Effect of not vortexing nasopharyngeal and throat swabs on SARS-CoV-2 nucleic acid detection: A pilot study.

The processing of swabs for respiratory virus detection involves vortexing while still in the viral transport medium (VTM). The effect of not vortexing swabs prior to analysis has not been studied extensively for SARS-CoV-2 detection, and presents an opportunity to improve pre-analytic laboratory workflow. We aimed to assess the impact of not vortexing nasopharyngeal/throat swabs submitted in VTM for SARS-CoV-2 testing. To assess the impact of not vortexing swabs, 277 swab samples were tested for SARS-CoV-2 RNA in paired vortexed and non-vortexed aliquots using eight routine nucleic acid amplification assays. We compared the qualitative (positive/negative) and semi-quantitative (cycle threshold, Ct) results. Following discordant analysis, all but one non-vortexed sample had the same qualitative result as the vortexed sample. 27.4 % of samples were SARS-CoV-2 positive. Comparison of Ct values revealed an apparent reduction in human cellular nucleic acid in the non-vortexed samples (mean Ct values of 24.0 and 26.5 for vortexed and non-vortexed samples, respectively, p < 0.0001) and increased Ct values for non-vortexed samples using a laboratory-developed SARS-CoV-2 assay (mean Ct values of 4.1 and 4.2 for vortexed and non-vortexed samples, respectively; p < 0.0001), but this was not observed for a more automated commercial SARS-CoV-2 assay (mean Ct values of 15.2 for both vortexed and non-vortexed samples, respectively; p = 0.68). While vortexing swabs appears to improve the recovery of cellular material, it does not have an appreciable impact on the qualitative sensitivity of SARS-CoV-2 nucleic acid tests, which may support omission of this step and simplification of front-end sample processing.

Development and validation of a multiplex reverse transcriptase-PCR assay for simultaneous testing of influenza A, influenza B and SARS-CoV-2.

In the current pandemic of coronavirus disease (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the co-circulation of SARS-CoV-2 and other respiratory viruses during the upcoming fall and winter seasons may present an unprecedented burden of respiratory disease in the population. Important respiratory viruses that will need to be closely monitored during this time include SARS-CoV-2, influenza A and influenza B. The epidemiology of these viruses is very similar in terms of susceptible populations, mode of transmission, and the clinical syndromes, thus the etiological agent will be difficult to differentiate without target specific assays. The availability of a sensitive and specific multiplex assay that can simultaneously detect all these targets will be valuable. Here we report the validation of a real-time reverse transciptase-PCR assay for the simultaneous detection of SARS-CoV-2, influenza A and influenza B. This multiplex assay is comparable to its singleplex counterparts with a limit-of-detection being less than 5 copies/reaction, 100 % specificity, over seven logs of dynamic range, less than 1 % coefficientof variation showing high precision, and equivalent accuracy using patient samples. It also offers the added benefits of savings in reagents and technologist time while improving testing efficiency and turn-around-times in order to respond effectively to the ongoing pandemic.

Development and validation of RT-PCR assays for testing for SARS-CoV-2.

Background: The recent emergence and rapid global spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) demonstrates the urgent need for laboratory-developed assays for clinical diagnosis and public health interventions in the absence of commercial assays. Methods: We outline the progression of reverse-transcriptase polymerase chain reaction (RT-PCR) assays that were developed and validated at the Alberta Precision Laboratories, Public Health Laboratory, Alberta, Canada, to respond to this pandemic. Initially, testing was performed using SARS-CoV-2-specific and pan-coronavirus gel-based assays that were soon superseded by real-time RT-PCR assays targeting the envelope and RNA-dependent RNA polymerase genes to accommodate the high anticipated volumes of samples. Throughput was further enhanced by multiplexing the different targets together with the co-detection of an internal extraction control. Results: These assays are comparable in sensitivity and specificity to the assays recommended by the World Health Organization and the US Centers for Disease Control and Prevention. Conclusions: The availability of real-time RT-PCR assays early in the pandemic was essential to provide valuable time to local health authorities to contain transmission and prepare for appropriate response strategies.

Historique: La récente émergence et la propagation mondiale rapide du coronavirus 2 du syndrome respiratoire aigu sévère (SARS-CoV-2) a démontré l'urgence de créer des dosages en laboratoire pour poser un diagnostic clinique et adopter des interventions sanitaires en l'absence de dosages commerciaux. Méthodologie: Les chercheurs exposent la progression des dosages d'amplification en chaîne par polymérase couplée à la transcriptase inverse (RT-PCR) mis au point et validés par les Alberta Precision Laboratories du Laboratoire de santé publique de l'Alberta, au Canada, pour répondre à cette pandémie. Les tests ont d'abord été effectués au moyen de dosages sur gel spécifiques au SARS-CoV-2 ou décelant tous les coronavirus, mais ont vite été remplacés par des dosages RT-PCR en temps réel ciblant l'enveloppe et les gènes d'ARN polymérase sous la dépendance d'ARN pour répondre au fort volume anticipé d'échantillons. Le criblage a également été renforcé par le multiplexage conjoint des différentes cibles et la codétection d'un contrôle d'extraction interne. Résultats: Ces dosages ont une sensibilité et une spécificité comparables à ceux recommandés par l'Organisation mondiale de la Santé et les Centers for Disease Control and Prevention des États-Unis. Conclusions: Il était essentiel de disposer de dosages RT-PCR au début de la pandémie pour que les autorités sanitaires locales puissent profiter de temps précieux pour contenir la transmission et préparer les stratégies de réponse appropriées.

A Public Health Laboratory Response to the Pandemic.

An outbreak of coronavirus disease 2019 (COVID-19) caused by a novel coronavirus (severe acute respiratory syndrome coronavirus 2 [SARS-CoV-2]) began in Wuhan, Hubei, China, in December 2019 and spread rapidly worldwide. The response by the Alberta Precision Laboratories, Public Health Laboratory (ProvLab), AB, Canada, included the development and implementation of nucleic acid detection-based assays and dynamic changes in testing protocols for the identification of cases as the epidemic curve increased exponentially. This rapid response was essential to slow down and contain transmission and provide valuable time to the local health authorities to prepare appropriate response strategies. As of May 24, 2020, 236,077 specimens were tested, with 6,475 (2.74%) positives detected in the province of Alberta, Canada. Several commercial assays are now available; however, the response from commercial vendors to develop and market validated tests is a time-consuming process. In addition, the massive global demand made it difficult to secure a reliable commercial supply of testing kits and reagents. A public health laboratory serves a unique and important role in the delivery of health care. One of its functions is to anticipate and prepare for novel emerging pathogens with a plan for pandemic preparedness. Here, we outline the response that involved the development and deployment of testing methodologies that evolved as SARS-CoV-2 spread worldwide, the challenges encountered, and mitigation strategies. We also provide insight into the organizational structure of how a public health response is coordinated in Alberta, Canada, and its benefits.

Filipinos Fit and Trim - A feasible and efficacious DPP-based intervention trial.

INTRODUCTION: Filipino Americans are at higher risk for obesity and related Type 2 diabetes (T2D) compared to other Asian subgroups and non-Hispanic whites. Yet, there are limited research studies to reduce health disparities and improve health outcomes for Filipinos. Weight loss lifestyle intervention trials such as the Diabetes Prevention Program (DPP) can reduce obesity and T2D risks through physical activity and healthy eating. METHODS: Thus, we conducted a pilot Fit&Trim (DPP-based) intervention study - a randomized controlled trial 3-month intervention augmented with mobile technology + 3-month maintenance follow-up with a waitlist control. The objective assessed the intervention feasibility and potential efficacy to reduce T2D risks in Filipino Americans with overweight/obesity. The overall study goal was a mean 5% weight reduction. RESULTS: Sixty-seven eligible Filipino men and women were enrolled and randomized to either an intervention or waitlist control group. Participant retention was 91%. In Phase 1 (baseline to 3-months), the intervention group had greater weight reduction compared to the waitlist control (-4.3% vs. -0.88%; cross-level interaction = -0.85 (-1.4, -0.35). In Phase 2 (3- to 6-months), after receiving the Fit&Trim intervention, the waitlist group also had similar significant weight reduction [-4.8% (- 0.75 (-0.92, -0.58)]. A majority of intervention group (57%) also maintained their weight loss. Overall, 41% of study participants achieved a 5% weight loss. CONCLUSION: The Fit&Trim intervention demonstrated feasibility and potential efficacy for Filipino Americans. Findings warrant a further larger, longer trial to test the Fit&Trim feasibility and effectiveness in a real-world Filipino community setting. CLINICALTRIALSGOV REGISTRATION NUMBER: NCT02278939.

Prevalence, Clinicopathological Characteristics, and Outcome of Human Papillomavirus-Associated Oropharyngeal Cancer in Southern Chinese Patients.

BACKGROUND: Although the global incidence of human papillomavirus (HPV)-associated oropharyngeal squamous cell carcinoma (OPSCC) is increasing, there is little information on southern Chinese population available. METHODS: We analyzed 207 patients which constituted 63.5% of all newly diagnosed OPSCC in Hong Kong during a 5-year period from 2005 to 2009. RESULTS: We used E6/7 mRNA as a marker of oncogenic involvement and found 20.8% (43/207) of OPSCC and 29.0% (36/124) of tonsillar SCC was associated with HPV. HPV-16 was identified in all cases except one (HPV-18). Patients with HPV-associated OPSCCs were significantly younger than HPV-negative patients (mean age: 59.8 vs. 63.9 years, P = 0.05). Multivariate analysis showed that HPV-associated OPSCC was more likely to occur in nonsmokers (39.5% vs. 15.1%, OR: 2.89, P = 0.05), nondrinkers (52.5% vs. 25.6%, OR: 2.72, P = 0.04), originate from the palatine tonsils (83.7% vs. 53.7%, OR: 3.88, P = 0.01), present with an early primary tumor (T1/2; 79.1% vs. 47.6%, OR: 3.81, P = 0.004), and exhibit basaloid differentiation (33.3% vs. 7.3%, OR: 19.74, P = 0.006). HPV positivity was an independent predictor for better prognosis for both 5-year overall and 5-year disease-specific survivals (DSS; 63.0% vs. 29.7%, HR: 0.33, P < 0.001, and 87.8% vs. 42.6%, HR: 0.16, P < 0.001, respectively). CONCLUSION: The estimated age-standardized incidence of OPSCC in Hong Kong during the period 2005-2009 was 0.12/100,000/year. IMPACT: This study has provided the most comprehensive clinical and pathologic information to date about this newly recognized disease in southern Chinese. In view of the global trend, we should anticipate and prepare for an increase in HPV-related OPSCC in southern China.