDBV infection as well as to Ebola virus infection and that no virus adaptation is required. Moreover, these animals develop a disease that is similar to that seen in humans and nonhuman primates. We believe that this will improve the ability to study BDBV and provide a platform to test vaccines and therapeutics.

ECO-EPIZOOTIOLOGIC STUDY OF FRANCISELLA TULARENSIS, THE AGENT OF TULAREMIA, IN QUÉBEC WILDLIFE.

In Canada, Francisella tularensis , the zoonotic bacterial agent of tularemia, affects mostly snowshoe hares ( Lepus americanus ), muskrats ( Ondatra zibethicus ), and beavers ( Castor canadensis ). Despite numerous studies, the ecologic cycle and natural reservoirs of F. tularensis are not clearly defined. We conducted a cross-sectional study to estimate the prevalence of F. tularensis in snowshoe hares, muskrats, and coyotes ( Canis latrans ) in four regions of Québec, Canada, and to describe the risk of infection in relation to host and environmental characteristics at three spatial scales. Between October 2012 and April 2013, trappers captured 345 snowshoe hares, 411 muskrats, and 385 coyotes. Blood samples were tested by microagglutination tests, and DNA extracts of liver, kidney, lung, and spleen of snowshoe hares and muskrats were tested by real-time PCR to detect past and active infection to F. tularensis , respectively. Individual host characteristics, including body condition, age, and sex, were evaluated as risk factors of infection, along with ecologic characteristics of the location of capture extracted from geographic databases. Prevalences of antibody to F. tularensis and 95% confidence intervals were 2.9% (1.4-5.1%) in coyotes, 0.6% (0.1-2.1%) in hares, and 0% (0.0-0.9%) in muskrats. Francisella tularensis DNA was not detected by real-time PCR in the pools of four organs from muskrats and hares, but F. tularensis type AI was detected during testing of the individual organs of two antibody-positive hares. Exact logistic regression analyses showed that age was a significant predictor of antibody detection in coyotes, as were the proportion of forest and the proportion of area considered as suitable habitat for hares in the environment around the location of capture of the coyotes. Our results suggest a terrestrial cycle of F. tularensis in the regions studied.

Verification of a Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry Method for Diagnostic Identification of High-Consequence Bacterial Pathogens.

We examined the utility of a single matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry method for the identification of security-sensitive biological agents (risk group 3 bacterial pathogens). The goal was 2-fold: to verify a method for inclusion into our scope of accreditation, and to assess the biological safety of extractions. We developed our sample flow to include a tube-based chemical extraction, followed by filtration, before processing on MALDI-TOF MS instruments in a containment level 2 laboratory.

Sylvatic plague in a Canadian black-tailed prairie dog (Cynomys ludovicianus).

In 2010, a black-tailed prairie dog (Cynomys ludovicianus) was found dead in Grasslands National Park, Saskatchewan, Canada. Postmortem gross and histologic findings indicated bacterial septicemia, likely due to Yersinia pestis, which was confirmed by molecular analysis. This is the first report of Y. pestis in the prairie dog population within Canada.