Real-Time RT-PCR Allelic Discrimination Assay for Detection of N501Y Mutation in the Spike Protein of SARS-CoV-2 Associated with B.1.1.7 Variant of Concern.

The N501Y amino acid mutation caused by a single point substitution A23063T in the spike gene of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is possessed by three variants of concern (VOCs), B.1.1.7, B.1.351, and P.1. A rapid screening tool using this mutation is important for surveillance during the coronavirus disease 2019 (COVID-19) pandemic. We developed and validated a single nucleotide polymorphism real-time reverse transcription PCR assay using allelic discrimination of the spike gene N501Y mutation to screen for potential variants of concern and differentiate them from SARS-CoV-2 lineages without the N501Y mutation. A total of 160 clinical specimens positive for SARS-CoV-2 were characterized as mutant (N501Y) or N501 wild type by Sanger sequencing and were subsequently tested with the N501Y single nucleotide polymorphism real-time reverse transcriptase PCR assay. Our assay, compared to Sanger sequencing for single nucleotide polymorphism detection, demonstrated positive percent agreement of 100% for all 57 specimens displaying the N501Y mutation, which were confirmed by Sanger sequencing to be typed as A23063T, including one specimen with mixed signal for wild type and mutant. Negative percent agreement was 100% in all 103 specimens typed as N501 wild type, with A23063 identified as wild type by Sanger sequencing. The identification of circulating SARS-CoV-2 lineages carrying an N501Y mutation is critical for surveillance purposes. Current identification methods rely primarily on Sanger sequencing or whole-genome sequencing, which are time consuming, labor intensive, and costly. The assay described herein is an efficient tool for high-volume specimen screening for SARS-CoV-2 VOCs and for selecting specimens for confirmatory Sanger or whole-genome sequencing. IMPORTANCE During the coronavirus disease 2019 (COVID-19) pandemic, several variants of concern (VOCs) have been detected, for example, B.1.1.7, B.1.351, P.1, and B.1.617.2. The VOCs pose a threat to public health efforts to control the spread of the virus. As such, surveillance and monitoring of these VOCs is of the utmost importance. Our real-time RT-PCR assay helps with surveillance by providing an easy method to quickly survey SARS-CoV-2 specimens for VOCs carrying the N501Y single nucleotide polymorphism (SNP). Samples that test positive for the N501Y mutation in the spike gene with our assay can be sequenced to identify the lineage. Thus, our assay helps to focus surveillance efforts and decrease turnaround times.

Impact of coronavirus disease 2019 (COVID-19) pre-test probability on positive predictive value of high cycle threshold severe acute respiratory coronavirus virus 2 (SARS-CoV-2) real-time reverse transcription polymerase chain reaction (RT-PCR) test results.

OBJECTIVES: Performance characteristics of SARS-CoV-2 nucleic acid detection assays are understudied within contexts of low pre-test probability, including screening asymptomatic persons without epidemiological links to confirmed cases, or asymptomatic surveillance testing. SARS-CoV-2 detection without symptoms may represent presymptomatic or asymptomatic infection, resolved infection with persistent RNA shedding, or a false-positive test. This study assessed the positive predictive value of SARS-CoV-2 real-time reverse transcription polymerase chain reaction (rRT-PCR) assays by retesting positive specimens from 5 pre-test probability groups ranging from high to low with an alternate assay. METHODS: In total, 122 rRT-PCR positive specimens collected from unique patients between March and July 2020 were retested using a laboratory-developed nested RT-PCR assay targeting the RNA-dependent RNA polymerase (RdRp) gene followed by Sanger sequencing. RESULTS: Significantly fewer (15.6%) positive results in the lowest pre-test probability group (facilities with institution-wide screening having ≤3 positive asymptomatic cases) were reproduced with the nested RdRp gene RT-PCR assay than in each of the 4 groups with higher pre-test probability (individual group range, 50.0%-85.0%). CONCLUSIONS: Large-scale SARS-CoV-2 screening testing initiatives among low pre-test probability populations should be evaluated thoroughly prior to implementation given the risk of false-positive results and consequent potential for harm at the individual and population level.

Characteristics of SARS-CoV-2 testing for rapid diagnosis of COVID-19 during the initial stages of a global pandemic.

Accurate SARS-CoV-2 diagnosis is essential to guide prevention and control of COVID-19. Here we examine SARS-CoV-2 molecular-based test performance characteristics and summarize case-level data related to COVID-19 diagnosis. From January 11 through April 22, 2020, Public Health Ontario conducted SARS-CoV-2 testing of 86,942 specimens collected from 80,354 individuals, primarily using real-time reverse-transcription polymerase chain reaction (rRT-PCR) methods. We analyzed test results across specimen types and for individuals with multiple same-day and multi-day collected specimens. Nasopharyngeal compared to throat swabs had a higher positivity (8.8% vs. 4.8%) and an adjusted estimate 2.9 Ct lower (SE = 0.5, p<0.001). Same-day specimens showed high concordance (98.8%), and the median Ct of multi-day specimens increased over time. Symptomatic cases had rRT-PCR results with an adjusted estimate 3.0 Ct (SE = 0.5, p<0.001) lower than asymptomatic/pre-symptomatic cases. Overall test sensitivity was 84.6%, with a negative predictive value of 95.5%. Molecular testing is the mainstay of SARS-CoV-2 diagnosis and testing protocols will continue to be dynamic and iteratively modified as more is learned about this emerging pathogen.

Correction: Evidence of transmission of Clostridium difficile in asymptomatic patients following admission screening in a tertiary care hospital.

[This corrects the article DOI: 10.1371/journal.pone.0207138.].

Evidence of transmission of Clostridium difficile in asymptomatic patients following admission screening in a tertiary care hospital.

BACKGROUND: Clostridium difficile (CD) is the leading cause of infectious health-care associated diarrhea. However, little is known regarding CD carriage and transmission amongst asymptomatic colonizers. We evaluated carriage, characterized strains and examined epidemiologic linkages in asymptomatic colonized CD patients. METHODS: Rectal swabs from asymptomatic patients admitted to the general medicine ward from April 1-June 30 2012 were collected. PCR-confirmed CD colonies were ribotyped and characterized by Modified-Multi Locus Variable Number Tandem Repeat Analysis (MMLVA). RESULTS: 1549-swabs were collected from 474-patients. Overall, 50/474(10.6%) were CD PCR-positive, 24/50 were colonized at admission, while 26/50 were first identified > = 72 hours after admission. Amongst the 50 CD PCR-positive patients, 90% were asymptomatically colonized and 80% of individuals carried toxigenic CD-strains, including ribotype-027 (5/45:11%). MMLVA revealed five-clusters involving 15-patients harboring toxigenic (4/5) and non-toxigenic CD strains (1/5). In two clusters, patients were CD positive on admission while in the other three clusters involving 10 patients, we observed CD transmission from asymptomatically colonized patients to 8 previously CD-negative patients. CONCLUSIONS: We identified increasing rates of colonization during admission to medical wards. MMLVA typing effectively discriminated between strains and suggests that 20% of patients with CD colonization acquired their strain(s) from asymptomatically colonized individuals in hospital.

Increased environmental sample area and recovery of Clostridium difficile spores from hospital surfaces by quantitative PCR and enrichment culture.

OBJECTIVE: Clostridium difficile spores play an important role in transmission and can survive in the environment for several months. Optimal methods for measuring environmental C. difficile are unknown. We sought to determine whether increased sample surface area improved detection of C. difficile from environmental samples. SETTING: Samples were collected from 12 patient rooms in a tertiary-care hospital in Toronto, Canada. METHODS: Samples represented small surface-area and large surface-area floor and bedrail pairs from single-bed rooms of patients with low (without prior antibiotics), medium (with prior antibiotics), and high (C. difficile infected) shedding risk. Presence of C. difficile in samples was measured using quantitative polymerase chain reaction (qPCR) with targets on the 16S rRNA and toxin B genes and using enrichment culture. RESULTS: Of the 48 samples, 64·6% were positive by 16S qPCR (geometric mean, 13·8 spores); 39·6% were positive by toxin B qPCR (geometric mean, 1·9 spores); and 43·8% were positive by enrichment culture. By 16S qPCR, each 10-fold increase in sample surface area yielded 6·6 times (95% CI, 3·2-13) more spores. Floor surfaces yielded 27 times (95% CI, 4·9-181) more spores than bedrails, and rooms of C. difficile-positive patients yielded 11 times (95% CI, 0·55-164) more spores than those of patients without prior antibiotics. Toxin B qPCR and enrichment culture returned analogous findings. CONCLUSIONS: Clostridium difficile spores were identified in most floor and bedrail samples, and increased surface area improved detection. Future research aiming to understand the role of environmental C. difficile in transmission should prefer samples with large surface areas.

Comparison of qPCR versus culture for the detection and quantification of Clostridium difficile environmental contamination.

Contaminated surfaces serve as an important reservoir for Clostridium difficile transmission. Current strategies to detect environmental contamination of C. difficile rely heavily on culture, and often only indicate presence versus absence of spores. The goal of this study was to compare quantitative PCR (qPCR) to culture for the detection and quantification of C. difficile from inert surfaces. First, we compared the limit of detection (LOD) of a 16S rRNA gene and toxin B gene qPCR assay for detection of C. difficile in solution. Second, we compared the LODs of 16S rRNA gene qPCR versus culture for detection of C. difficile from surfaces. Solution experiments were performed by direct seeding of spores into neutralizing broth, whereas surface experiments involved seeding of spores onto plastic test surfaces, and recovery using sponge swabs. Both experiments were conducted using spores expressing short (NAP1) and long (NAP4) hair lengths. Combining data from both strains, the overall LOD for C. difficile cells in solution was 1.4 cells for 16S rRNA gene and 23.6 cells for toxin B gene qPCR (p<0.001). The overall LOD for C. difficile cells from surfaces was 17.1 cells for 16S rRNA gene qPCR and 54.5 cells for culture (p = 0.05), and was not statistically different between strains for each method (p = 0.52). Overall, the proportion of C. difficile cells recovered from surfaces was good when detected by 16S rRNA gene qPCR and culture (qPCR: 76%, culture: 67%, p = 0.36), but, 16S rRNA gene qPCR was capable of detecting lower levels of surface contamination. Future work attempting to measure the presence of C. difficile on environmental surfaces should consider using qPCR.

Using Dog Scent Detection as a Point-of-Care Tool to Identify Toxigenic Clostridium difficile in Stool.

We evaluated the operating characteristics of 2 comparably trained dogs as a "point-of-care" diagnostic tool to detect toxin gene-positive Clostridium difficile. Although each dog could detect toxin gene-positive C difficile in stool specimens with sensitivities of 77.6 and 92.6 and specificities of 85.1 and 84.5, respectively, interrater reliability is only modest (Cohen's kappa 0.52), limiting widespread application.

Indeterminate tcdB using a Clostridium difficile PCR assay: a retrospective cohort study.

BACKGROUND: C. difficile (CD) real-time polymerase chain reaction (PCR) for toxin B gene (tcdB) is more sensitive, and reduces turnaround time when compared to toxin immunoassay. We noted typical amplification curves with high tcdB cycle thresholds (Ct) and low endpoints (Ept) that are labeled negative by the Xpert(®) C. difficile assay (Cepheid) and undertook this study to determine their significance. METHODS: We defined an indeterminate CD assay result as detection of a typical PCR amplification curve with an Ept >10 that was interpreted as negative by the Xpert(®) assay. Samples with indeterminate Xpert(®) result were collected for 5 months and retested by Xpert(®), cultured for toxigenic CD, and isolates subjected to PCR ribotyping, detection of toxin genes and multilocus variable-number tandem repeat analysis (MLVA) typing. Chart reviews were completed to assess if patients met the Society of Healthcare Epidemiology of America and the Infectious Diseases Society of America CD infection (CDI) clinical case definition. Illness severity was compared with tcdB Ct and culture results. RESULTS: During the 5-month study period, 48/3620 (1%) of specimens were indeterminate and 387/3620 (11%) were positive. Of the 48 patients with indeterminate results, 39 (81%) met the clinical case definition of CDI, and 7 of these (18%) met criteria for severe CDI. Toxigenic stool cultures were positive for 86% (6/7) of patients with severe CDI, 19% (6/32) of patients with non-severe CDI, and 44% (4/9) of patients who did not meet the clinical case definition of CDI (p = 0.002). Lower tcdB Ct and higher Ept were associated with greater likelihood of toxigenic culture positivity (p = 0.03) and more severe symptoms (p = 0.06). Indeterminate results were not associated with a particular technologist or instrument module, or CD strain type. CONCLUSIONS: A subset of specimens (1%) using the Xpert(®) C. difficile assay have typical amplification curves and are interpreted as negative. At least one-third of these results are associated with positive CD culture. The mechanism of these indeterminate results is not technique-related, equipment-related, or due to particular CD strains. Clinicians should be aware that even PCR testing has the potential to miss CDI cases and further highlights the importance of clinical context when interpreting results.

Real-time polymerase chain reaction method for detection of toxigenic Clostridium difficile from stools and presumptive identification of NAP1 clone.

This study describes the development of a cost-effective, multiplex real-time polymerase chain reaction (RTPCR) method for detection of toxigenic Clostridium difficile from stools and presumptive identification of the NAP-1 strain. The diagnostic value of the new method is for the detection of toxigenic C. difficile which has the following performance characteristics: 99.8% specificity, 95.1% sensitivity, 97.5% positive predictive value, and 99.5% negative predictive value. Examination of 24 specimens presumptively identified as NAP1 strain by RTPCR with Pulsed-field gel electrophoresis performed on C. difficile isolated from those specimens showed 100% agreement. This RTPCR showed equivalent test performance characteristics as the 2 commercially available assays which were evaluated. The estimated cost per test is CAD$9.50 and which is significantly less than the commercial assays. The average turnaround time from setup to detection is 3.5 h. The RTPCR method described here is a cost-effective and highly sensitive test which can be implemented in a clinical laboratory to assist clinicians in establishing the diagnosis of C. difficile infection and indirectly determine the presence of the hypervirulent epidemic binary toxin (BI)/NAP 1 strain for prompt infection control interventions.

Leprosy-like illness in a patient with Mycobacterium lepromatosis from Ontario, Canada.

Here we present the first case of a patient from Ottawa Canada, presenting with leprosy-like illness associated with Mycobacterium lepromatosis. The patient had no history of travel to leprosy-endemic areas or any obvious risk factors. Clinically, the patient presented with an anesthetic maculopapular rash on the trunk, back, and extremities. A skin biopsy of a lesion revealed a dermal lymphohistiocytic infiltration involving the vessels with an inflammatory process extending to the nerves. A neurological exam also identified a severe sensorimotor polyneuropathy. Concurrently, the patient was diagnosed with non-resectable, non small cell carcinoma of the lung, further complicating his clinical presentation. A Kinyoun stain of nasal blows and a Fite stain of the skin biopsy revealed few to moderate acid fast bacilli respectively. Cultures of the skin biopsy and multiple nasal blows were negative. Molecular studies of a skin biopsy sample including sequence analysis of a 765 bp region of the 16s rRNA gene eventually identified the organism with 100% homology to M. lepromatosis. The patient was treated for leprosy and appeared to improve slightly on therapy but died as a result of his malignancy approximately five months after the initiation of therapy. This represents the first case of a patient with M. lepromatosis like illness outside of Mexico and Singapore.

Defining criteria to interpret multilocus variable-number tandem repeat analysis to aid Clostridium difficile outbreak investigation.

PFGE is currently the North American standard for surveillance for Clostridium difficile but lacks discriminatory power to aid outbreak investigation. A further limitation to PFGE is the high baseline rate of the epidemic North American pulsotype (NAP) 1 strain in hospitals. Multilocus variable-number tandem repeat analysis (MLVA) appears to have superior discriminatory power but criteria to define clonality have not been set. We conducted surveillance for toxin-positive C. difficile infection (CDI) at a single academic health sciences centre between September 2009 and April 2010. Seventy-four patient specimens resulting in 86 discrete CDI episodes were subjected to PFGE and MLVA. Results were analysed using Bionumerics software to generate phylogenetic trees and coupled to patient demographic data. Amongst the NAP1 strains, two distinct clusters were identified by MLVA using 90 % similarity as a cut-off by Manhattan distance-based clustering, four clusters using 95 % and seven clusters using 97 %. Population analysis conducted on multiple colonies (n = 25) demonstrated that 1-3 % difference in MLVA types was typical for a single individual. Typing was also conducted in the context of institutional outbreaks (n = 42, three outbreaks) in order to determine clusters within the NAP1 strain. By combining longitudinal surveillance with epidemiological information, single specimen population analysis and typing in the context of institutional outbreaks, we conclude that the use of the Manhattan distance-based clustering with a cut-off of 95-97 % is capable of distinguishing outbreak clones from sporadic isolates.

Modified multiple-locus variable-number tandem-repeat analysis for rapid identification and typing of Clostridium difficile during institutional outbreaks.

A modified multiple-locus variable-number tandem-repeat analysis (MMLVA) method was validated on Clostridium difficile-infected stool specimens from institutional outbreaks. The method allows simultaneous detection of toxin genes, deletions, and tandem repeats from cultured isolates or stool specimens. Results were used to aid institutional outbreak investigation by identifying clusters of NAP1/027.

The relative test performance characteristics of two commercial assays for the detection of Mycobacterium tuberculosis complex in paraffin-fixed human biopsy specimens.

The Seeplex TB Detection-2 assay (Rockville, MD) is a nested endpoint PCR for the Mycobacterium tuberculosis complex (MTBC) targets IS6110 and MPB64 that utilizes dual priming oligonucleotide technology. When used to detect the presence of MTBC DNA in formalin-fixed paraffin-embedded tissue specimens, the sensitivity and specificity of this assay is equivalent to a labor-intensive traditional endpoint PCR assay and is more sensitive than a commercial real-time PCR assay.

Comparison of a commercial qualitative real-time RT-PCR kit with direct immunofluorescence assay (DFA) and cell culture for detection of influenza A and B in children.

BACKGROUND: Institutional pandemic planning prompted a study of the molecular detection of influenza virus from respiratory specimens in children, compared to conventional diagnostics. OBJECTIVE: To evaluate the performance of a commercial qualitative real-time RT-PCR kit (rRT-PCR), the artus Influenza LC RT-PCR (Qiagen). STUDY DESIGN (METHODS): Specimens were pre-selected to include a high percentage of positives by direct immunofluorescence assay (DFA) or culture. The sensitivity and specificity of the kit for detection of influenza A and B in children were determined against the gold standard, DFA and culture. Specimens yielding discordant results between artus and the gold standard were tested against a reference rRT-PCR assay (Centers for Disease Control) to create an "expanded gold standard". RESULTS: When compared to DFA or cell culture, the sensitivity of the rRT-PCR artus kit was 96.2% and the specificity was 94%. It detected influenza RNA in 6.0% of clinical samples negative by DFA or culture. Using the expanded gold standard, the revised sensitivity was 98.7% (98.6% for influenza A and 97.6% for influenza B) and the specificity was 100%. CONCLUSION: The artus Influenza LC RT-PCR kit is an effective alternative to virus isolation and DFA for the detection of influenza A and B in pediatric clinical specimens.

Molecular typing of a Legionella pneumophila outbreak in Ontario, Canada.

An outbreak of Legionnaires' disease at a long-term care facility in Ontario, Canada from September to October 2005 resulted in the death of 23 residents and the illness of 112 other people. In response, molecular methods were developed to detect Legionella pneumophila in clinical lung samples and to subtype isolates from clinical and environmental samples. The targeted genetic loci included Legionella-specific virulence determinants (mip, icmO, sidA and lidA) and core bacterial determinants (ftsZ, trpS and dnaX). An established amplified fragment length polymorphism typing method provided the first indication of genetic relatedness between strains recovered from clinical samples and strains cultured from environmental samples taken from the outbreak site. These associations were verified using the European Working Group for Legionella Infections sequence-based typing protocol targeting the flaA, pilE, asd, mip, mompS and proA loci. These molecular typing methods confirmed the outbreak source as a contaminated air conditioning cooling tower.

Use of sequence-based typing for investigation of a case of nosocomial legionellosis.

A fatal case of nosocomial legionellosis in a low prevalence region (Calgary, Alberta, Canada) prompted investigation into the source of infection. Hospital water systems contaminated with Legionella pneumophila have been shown to pose a risk to compromised patients. Typing of an L. pneumophila serogroup 1 strain isolated from the patient using sequence-based typing (SBT) and amplified fragment length polymorphism (AFLP) analysis linked it to a persistent and widespread strain isolated from the hospital water system establishing a nosocomial mode of acquisition. Different SBT and AFLP patterns were determined for non-epidemiologically linked cases and isolates from different hospitals.

Triple reassortant H3N2 influenza A viruses, Canada, 2005.

Since January 2005, H3N2 influenza viruses have been isolated from pigs and turkeys throughout Canada and from a swine farmer and pigs on the same farm in Ontario. These are human/classical swine/avian reassortants similar to viruses that emerged in US pigs in 1998 but with a distinct human-lineage neuraminidase gene.

False-positive amplified Mycobacterium tuberculosis direct test results for samples containing Mycobacterium leprae.

Nucleic acid amplification tests are widely used in mycobacteriology laboratories to rapidly detect Mycobacterium tuberculosis complex directly in clinical specimens. A positive result provides an early diagnosis of tuberculosis, allowing initiation of appropriate therapy and public health measures.

Outbreak of acupuncture-associated cutaneous Mycobacterium abscessus infections.

BACKGROUND: Cutaneous atypical mycobacterial infections have been increasingly described in association with cosmetic and alternative procedures. OBJECTIVE: We report an outbreak of acupuncture-associated mycobacteriosis. Between April and December 2002, 32 patients developed cutaneous mycobacteriosis after visiting an acupuncture practice in Toronto, Canada. RESULTS: Of 23 patients whose lesions were biopsied, 6 (26.1%) had culture-confirmed infection with Mycobacterium abscessus. These isolates were genetically indistinguishable by amplified fragment length polymorphism. The median incubation period was 1 month. Of 24 patients for whom clinical information was available, 23 (95.8%) had resolution of their infection. All patients developed residual scarring or hyperpigmentation. CONCLUSION: Nontuberculous mycobacteria should be recognized as an emerging, but preventable, cause of acupuncture-associated infections.