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BackgroundPerformance 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 resolved infection with persistent RNA shedding, presymptomatic or asymptomatic infection, or a false positive test. This study assessed clinical specificity of SARS-CoV-2 real-time reverse transcription polymerase chain reaction (rRT-PCR) assays by retesting positive specimens from five pre-test probability groups ranging from high to low with an alternate assay.Materials and MethodsA total of 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.ResultsSignificantly less 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 all other groups combined (5/32, 15·6% vs 61/90, 68%; p ConclusionsA higher proportion of false-positive test results are likely with lower pre-test probability. Positive SARS-CoV-2 PCR results should be interpreted within the context of patient history, clinical setting, known exposure, and estimated community disease prevalence. 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 positives and consequent potential for harm at the individual and population level.

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, pt of multi-day specimens increased over time. Symptomatic cases had rRT-PCR results with an adjusted estimate 3.0 Ct (SE = 0.5, p

ObjectiveClostridium difficilespores play an important role in transmission and can survive in the environment for several months. Optimal methods for measuring environmentalC. difficileare unknown. We sought to determine whether increased sample surface area improved detection ofC. difficilefrom environmental samples.SettingSamples were collected from 12 patient rooms in a tertiary-care hospital in Toronto, Canada.MethodsSamples 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. difficileinfected) shedding risk. Presence ofC. difficilein samples was measured using quantitative polymerase chain reaction (qPCR) with targets on the 16S rRNA and toxin B genes and using enrichment culture.ResultsOf 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 ofC. 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.ConclusionsClostridium difficilespores were identified in most floor and bedrail samples, and increased surface area improved detection. Future research aiming to understand the role of environmentalC. difficilein transmission should prefer samples with large surface areas.

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.

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.

Clostridium difficile infection (CDI) is one of the most frequent causes of healthcare-associated infections, and its rates are also increasing in the community. The management of CDI has become a major challenge, given growing rates of recurrences and failures with standard antibiotic therapy. Mounting evidence suggests that fecal microbiota transplantation (FMT) may be effective; however, as there is a paucity of data with regard to repeat FMT for primary non-response to this treatment, this study examined the outcome of multiple FMTs for recurrent CDI. Case records were reviewed for 94 patients who underwent FMT via retention enema for recurrent or refractory CDI during the period 2008-2012. Demographic information, treatment data, and clinical resolution rates were examined for single FMT and cumulative resolution was assessed for multiple FMTs in the context of ongoing symptoms. The cumulative clinical resolution following four or more FMTs was 86%. When antibiotic therapy was used between FMTs, the clinical resolution rate increased to 92%. There were no reported adverse events and no patients who were cured with FMT had further episodes of CDI at 6-24 months follow-up. Multiple FMTs administered through enemas is an effective, safe, and simple therapy for the management of recurrent or refractory CDI.

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.

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.

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.

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.

Three commercially available real-time reverse transcriptase PCR assays (the Artus RealArt HPA coronavirus LightCycler, the Artus RealArt HPA coronavirus Rotor-Gene, and the EraGen severe acute respiratory syndrome coronavirus POL assay) and three RNA extraction methodologies were evaluated for the detection of severe acute respiratory syndrome coronavirus RNA from 91 stool specimens. The assays' sensitivities were highest (58% to 75%) for specimens obtained 8 to 21 days after symptom onset. The assays were less sensitive when specimens were obtained less than 8 days or more than 21 days after the onset of symptoms. All assays were 100% specific.

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.

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.

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.

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.

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.

A strain of a novel non-chromogenic mycobacterium was isolated from synovial tissue from a 68-year-old female with bursitis of her right elbow. The slowly growing strain had a unique PCR-restriction enzyme analysis (PRA) profile of the hsp65 gene and 16S rRNA gene sequence in comparison with other mycobacterium species. The most closely related species, as determined by 16S rRNA gene sequence analysis, are Mycobacterium malmoense, Mycobacterium marinum, Mycobacterium ulcerans and members of the Mycobacterium tuberculosis complex. The HPLC and biochemical profiles resembled those of Mycobacterium gastri, although differences were noted in the peak-height ratio of the HPLC pattern and the nitrate and pyrazinamidase tests. On the basis of PRA, HPLC, biochemical and 16S rRNA gene sequence analyses, the name Mycobacterium lacus sp. nov. is proposed for this potential pathogen. The type strain is strain NRCM 00-255(T) (= ATCC BAA-323(T) = DSM 44577(T)).

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.