Robust Saliva-Based RNA Extraction-Free One-Step Nucleic Acid Amplification Test for Mass SARS-CoV-2 Monitoring.

Early diagnosis with rapid detection of the virus plays a key role in preventing the spread of infection and in treating patients effectively. In order to address the need for a straightforward detection of SARS-CoV-2 infection and assessment of viral spread, we developed rapid, sensitive, extraction-free one-step reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and reverse transcription loop-mediated isothermal amplification (RT-LAMP) tests for detecting SARS-CoV-2 in saliva. We analyzed over 700 matched pairs of saliva and nasopharyngeal swab (NSB) specimens from asymptomatic and symptomatic individuals. Saliva, as either an oral cavity swab or passive drool, was collected in an RNA stabilization buffer. The stabilized saliva specimens were heat-treated and directly analyzed without RNA extraction. The diagnostic sensitivity of saliva-based RT-qPCR was at least 95% in individuals with subclinical infection and outperformed RT-LAMP, which had at least 70% sensitivity when compared to NSBs analyzed with a clinical RT-qPCR test. The diagnostic sensitivity for passive drool saliva was higher than that of oral cavity swab specimens (95% and 87%, respectively). A rapid, sensitive one-step extraction-free RT-qPCR test for detecting SARS-CoV-2 in passive drool saliva is operationally simple and can be easily implemented using existing testing sites, thus allowing high-throughput, rapid, and repeated testing of large populations. Furthermore, saliva testing is adequate to detect individuals in an asymptomatic screening program and can help improve voluntary screening compliance for those individuals averse to various forms of nasal collections.

Seasonality and Genotype Diversity of Human Rhinoviruses during an Eight-Year Period in Slovenia.

Due to the high socioeconomic burden of rhinoviruses, the development of prevention and treatment strategies is of high importance. Understanding the epidemiological and clinical features of rhinoviruses is essential in order to address these issues. Our study aimed to define the seasonality and molecular epidemiology of rhinoviruses in Slovenia. Over a period of eight years, a total of 20,425 patients from sentinel primary healthcare settings and sentinel hospitals were examined for a panel of respiratory viruses in the national programme for the surveillance of influenza-like illnesses and acute respiratory infections. The patients were from all age groups and had respiratory infections of various severity. Infection with a rhinovirus was confirmed using an RT-rPCR in 1834 patients, and 1480 rhinoviruses were genotyped. The molecular analysis was linked to demographical and meteorological data. We confirmed the year-round circulation of rhinoviruses with clear seasonal cycles, resulting in two seasonal waves with peaks in spring and autumn. High levels of genotype variability and co-circulation were confirmed between and within seasons and were analysed in terms of patient age, the patient source reflecting disease severity, and meteorological factors. Our study provides missing scientific information on the genotype diversity of rhinoviruses in Slovenia. As most previous investigations focused on exclusive segments of the population, such as children or hospitalised patients, and for shorter study periods, our study, with its design, size and length, contributes complementary aspects and new evidence-based knowledge to the regional and global understanding of rhinovirus seasonality and molecular epidemiology.

Determining the seasonality of respiratory syncytial virus in Slovenia.

BACKGROUND: In Slovenia, the respiratory syncytial virus (RSV) surveillance is based on national laboratory data. The weeks with more than 10% of samples tested positive compose RSV epidemic season. The use of real-time multiplex PCR, which identifies other respiratory pathogens in parallel with RSV, caused more testing but the percentage of RSV positives lowered. The 10% threshold was reached with delay, which raised concern about its suitability for defining RSV seasonality. METHODS: To describe the seasonality of RSV, the onset, offset and duration of the RSV epidemic season across 10 years (from week 40, 2008/2009 to week 39, 2017/2018), four calculative methods were deployed including moving epidemic method, MEM, and epidemiological parameters were compared. RESULTS: In 10 years, 10 969 (12%) out of 90 264 samples tested positive for RSV. The number of tested samples increased remarkably from the first to last season with a drop in the percentage of positive samples from 23% to 10%. The onset of RSV epidemic varied considerably regardless of the calculative method used (from 10 to 13 weeks). The unevenness in the RSV epidemic season end was also observed. The average duration of RSV epidemic season was the shortest when moving epidemic method has been used (15.7 weeks) and longest with ≥3% method (22.9 weeks). CONCLUSION: The ≥3% calculative method could be used as an early warning of the RSV season. However, ≥7% calculative method was found to be reliable enough to define the epidemiological parameters of an ongoing season and to support public health response. The potential of the moving epidemic method should be further explored.