Tracking SARS-CoV-2 Omicron lineages using real-time reverse transcriptase PCR assays and prospective comparison with genome sequencing.

Omicron has become the dominant SARS-CoV-2 variant globally since December 2021, with distinct waves being associated with separate Omicron sublineages. Rapid detection of BA.1, BA.2, BA.4, and BA.5 was accomplished in the province of Alberta, Canada, through the design and implementation of real-time reverse transcriptase PCR assays targeting S:N501Y, S:ins214EPE, S:H69/V70, ORF7b:L11F, and M:D3N. Using the combination of results for each of these markers, samples could be designated as belonging to sublineages within BA.1, BA.2, BA.4, or BA.5. The analytical sensitivity of these markers ranged from 132 to 2229 copies/mL and in-laboratory accuracy was 98.9-100%. A 97.3% agreement using 12,592 specimens was demonstrated for the assays compared to genome sequencing. The use of these assays, combined with genome sequencing, facilitated the surveillance of SARS-CoV-2 lineages throughout a BA.5-dominated period.

Evolving strategy for an evolving virus: Development of real-time PCR assays for detecting all SARS-CoV-2 variants of concern.

In order to detect the SARS-CoV-2 variants of concern (VOCs), five real-time reverse transcriptase PCR (rRT-PCR) assays were designed to target the critical discriminatory mutations responsible for the following amino acid changes in the spike protein: two Δ69-70 + N501Y + E gene triplexes (one optimized for Alpha [B.1.1.7] and one optimized for Omicron [B.1.1.529]), a K417N + 242-244 wild-type duplex, a K417T + E484K duplex, and a L452R + P681 + E484Q triplex. Depending on the assay, sensitivity was 98.97-100% for the detection of known VOC-positive samples, specificity was 97.2-100%, limit of detection was 2-116 copies/reaction, intra- and interassay variability was less than 5%, and no cross-reactivity with common respiratory pathogens was observed with any assay. A subset of rRT-PCR- positive VOC samples were further characterized by genome sequencing. A comparison of the lineage designation by the VOC rRT-PCR assays and genome sequencing for the detection of the Alpha, Beta, Gamma, Delta and Omicron variants showed clinical sensitivities of 99.97-100 %, clinical specificities of 99.6-100 %, positive predictive values of 99.8-100%, and negative predictive values of 99.98-100 %. We have implemented these rRT-PCR assays targeting discriminatory single nucleotide polymorphisms for ongoing VOC screening of SARS-CoV-2 positive samples for surveillance purposes. This has proven extremely useful in providing close to real-time molecular surveillance to monitor the emergence of Alpha, the replacement of Alpha by Delta, and the replacement of Delta by Omicron. While the design, validation and implementation of the variant specific PCR targets is an ever-evolving approach, we find the turn-around-time, high throughput and sensitivity to be a useful complementary approach for SARS-CoV-2 genome sequencing for surveillance purposes in the province of Alberta, Canada.

One Swab Fits All: Performance of a Rapid, Antigen-Based SARS-CoV-2 Test Using a Nasal Swab, Nasopharyngeal Swab for Nasal Collection, and RT-PCR Confirmation from Residual Extraction Buffer.

BACKGROUND: Point-of-care SARS-CoV-2 antigen tests have great potential to help combat the COVID-19 pandemic. In the performance of a rapid, antigen-based SARS-CoV-2 test (RAT), our study had 3 main objectives: to determine the accuracy of nasal swabs, the accuracy of using nasopharyngeal swabs for nasal collection (nasalNP), and the effectiveness of using residual extraction buffer for real-time reverse-transcriptase PCR (RT-PCR) confirmation of positive RAT (rPan). METHODS: Symptomatic adults recently diagnosed with COVID-19 in the community were recruited into the study. Nasal samples were collected using either a nasalNP or nasal swab and tested immediately with the RAT in the individual's home by a health care provider. 500 µL of universal transport media was added to the residual extraction buffer after testing and sent to the laboratory for SARS-CoV-2 testing using RT-PCR. Parallel throat swabs tested with RT-PCR were used as the reference comparators. RESULTS: One hundred and fifty-five individuals were included in the study (99 nasal swabs, 56 nasalNP). Sensitivities of nasal samples tested on the RAT using either nasal or nasalNP were 89.0% [95% confidence interval (CI) 80.7%-94.6%] and 90.2% (95% CI 78.6%-96.7%), respectively. rPan positivity agreement compared to throat RT-PCR was 96.2%. CONCLUSIONS: RAT reliably detect SARS-CoV-2 from symptomatic adults in the community presenting within 7 days of symptom onset using nasal swabs or nasalNP. High agreement with rPan can avoid the need for collecting a second swab for RT-PCR confirmation or testing of variants of concern from positive RAT in this population.

Global phylogeny of Treponema pallidum lineages reveals recent expansion and spread of contemporary syphilis.

Syphilis, which is caused by the sexually transmitted bacterium Treponema pallidum subsp. pallidum, has an estimated 6.3 million cases worldwide per annum. In the past ten years, the incidence of syphilis has increased by more than 150% in some high-income countries, but the evolution and epidemiology of the epidemic are poorly understood. To characterize the global population structure of T. pallidum, we assembled a geographically and temporally diverse collection of 726 genomes from 626 clinical and 100 laboratory samples collected in 23 countries. We applied phylogenetic analyses and clustering, and found that the global syphilis population comprises just two deeply branching lineages, Nichols and SS14. Both lineages are currently circulating in 12 of the 23 countries sampled. We subdivided T. p. pallidum into 17 distinct sublineages to provide further phylodynamic resolution. Importantly, two Nichols sublineages have expanded clonally across 9 countries contemporaneously with SS14. Moreover, pairwise genome analyses revealed examples of isolates collected within the last 20 years from 14 different countries that had genetically identical core genomes, which might indicate frequent exchange through international transmission. It is striking that most samples collected before 1983 are phylogenetically distinct from more recently isolated sublineages. Using Bayesian temporal analysis, we detected a population bottleneck occurring during the late 1990s, followed by rapid population expansion in the 2000s that was driven by the dominant T. pallidum sublineages circulating today. This expansion may be linked to changing epidemiology, immune evasion or fitness under antimicrobial selection pressure, since many of the contemporary syphilis lineages we have characterized are resistant to macrolides.

Precision Response to the Rise of the SARS-CoV-2 B.1.1.7 Variant of Concern by Combining Novel PCR Assays and Genome Sequencing for Rapid Variant Detection and Surveillance.

SARS-CoV-2 variants of concern (VOCs) have emerged as a global threat to the COVID-19 pandemic response. We implemented a combined approach to quickly detect known VOCs while continuously monitoring for evolving mutations of the virus. To rapidly detect VOCs, two real-time reverse transcriptase PCR assays were designed and implemented, targeting the spike gene H69/V70 deletion and the N501Y mutation. The H69/V70 deletion and N501Y mutation assays demonstrated accuracies of 98.3% (95% CI 93.8 to 99.8) and 100% (95% CI 96.8 to 100), limits of detection of 1,089 and 294 copies/ml, and percent coefficients of variation of 0.08 to 1.16% and 0 to 2.72% for the two gene targets, respectively. No cross-reactivity with common respiratory pathogens was observed with either assay. Implementation of these tests allowed the swift escalation in testing for VOCs from 2.2% to ∼100% of all SARS-CoV-2-positive samples over 12 January to 9 February 2021, and resulted in the detection of a rapid rise of B.1.1.7 cases within the province of Alberta, Canada. A prospective comparison of the VOC assays to genome sequencing for the detection of B.1.1.7, combined detection of P.1 and B.1.351, and wild-type (i.e., non-VOC) lineages showed sensitivities of 98.2 to 100%, specificities of 98.9 to 100%, positive predictive values of 76.9% to 100%, and negative predictive values of 96 to 100%. Variant screening results inform sampling strategies for regular surveillance by genome sequencing, thus allowing rapid identification of known VOCs while continuously monitoring the evolution of SARS-CoV-2 in the province. IMPORTANCE Different strains, or variants, of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2, the virus that causes COVID-19) have emerged that have higher levels of transmission, less susceptibility to our immune response, and possibly cause more severe disease than previous strains of the virus. Rapid detection of these variants of concern is important to help contain them and prevent them from spreading widely within the population. This study describes two newly developed tests that are able to identify and differentiate the variants of concern from regular strains of SARS-CoV-2. These tests are faster and simpler than the main, gold standard method of identifying variants of concern (genome sequencing). These tests also demonstrated a high correlation with genome sequencing and allowed for the rapid and accurate detection of the rise of B.1.1.7 (one of the variants of concern) in the province of Alberta, Canada.

Acceptable performance of the Abbott ID NOW among symptomatic individuals with confirmed COVID-19.

Introduction. The ID NOW is FDA approved for the detection of SARS-CoV-2 in symptomatic individuals within the first 7 days of symptom onset for COVID-19 if tested within 1 h of specimen collection.Gap statement. Clinical data on the performance of the ID NOW are limited, with many studies varying in their study design and/or having small sample size.Aim. In this study we aimed to determine the clinical performance of the ID NOW compared to conventional RT-PCR testing.Methodology. Adults with COVID-19 in the community or hospital were recruited into the study. Paired throat swabs were collected, with one throat swab transported immediately in an empty sterile tube to the laboratory for ID NOW testing, and the other transported in universal transport media and tested by an in-house SARS-CoV-2 RT-PCR assay targeting the E gene.Results. In total, 133 individuals were included in the study; 129 samples were positive on either the ID NOW and/or RT-PCR. Assuming any positive result on either assay represents a true positive, positive per cent agreement (PPA) of the ID NOW compared to RT-PCR with 95 % confidence intervals was 89.1 % (82.0-94.1%) and 91.6 % (85.1-95.9%), respectively. When analysing individuals with symptom duration ≤7 days and who had the ID NOW performed within 1 h (n=62), ID NOW PPA increased to 98.2 %.Conclusion. Results from the ID NOW were reliable, especially when adhering to the manufacturer's recommendations for testing.

Clinical performance of the Abbott Panbio with nasopharyngeal, throat, and saliva swabs among symptomatic individuals with COVID-19.

SARS-CoV-2 antigen tests used at the point-of-care, such as the Abbott Panbio, have great potential to help combat the COVID-19 pandemic. The Panbio is Health Canada approved for the detection of SARS-CoV-2 in symptomatic individuals within the first 7 days of COVID-19 symptom onset(s). Symptomatic adults recently diagnosed with COVID-19 in the community were recruited into the study. Paired nasopharyngeal (NP), throat, and saliva swabs were collected, with one paired swab tested immediately with the Panbio, and the other transported in universal transport media and tested using real-time reverse-transcriptase polymerase chain reaction (RT-PCR). We also prospectively evaluated results from assessment centers within the community. For those individuals, an NP swab was collected for Panbio testing and paired with RT-PCR results from parallel NP or throat swabs. One hundred and forty-five individuals were included in the study. Collection of throat and saliva was stopped early due to poorer performance (throat sensitivity 57.7%, n=61, and saliva sensitivity 2.6%, n=41). NP swab sensitivity was 87.7% [n=145, 95% confidence interval (CI) 81.0-92.7%]. There were 1641 symptomatic individuals tested by Panbio in assessment centers with 268/1641 (16.3%) positive for SARS-CoV-2. There were 37 false negatives and 2 false positives, corresponding to a sensitivity and specificity of 86.1% [95% CI 81.3-90.0%] and 99.9% [95% CI 99.5-100.0%], respectively. The Panbio test reliably detects most cases of SARS-CoV-2 from adults in the community setting presenting within 7 days of symptom onset using nasopharyngeal swabs. Throat and saliva swabs are not reliable specimens for the Panbio.

Evaluation of 5 commercial assays for the detection of Mycoplasma genitalium and other Urogenital Mycoplasmas.

The focus on urogenital mycoplasmas as the possible etiologic agents of urogenital infections and syndromes, has increased in the last decade. Of these, Mycoplasma genitalium is proven to be pathogenic and sexually transmitted. We compared five commercially available assays for the detection of these organisms in urogenital mycoplasma culture specimen remnants. Stored specimen remnants were tested on Aptima Mycoplasma genitalium, Allplex™ STI Essential and CGMT, ResitancePlus®MG and Allplex™ MG & AziR Assays. All positive M. genitalium specimens and culture negative, nucleic acid positive Ureaplasmas were sent to the National Microbiology Laboratory for confirmation. The Aptima Mycoplasma genitalium assay detected 7 M. genitalium infections, the Allplex™ STI-EA and the Allplex™ CGMT detected 6 M. genitalium positives, and the Allplex™MG and AziR and SpeeDx ResistancePlus® MG detected 5 M. genitalium positives, four with macrolide resistant genes. The Allplex™ STI Essential assay was 100% sensitive and specific for Mycoplasma hominis and Ureaplasma targets. As seen in other studies, the Aptima Mycoplasma genitalium assay was 100% sensitive and specific for the detection of M. genitalium. The multiplex assays had lower sensitivities for M. genitalium detection (Allplex™ STI Essential and CGMT sensitivity of 85.71%; Allplex™ MG & AziR and SpeeDx ResistancePlus® MG sensitivity of 71.43%) with high specificities of 100%. Assays tested have high sensitivities and specificities for the detection of urogenital mycoplasmas especially M. genitalium macrolide resistance markers. All labs wanting to perform onsite detection of these organisms will find an assay to easily fit into their workflow.

Histoplasmosis acquired in Alberta, Canada: an epidemiological and genomic study.

BACKGROUND: The classic geographical range of histoplasmosis in North America primarily includes the states and provinces adjacent to the Ohio, Mississippi, and St Lawrence riverways. Although Alberta, Canada is not typically considered a region of risk for histoplasmosis, cases with suspected local acquisition have been reported. We aimed to investigate the epidemiology and geographical distribution of cases of histoplasmosis in Alberta to assess evidence for local acquisition of infections, using genomic analysis for corroboration. METHODS: We did an epidemiological and genomic investigation, in which laboratory-confirmed cases of histoplasmosis were reviewed in Alberta from 2011, when the disease became reportable, until 2018. We used data attained from Alberta Health. Travel and exposure histories and clinical features were reviewed. Definite local acquisition was defined as a case without previous travel outside Alberta or associated with a common-source outbreak within the province, whereas probable local acquisition was a sporadic case with travel outside Alberta but compelling local exposures. Genomes of selected case isolates were analysed, including those from cases suspected to have been locally acquired and imported. FINDINGS: Between Jan 1, 2011, and June 30, 2018, 45 cases of histoplasmosis were identified. Participants had a median age of 53 years (range 17-77) and 32 [71%] were male. Among 34 patients with documented travel histories, ten (29%) had never left the province. 11 cases were of definite local acquisition, including eight cases from three common-source outbreaks and three sporadic cases in patients who had never travelled outside Alberta. The common-source outbreaks all involved exposure to bats or their droppings in chimneys or attics of private dwellings or churches. Four patients had travelled outside Alberta but compelling evidence was seen for local exposure to bat guano. Genome sequencing showed that isolates from cases of definite and probable local acquisition clustered together and were genetically distinct from isolates from suspected imported cases and other published isolates. INTERPRETATION: Using epidemiological and genomic analyses, we established that cases of histoplasmosis have been acquired in Alberta, thus expanding the geographical range of Histoplasma spp much further northwest than was previously appreciated. Histoplasmosis should be considered in patients with compatible symptoms outside areas of classic geographical risk. FUNDING: None.

Development of an Ordinal Scale Treatment Endpoint for Adults Hospitalized With Influenza.

BACKGROUND: An obstacle in influenza therapeutics development is the lack of clinical endpoints, especially in hospitalized patients. A single time-point binary outcome measure is limited by patients' diverse clinical trajectories and low event rates. METHODS: A 6-point ordinal scale with ascending clinical status severity (scoring: discharged; subacute care; acute care without/with respiratory failure; intensive care unit [ICU]; death) was proposed to study outcomes of adults hospitalized with influenza. Individual patient data from 2 active surveillance cohorts' datasets (2015/2016-2017/2018; Edmonton, Hong Kong) was used for evaluation. The impact of neuraminidase inhibitor (NAI) treatment on longitudinal ordinal outcome changes over 30 days was analyzed using mixed-effects ordinal logistic regression and group-based trajectory models. RESULTS: Patient (n = 1226) baseline characteristics included age (mean 68.0 years), virus-type (A 78.1%, B 21.9%), respiratory failure (57.2%), ICU admittance (14.4%), and NAI treatment within 5 days of illness (69.2%). Outcomes at 30 days included discharged (75.2%), subacute care (13.7%), acute care (4.5%), and death (6.6%). Two main clinical trajectories were identified, predictive by baseline scoring (mean ±â€…SD, 4.3 ±â€…0.6 vs 3.5 ±â€…0.6, P < .001). Improved outcomes with NAI treatment within 5 days were indicated by significantly lower clinical status scores over time (unadjusted odds ratio [OR], 0.53; 95% confidence interval [CI], .41-.69; P < .001; adjusted OR, 0.62; 95% CI, .50-.77; P < .001, for baseline score, age, and within-patient correlations). In subanalysis, influenza vaccination was also associated with lower scores (adjusted OR, 0.67; 95% CI, .50-.90; P = .007). Analyses of binary endpoints showed insignificant results. CONCLUSIONS: The ordinal outcome scale is a potentially useful clinical endpoint for influenza therapeutic trials, which could account for the diverse clinical trajectories of hospitalized patients, warranting further development.

Species distribution and antifungal susceptibility of invasive Candida isolates from Canadian hospitals: results of the CANWARD 2011-16 study.

OBJECTIVES: Understanding the epidemiology of invasive Candida infections is essential to patient management decisions and antifungal stewardship practices. This study characterized the species distribution and antifungal susceptibilities of prospectively collected isolates of Candida species causing bloodstream infections (BSIs) in patients admitted to tertiary care hospitals located in 14 cities across 8 of the 10 Canadian provinces between 2011 and 2016. METHODS: Antifungal susceptibility testing was performed by broth microdilution using CLSI methods, breakpoints and epidemiological cut-off values. DNA sequencing of fks loci was performed on all echinocandin-non-susceptible isolates. RESULTS: Candida albicans (49.6%), Candida glabrata (20.8%) and Candida parapsilosis complex (12.0%) were the most common species out of 1882 isolates associated with BSIs. Candida tropicalis (5.2%), Candida krusei (4.3%), Candida dubliniensis (4.1%), Candida lusitaniae (1.4%) and Candida guilliermondii (1.1%) were less frequently isolated. Between 2011 and 2016, the proportion of C. albicans significantly decreased from 60.9% to 42.1% (P < 0.0001) while that of C. glabrata significantly increased from 16.4% to 22.4% (P = 0.023). C. albicans (n = 934), C. glabrata (n = 392) and C. parapsilosis complex (n = 225) exhibited 0.6%, 1.0% and 4.9% resistance to fluconazole and 0.1%, 2.5% and 0% resistance to micafungin, respectively. Mutations in fks hot-spot regions were confirmed in all nine micafungin non-susceptible C. glabrata. CONCLUSIONS: Antifungal resistance in contemporary isolates of Candida causing BSIs in Canada is uncommon. However, the proportion of C. glabrata isolates has increased and echinocandin resistance in this species has emerged. Ongoing surveillance of local hospital epidemiology and appropriate antifungal stewardship practices are necessary to preserve the utility of available antifungal agents.

Prevalence and antibiotic resistance of Mycoplasma genitalium among STI clinic attendees in Western Canada: a cross-sectional analysis.

OBJECTIVES: To determine the prevalence and correlates of Mycoplasma genitalium (MG) infection among men and women, determine the prevalence of gene mutations conferring resistance and compare test performance of female specimen types. METHODS: A cross-sectional study was conducted on specimens collected for gonorrhoea (NG, Neisseria gonorrhoeae) and chlamydia (CT, Chlamydia trachomatis) among male and female Alberta STI clinic attendees using the M. genitalium transcription-mediated amplification-research use only test. Positive specimens were sequenced for 23SrRNA, parC and gyrA genes. Gender-stratified analysis compared test results using χ2 or Fisher's exact test, Mann-Whitney U test and logistic regression. Female endocervical and urine specimens were compared. RESULTS: A total of 2254 individuals were tested; 53.8% (n=1212) were male. Male prevalence of MG was 5.3%; CT was 5.9% and NG was 1.8%. Correlates of male infection were a non-gonococcal urethritis diagnosis and NG coinfection. MG prevalence for women was 7.2%; CT was 5.8% and NG was 1.8%. Correlates of female infection were younger age, Indigenous/Other ethnicity and CT/NG coinfection. Nearly two-thirds of eligible specimens had mutations associated with macrolide resistance and 12.2% of specimens had a parC mutation signifying possible moxifloxacin resistance. There was high concordance (98.1%) of results between urine and endocervical swabs. CONCLUSIONS: The high prevalence of MG relative to CT and NG supports the incorporation of MG testing into routine sexually transmissible infection screening. The high rate of resistance to macrolides and moxifloxacin raises concerns about treatment options. The good concordance of results between urine and endocervical swabs supports the use of female urine specimens for testing.

Multicenter evaluation of the new Vitek 2 Neisseria-Haemophilus identification card.

The new Neisseria-Haemophilus identification (NH) card for Vitek 2 was compared with 16S rRNA gene sequencing (16S) as the reference method for accurate identification of Neisseria spp., Haemophilus spp., and other fastidious gram-negative bacteria. Testing was performed on the Vitek 2 XL system with modified software at three clinical trial laboratories. Reproducibility was determined with nine ATCC quality control strains tested 20 times over a minimum of 10 days at all three sites. A challenge set of 30 strains with known identifications and 371 recent fresh and frozen clinical isolates were also tested. Expected positive and negative biochemical reactions were also evaluated for substrate reproducibility. All microorganisms were tested on the NH card, and all clinical and stock isolates were saved for 16S testing. All reproducibility tests yielded expected results within a 95% confidence interval. For challenge microorganisms, there was 98% overall correct identification, including 8% low discrimination, 2% incorrect identification, and 0% unidentified. For clinical strains, there was 96.5% overall correct identification, including 10.2% low discrimination, 2.7% incorrect identification, and 0.8% unidentified. The 2.7% (10/371) of clinical isolates that gave an incorrect identification consisted of 7 isolates correct to genus and 3 strains incorrect to genus. There were an additional 27 strains (primarily Neisseria species) for which the 16S identification result was different from the NH card result. These were all unclaimed species by the system. The new NH card met all performance criteria within a 95% confidence interval compared to identification of clinical isolates by 16S.

Adamantane resistance in circulating human influenza A viruses from Alberta, Canada (1970-2007).

Mutation in one of five key amino acid residues (positions 26, 27, 30, 31 and 34) within the M2 protein of influenza A viruses, leads to resistance against the adamantane class of anti-influenza drugs. To investigate the emergence and prevalence of adamantane resistance in Alberta, Canada (between 1970 and 2007), 381 influenza A positive samples (original patient specimens) or isolates (virus cultured from patient specimens) were analyzed for changes in these critical amino acid residues. Our results show a significant increase in adamantane resistance in circulating H3N2 viruses in Alberta from 2005 and 2006 when compared with those from 2004 (p<0.001). Adamantane resistance peaked at 74% in 2006 and then decreased (to 38%) in 2007 (p=0.001). All resistant H3N2 viruses contained the substitution Ser to Asn at amino acid position 31 of the M2 protein with two viruses having an additional Ala to Val substitution at position 30. Resistance was not observed in the H1N1 viruses tested. Results presented here are concordant with, and extend, previous reports of increased resistance to adamantanes in Asia and North America in recent years. It is important to continue studies to evaluate circulating influenza A viruses for antiviral resistance markers to ensure their optimal use for prophylaxis and treatment of influenza.

Bacterial contamination of health care workers' pagers and the efficacy of various disinfecting agents.

Health care workers in our facility were surveyed, and their pagers were cultured before and after disinfection with various agents. All pagers were contaminated with bacteria, including the pathogens Staphylococcus aureus, Pseudomonas aeruginosa, Acinetobacter baumannii, Klebsiella spp. and methicillin-resistant S. aureus. Disinfection reduced bacterial contamination. No risk factors for pager contamination with pathogens were identified.