Severe acute respiratory coronavirus virus 2 (SARS-CoV-2) RNA and viable virus contamination of hospital emergency department surfaces and association with patient coronavirus disease 2019 (COVID-19) status and aerosol-generating procedures.

Emergency departments are high-risk settings for severe acute respiratory coronavirus virus 2 (SARS-CoV-2) surface contamination. Environmental surface samples were obtained in rooms with patients suspected of having COVID-19 who did or did not undergo aerosol-generating procedures (AGPs). SARS-CoV-2 RNA surface contamination was most frequent in rooms occupied by coronavirus disease 2019 (COVID-19) patients who received no AGPs.

Saliva as an alternative sample type for detection of pneumococcal carriage in young children.

For children, the gold standard for the detection of pneumococcal carriage is conventional culture of a nasopharyngeal swab. Saliva, however, has a history as one of the most sensitive methods for surveillance of pneumococcal colonization and has recently been shown to improve carriage detection in older age groups. Here, we compared the sensitivity of paired nasopharyngeal and saliva samples from PCV7-vaccinated 24-month-old children for pneumococcal carriage detection using conventional and molecular detection methods. Nasopharyngeal and saliva samples were collected from 288 24-month-old children during the autumn/winter, 2012/2013. All samples were first processed by conventional diagnostic culture. Next, DNA extracted from all plate growth was tested by qPCR for the presence of the pneumococcal genes piaB and lytA and a subset of serotypes. By culture, 161/288 (60 %) nasopharyngeal swabs tested positive for pneumococcus, but detection was not possible from saliva due to abundant polymicrobial growth on culture plates. By qPCR, 155/288 (54 %) culture-enriched saliva samples and 187/288 (65 %) nasopharyngeal swabs tested positive. Altogether, 219/288 (76 %) infants tested positive for pneumococcus, with qPCR-based carriage detection of culture-enriched nasopharyngeal swabs detecting significantly more carriers compared to either conventional culture (P<0.001) or qPCR detection of saliva (P=0.002). However, 32/219 (15 %) carriers were only positive in saliva, contributing significantly to the overall number of carriers detected (P=0.002). While testing nasopharyngeal swabs by qPCR proved most sensitive for pneumococcal detection in infants, saliva sampling could be considered as complementary to provide additional information on carriage and serotypes that may not be detected in the nasopharynx and may be particularly useful in longitudinal studies, requiring repeated sampling of study participants.

The potential of saliva as an accessible and sensitive sample type for the detection of respiratory pathogens and host immunity.

Despite its prominence in early scientific records, the usefulness of saliva as a respiratory specimen has been de-emphasised over the past century. However, due to its low cost and reliance on specific supply chains and the non-invasive nature of its collection, its benefits over swab-based specimens are again becoming increasingly recognised. These benefits were highlighted over the course of the COVID-19 pandemic, where saliva emerged as a more practical, clinically non-inferior sample type for the detection of SARS-CoV-2 and saw numerous saliva-based diagnostic tests approved for clinical use. Looking forward, as saliva uniquely contains both respiratory secretions and immunological components, it has potentially wide applications, ranging from clinical diagnostics to post-vaccine disease burden and immunity surveillance. This Personal View seeks to summarise the existing evidence for the use of saliva in detecting respiratory pathogens, beyond SARS-CoV-2, as well as detailing methodological factors that can influence sample quality and thus, clinical utility.

Method versatility in RNA extraction-free PCR detection of SARS-CoV-2 in saliva samples.

Early in the pandemic, a simple, open-source, RNA extraction-free RT-qPCR protocol for SARS-CoV-2 detection in saliva was developed and made widely available. This simplified approach (SalivaDirect) requires only sample treatment with proteinase K prior to PCR testing. However, feedback from clinical laboratories highlighted a need for a flexible workflow that can be seamlessly integrated into their current health and safety requirements for the receiving and handling of potentially infectious samples. To address these varying needs, we explored additional pre-PCR workflows. We built upon the original SalivaDirect workflow to include an initial incubation step (95 °C for 30 min, 95 °C for 5 min or 65 °C for 15 min) with or without addition of proteinase K. The limit of detection for the workflows tested did not significantly differ from that of the original SalivaDirect workflow. When tested on de-identified saliva samples from confirmed COVID-19 individuals, these workflows also produced comparable virus detection and assay sensitivities, as determined by RT-qPCR analysis. Exclusion of proteinase K did not negatively affect the sensitivity of the assay. The addition of multiple heat pretreatment options to the SalivaDirect protocol increases the accessibility of this cost-effective SARS-CoV-2 test as it gives diagnostic laboratories the flexibility to implement the workflow which best suits their safety protocols.

Pooled RNA-extraction-free testing of saliva for the detection of SARS-CoV-2.

The key to limiting SARS-CoV-2 spread is to identify virus-infected individuals (both symptomatic and asymptomatic) and isolate them from the general population. Hence, routine weekly testing for SARS-CoV-2 in all asymptomatic (capturing both infected and non-infected) individuals is considered critical in situations where a large number of individuals co-congregate such as schools, prisons, aged care facilities and industrial workplaces. Such testing is hampered by operational issues such as cost, test availability, access to healthcare workers and throughput. We developed the SalivaDirect RT-qPCR assay to increase access to SARS-CoV-2 testing via a low-cost, streamlined protocol using self-collected saliva. To expand the single sample testing protocol, we explored multiple extraction-free pooled saliva testing workflows prior to testing with the SalivaDirect RT-qPCR assay. A pool size of five, with or without heat inactivation at 65 °C for 15 min prior to testing resulted in a positive agreement of 98% and 89%, respectively, and an increased Ct value shift of 1.37 and 1.99 as compared to individual testing of the positive clinical saliva specimens. Applying this shift in Ct value to 316 individual, sequentially collected, SARS-CoV-2 positive saliva specimen results reported from six clinical laboratories using the original SalivaDirect assay, 100% of the samples would have been detected (Ct value < 45) had they been tested in the 1:5 pool strategy. The availability of multiple pooled testing workflows for laboratories can increase test turnaround time, permitting results in a more actionable time frame while minimizing testing costs and changes to laboratory operational flow.

Persistence of Pneumococcal Carriage among Older Adults in the Community despite COVID-19 Mitigation Measures.

Reported rates of invasive pneumococcal disease were markedly lower than normal during the 2020/2021 winter in the Northern Hemisphere, the first year after the start of the COVID-19 pandemic. However, little is known about rates of carriage of pneumococcus among adults during this period. Between October 2020-August 2021, couples in the Greater New Haven Area, USA, were enrolled if both individuals were aged 60 years and above and did not have any individuals under the age of 60 years living in the household. Saliva samples and questionnaires regarding social activities and contacts and medical history were obtained every 2 weeks for a period of 10 weeks. Following culture-enrichment, extracted DNA was tested using qPCR for pneumococcus-specific sequences piaB and lytA. Individuals were considered positive for pneumococcal carriage when Ct values for piaB were ≤40. Results. We collected 567 saliva samples from 95 individuals (47 household pairs and 1 singleton). Of those, 7.1% of samples tested positive for pneumococcus, representing 22/95 (23.2%) individuals and 16/48 (33.3%) households. Study participants attended few social events during this period. However, many participants continued to have regular contact with children. Individuals who had regular contact with preschool and school-aged children (i.e., 2 to 9 year olds) had a higher prevalence of carriage (15.9% versus 5.4%). Despite COVID-19-related disruptions, a large proportion of older adults continued to carry pneumococcus. Prevalence was particularly high among those who had contact with school-aged children, but carriage was not limited to this group. IMPORTANCE Carriage of Streptococcus pneumoniae (pneumococcus) in the upper respiratory tract is considered a prerequisite to invasive pneumococcal disease. During the first year of the COVID-19 pandemic, markedly lower rates of invasive pneumococcal disease were reported worldwide. Despite this, by testing saliva samples with PCR, we found that older adults continued to carry pneumococcus at pre-pandemic levels. Importantly, this study was conducted during a period when transmission mitigation measures related to the COVID-19 pandemic were in place. However, our observations are in line with reports from Israel and Belgium where carriage was also found to persist in children. In line with this, we observed that carriage prevalence was particularly high among the older adults in our study who maintained contact with school-aged children.

High Levels of Detection of Nonpneumococcal Species of Streptococcus in Saliva from Adults in the United States.

While the sensitivity of detection of pneumococcal carriage can be improved by testing respiratory tract samples with quantitative PCR (qPCR), concerns have been raised regarding the specificity of this approach. We therefore investigated the reliability of the widely used lytA qPCR assay when applied to saliva samples from older adults in relation to a more specific qPCR assay (piaB). During the autumn/winter seasons of 2018/2019 and 2019/2020, saliva was collected at multiple time points from 103 healthy adults aged 21 to 39 (n = 34) and >64 (n = 69) years (n = 344 total samples). Following culture enrichment, extracted DNA was tested using qPCR for piaB and lytA. By sequencing the variable region of rpsB (S2 typing), we identified the species of bacteria isolated from samples testing lytA-positive only. While 30 of 344 (8.7%) saliva samples (16.5% individuals) tested qPCR-positive for both piaB and lytA, 52 (15.1%) samples tested lytA-positive only. No samples tested piaB-positive only. Through extensive reculture attempts of the lytA-positive samples collected in 2018/2019, we isolated 23 strains (in 8 samples from 5 individuals) that were also qPCR-positive for only lytA. Sequencing determined that Streptococcus mitis and Streptococcus infantis were predominantly responsible for this lytA-positive qPCR signal. We identified a comparatively large proportion of samples generating positive signals with the widely used lytA qPCR and identified nonpneumococcal Streptococcus species responsible for this signal. This highlights the importance of testing for the presence of multiple gene targets in tandem for reliable and specific detection of pneumococcus in polymicrobial respiratory tract samples. IMPORTANCE Testing saliva samples with quantitative PCR (qPCR) improves the sensitivity of detection of pneumococcal carriage. The qPCR assay targeting lytA, the gene encoding the major pneumococcal autolysin, has become widely accepted for the identification of pneumococcus and is even considered the "gold standard" by many. However, when applying this approach to investigate the prevalence of pneumococcal carriage in adults in New Haven, CT, USA, we identified nonpneumococcal Streptococcus spp. that generate positive signals in this widely used assay. By testing also for piaB (encoding the iron acquisition ABC transporter lipoprotein, PiaB), our findings demonstrate the importance of testing for the presence of multiple gene targets in tandem for reliable molecular detection of pneumococcus in respiratory tract samples; targeting only lytA may lead to an overestimation of true carriage rates.

Magnetic bead-based separation of pneumococcal serotypes.

The separation of pneumococcal serotypes from a complex polymicrobial mixture may be required for different applications. For instance, a minority strain could be present at a low frequency in a clinical sample, making it difficult to identify and isolate by traditional culture-based methods. We therefore developed an assay to separate mixed pneumococcal samples using serotype-specific antiserum and a magnetic bead-based separation method. Using qPCR and colony counting methods, we first show that serotypes (12F, 23F, 3, 14, 19A, and 15A) present at ∼0.1% of a dual serotype mixture can be enriched to between 10% and 90% of the final sample. We demonstrate two applications for this method: extraction of known pneumococcal serotypes from saliva samples and efficient purification of capsule switch variants from experimental transformation experiments. This method may have further laboratory or clinical applications when the selection of specific serotypes is required.

Routine saliva testing for SARS-CoV-2 in children: Methods for partnering with community childcare centers.

While considerable attention was placed on SARS-CoV-2 testing and surveillance programs in the K-12 setting, younger age groups in childcare centers were largely overlooked. Childcare facilities are vital to communities, allowing parents/guardians to remain at work and providing safe environments for both children and staff. Therefore, early in the COVID-19 pandemic (October 2020), we established a PCR-based COVID-19 surveillance program in childcare facilities, testing children and staff with the goal of collecting actionable public health data and aiding communities in the progressive resumption of standard operations and ways of life. In this study we describe the development of a weekly saliva testing program and provide early results from our experience implementing this in childcare centers. We enrolled children (aged 6 months to 7 years) and staff at seven childcare facilities and trained participants in saliva collection using video chat technology. Weekly surveys were sent out to assess exposures, symptoms, and vaccination status changes. Participants submitted weekly saliva samples at school. Samples were transported to a partnering clinical laboratory or RT-PCR testing using SalivaDirect and results were uploaded to each participant's online patient portal within 24 h. SARS-CoV-2 screening and routine testing programs have focused less on the childcare population, resulting in knowledge gaps in this critical age group, especially as many are still ineligible for vaccination. SalivaDirect testing for SARS-CoV-2 provides a feasible method of asymptomatic screening and symptomatic testing for children and childcare center staff. Given the relative aversion to nasal swabs in younger age groups, an at-home saliva collection method provides an attractive alternative, especially as a routine surveillance tool. Results can be shared rapidly electronically through participants' private medical chart portals, and video chat technology allows for discussion and instruction between investigators and participants. This study fosters a cooperative partnership with participating childcare centers, parents/guardians, and staff with the goal of mitigating COVID-19 transmission in childcare centers. Age-related challenges in saliva collection can be overcome by working with parents/guardians to conceptualize new collection strategies and by offering parents/guardians continued virtual guidance and support.

Association of Upper Respiratory Streptococcus pneumoniae Colonization With Severe Acute Respiratory Syndrome Coronavirus 2 Infection Among Adults.

BACKGROUND: Streptococcus pneumoniae interacts with numerous viral respiratory pathogens in the upper airway. It is unclear whether similar interactions occur with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). METHODS: We collected saliva specimens from working-age adults undergoing SARS-CoV-2 molecular testing at outpatient clinics and via mobile community-outreach testing between July and November 2020 in Monterey County, California. After bacterial culture enrichment, we tested for pneumococci by means of quantitative polymerase chain reaction targeting the lytA and piaB genes, and we measured associations with SARS-CoV-2 infection using conditional logistic regression. RESULTS: Analyses included 1278 participants, with 564 enrolled in clinics and 714 enrolled through outreach-based testing. The prevalence of pneumococcal carriage was 9.2% (117 of 1278) among all participants (11.2% [63 of 564] in clinic-based testing and 7.6% [54 of 714] in outreach-based testing). The prevalence of SARS-CoV-2 infection was 27.4% (32 of 117) among pneumococcal carriers and 9.6% (112 of 1161) among noncarriers (adjusted odds ratio [aOR], 2.73 [95% confidence interval (CI): 1.58-4.69). Associations between SARS-CoV-2 infection and pneumococcal carriage were enhanced in the clinic-based sample (aOR, 4.01 [95% CI: 2.08-7.75]) and among symptomatic participants (3.38 [1.35-8.40]), compared with findings within the outreach-based sample and among asymptomatic participants. The adjusted odds of SARS-CoV-2 coinfection increased 1.24-fold (95% CI: 1.00-1.55-fold) for each 1-unit decrease in piaB quantitative polymerase chain reaction cycle threshold value among pneumococcal carriers. Finally, pneumococcal carriage modified the association of SARS-CoV-2 infection with recent exposure to a suspected coronavirus disease 2019 case (aOR, 7.64 [95% CI: 1.91-30.7] and 3.29 [1.94-5.59]) among pneumococcal carriers and noncarriers, respectively). CONCLUSIONS: Associations of pneumococcal carriage detection and density with SARS-CoV-2 suggest a synergistic relationship in the upper airway. Longitudinal studies are needed to determine interaction mechanisms between pneumococci and SARS-CoV-2.

Saliva-based methods for SARS-CoV-2 testing in low- and middle-income countries.

As the coronavirus disease 2019 (COVID-19) continues to disproportionately affect low- and middle-income countries, the need for simple, accessible and frequent diagnostic testing grows. In lower-resource settings, case detection is often limited by a lack of available testing for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). To address global inequities in testing, alternative sample types could be used to increase access to testing by reducing the associated costs. Saliva is a sensitive, minimally invasive and inexpensive diagnostic sample for SARS-CoV-2 detection that is appropriate for asymptomatic surveillance, symptomatic testing and at-home collection. Saliva testing can lessen two major challenges faced by lower- and middle-income countries: constrained resources and overburdened health workers. Saliva sampling enables convenient self-collection and requires fewer resources than swab-based methods. However, saliva testing for SARS-CoV-2 diagnostics has not been implemented on a large scale in low- and middle-income countries. While numerous studies based in these settings have demonstrated the usefulness of saliva sampling, there has been insufficient attention on optimizing its implementation in practice. We argue that implementation science research is needed to bridge this gap between evidence and practice. Low- and middle-income countries face many barriers as they continue their efforts to provide mass COVID-19 testing in the face of substantial inequities in global access to vaccines. Laboratories should look to replicate successful approaches for sensitive detection of SARS-CoV-2 in saliva, while governments should act to facilitate mass testing by lifting restrictions that limit implementation of saliva-based methods.

La maladie à coronavirus 2019 (COVID-19) continue à affecter les pays à revenu faible et intermédiaire de manière disproportionnée, accentuant le besoin en tests diagnostiques simples, accessibles et fréquents. Dans les endroits disposant de ressources limitées, la détection des cas se heurte souvent au manque de tests disponibles pour le syndrome respiratoire aigu sévère (SARS-CoV-2). Afin de lutter contre les inégalités mondiales en la matière, d'autres types d'échantillons pourraient être exploités, dans le but d'améliorer l'accès au dépistage tout en diminuant les frais qu'il engendre. Les échantillons de salive offrent une méthode de diagnostic fiable, peu invasive et peu coûteuse pour détecter le SARS-CoV-2. Cette méthode est compatible avec le suivi des personnes asymptomatiques, le dépistage des personnes symptomatiques et la collecte d'échantillons à domicile. Les tests salivaires permettent d'atténuer deux problèmes majeurs rencontrés par les pays à revenu faible et intermédiaire: une pénurie de ressources et des soignants surmenés. En outre, les patients peuvent effectuer le prélèvement eux-mêmes et cette méthode nécessite moins de moyens que celle reposant sur l'écouvillonnage. Pourtant, les tests salivaires de détection du SARS-CoV-2 n'ont pas été déployés à grande échelle dans les pays à revenu faible et intermédiaire. Malgré les nombreuses études démontrant l'utilité des tests salivaires dans ces régions, les perspectives d'optimisation de leur mise en œuvre n'ont suscité que peu d'attention. Dans le présent document, nous affirmons que des recherches scientifiques sur leur exécution sont requises pour combler ce fossé entre les faits et la pratique. Les pays à revenu faible et intermédiaire sont confrontés à une multitude d'obstacles dans leurs efforts de dépistage massif de la COVID-19. Et ce, en dépit des profondes inégalités qu'ils subissent dans le monde en matière d'accès aux vaccins. Les laboratoires devraient tenter de reproduire les approches les plus efficaces pour détecter le SARS-CoV-2 dans la salive, tandis que les gouvernements devraient prendre des mesures favorisant un dépistage de masse en levant les restrictions qui entravent le déploiement des tests salivaires.

A medida que la enfermedad por coronavirus de 2019 (COVID-19) sigue afectando de manera desproporcionada a los países de ingresos bajos y medios, crece la necesidad de realizar pruebas de diagnóstico sencillas, accesibles y frecuentes. En entornos de bajos recursos, la detección de casos suele estar limitada por la falta de pruebas disponibles para diagnosticar el coronavirus del síndrome respiratorio agudo grave de tipo 2 (SARS-CoV-2). Para abordar las desigualdades globales en las pruebas, se podrían utilizar tipos de muestra alternativos para aumentar el acceso a las pruebas reduciendo los costes asociados. La saliva es una muestra de diagnóstico sensible, poco invasiva y económica para la detección del SARS-CoV-2 que es apropiada para la vigilancia asintomática, las pruebas sintomáticas y la obtención en el hogar. Las pruebas de saliva pueden reducir dos de los principales problemas a los que se enfrentan los países de ingresos bajos y medios: la escasez de recursos y la sobrecarga de trabajo del personal sanitario. La toma de muestras de saliva permite realizar fácilmente la obtención por cuenta propia y requiere menos recursos que los métodos con hisopos. Sin embargo, las pruebas de saliva para el diagnóstico del SARS-CoV-2 no se han aplicado a gran escala en los países de ingresos bajos y medios. Aunque varios estudios realizados en estos entornos han demostrado la utilidad del muestreo de saliva, no se ha prestado suficiente atención a la optimización de su aplicación en la práctica. En este sentido, se considera que la investigación científica sobre la implementación es necesaria para subsanar esta deficiencia entre la evidencia y la práctica. Los países de ingresos bajos y medios se enfrentan a muchas dificultades en sus esfuerzos por realizar pruebas masivas en relación con la COVID-19, a pesar de las grandes desigualdades en el acceso global a las vacunas. Los laboratorios deberían intentar reproducir los enfoques que han tenido éxito para la detección sensible de la infección por el SARS-CoV-2 en la saliva, mientras que los gobiernos deberían actuar para facilitar las pruebas masivas eliminando las restricciones que limitan la aplicación de los métodos de diagnóstico salival.

Impact of Temporary Storage Conditions on the Viability of Streptococcus pneumoniae in Saliva.

Nasopharyngeal swabs are considered the gold-standard sample type for the detection of Streptococcus pneumoniae carriage, but recent studies have demonstrated the utility of saliva in improving the detection of carriage in adults. Saliva is generally collected in its raw, unsupplemented state, unlike nasopharyngeal swabs, which are collected into stabilizing transport media. Few data exist regarding the stability of pneumococci in unsupplemented saliva during transport and laboratory storage. We therefore evaluated the effect of storage conditions on the detection of pneumococci in saliva samples using strains representing eight pneumococcal serotypes. The bacteria were spiked into raw saliva from asymptomatic individuals, and we assessed sample viability after storage at 4°C, room temperature, and 30°C for up to 72 h; at 40°C for 24 h; and following three freeze-thaw cycles. We observed little decrease in pneumococcal detection following culture enrichment and quantitative PCR (qPCR) detection of the piaB and lytA genes compared to testing fresh samples, indicating the prolonged viability of pneumococci in neat saliva samples. This sample stability makes saliva a viable sample type for pneumococcal carriage studies conducted in remote or low-resource settings and provides insight into the effect of the storage of saliva samples in the laboratory. IMPORTANCE For pneumococcal carriage studies, saliva is a sample type that can overcome some of the issues typically seen with nasopharyngeal and oropharyngeal swabs. Understanding the limitations of saliva as a sample type is important for maximizing its use. This study sought to better understand how different storage conditions and freeze-thaw cycles affect pneumococcal survival over time. These findings support the use of saliva as an alternative sample type for pneumococcal carriage studies, particularly in remote or low-resource settings with reduced access to health care facilities.

Association of upper respiratory Streptococcus pneumoniae colonization with SARS-CoV-2 infection among adults.

Background: Streptococcus pneumoniae interacts with numerous viral respiratory pathogens in the upper airway. It is unclear whether similar interactions occur with SARS-CoV-2. Methods: We collected saliva specimens from working-age adults receiving SARS-CoV-2 molecular testing at outpatient clinics and via mobile community-outreach testing between July and November 2020 in Monterey County, California. Following bacterial culture enrichment, we tested for pneumococci by quantitative polymerase chain reaction (qPCR) targeting the lytA and piaB genes, and measured associations with SARS-CoV-2 infection via conditional logistic regression. Results: Analyses included 1,278 participants, with 564 enrolled in clinics and 714 enrolled through outreach-based testing. Prevalence of pneumococcal carriage was 9.2% (117/1,278) among all participants (11.2% [63/564] clinic-based testing; 7.6% [54/714] outreach testing). Prevalence of SARS-CoV-2 infection was 27.4% (32/117) among pneumococcal carriers and 9.6% (112/1,161) among non-carriers (adjusted odds ratio [aOR]: 2.73; 95% confidence interval: 1.58-4.69). Associations between SARS-CoV-2 infection and pneumococcal carriage were enhanced in the clinic-based sample (aOR=4.01 [2.08-7.75]) and among symptomatic participants (aOR=3.38 [1.35-8.40]), when compared to findings within the outreach-based sample and among asymptomatic participants. Adjusted odds of SARS-CoV-2 co-infection increased 1.24 (1.00-1.55)-fold for each 1-unit decrease in piaB qPCR C T value among pneumococcal carriers. Last, pneumococcal carriage modified the association of SARS-CoV-2 infection with recent exposure to a suspected COVID-19 case (aOR=7.64 [1.91-30.7] and 3.29 [1.94-5.59]) among pneumococcal carriers and non-carriers, respectively). Conclusions: Associations of pneumococcal carriage detection and density with SARS-CoV-2 suggest a synergistic relationship in the upper airway. Longitudinal studies are needed to determine interaction mechanisms between pneumococci and SARS-CoV-2. Key points: In an adult ambulatory and community sample, SARS-CoV-2 infection was more prevalent among pneumococcal carriers than non-carriers.Associations between pneumococcal carriage and SARS-CoV-2 infection were strongest among adults reporting acute symptoms and receiving SARS-CoV-2 testing in a clinical setting.

Pneumococcal genetic variability in age-dependent bacterial carriage.

The characteristics of pneumococcal carriage vary between infants and adults. Host immune factors have been shown to contribute to these age-specific differences, but the role of pathogen sequence variation is currently less well-known. Identification of age-associated pathogen genetic factors could leadto improved vaccine formulations. We therefore performed genome sequencing in a large carriage cohort of children and adults and combined this with data from an existing age-stratified carriage study. We compiled a dictionary of pathogen genetic variation, including serotype, strain, sequence elements, single-nucleotide polymorphisms (SNPs), and clusters of orthologous genes (COGs) for each cohort - all of which were used in a genome-wide association with host age. Age-dependent colonization showed weak evidence of being heritable in the first cohort (h2 = 0.10, 95% CI 0.00-0.69) and stronger evidence in the second cohort (h2 = 0.56, 95% CI 0.23-0.87). We found that serotypes and genetic background (strain) explained a proportion of the heritability in the first cohort (h2serotype = 0.07, 95% CI 0.04-0.14 and h2GPSC = 0.06, 95% CI 0.03-0.13) and the second cohort (h2serotype = 0.11, 95% CI 0.05-0.21 and h2GPSC = 0.20, 95% CI 0.12-0.31). In a meta-analysis of these cohorts, we found one candidate association (p=1.2 × 10-9) upstream of an accessory Sec-dependent serine-rich glycoprotein adhesin. Overall, while we did find a small effect of pathogen genome variation on pneumococcal carriage between child and adult hosts, this was variable between populations and does not appear to be caused by strong effects of individual genes. This supports proposals for adaptive future vaccination strategies that are primarily targeted at dominant circulating serotypes and tailored to the composition of the pathogen populations.

Saliva as a sample type for SARS-CoV-2 detection: implementation successes and opportunities around the globe.

INTRODUCTION: Symptomatic testing and asymptomatic screening for SARS-CoV-2 continue to be essential tools for mitigating virus transmission. Though COVID-19 diagnostics initially defaulted to oropharyngeal or nasopharyngeal sampling, the worldwide urgency to expand testing efforts spurred innovative approaches and increased diversity of detection methods. Strengthening innovation and facilitating widespread testing remains critical for global health, especially as additional variants emerge and other mitigation strategies are recalibrated. AREAS COVERED: A growing body of evidence reflects the need to expand testing efforts and further investigate the efficiency, sensitivity, and acceptability of saliva samples for SARS-CoV-2 detection. Countries have made pandemic response decisions based on resources, costs, procedures, and regional acceptability - the adoption and integration of saliva-based testing among them. Saliva has demonstrated high sensitivity and specificity while being less invasive relative to nasopharyngeal swabs, securing saliva's position as a more acceptable sample type. EXPERT OPINION: Despite the accessibility and utility of saliva sampling, global implementation remains low compared to swab-based approaches. In some cases, countries have validated saliva-based methods but face challenges with testing implementation or expansion. Here, we review the localities that have demonstrated success with saliva-based SARS-CoV-2 testing approaches and can serve as models for transforming concepts into globally-implemented best practices.

Sequencing SARS-CoV-2 genomes from saliva.

Genomic sequencing is crucial to understanding the epidemiology and evolution of Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Often, genomic studies rely on remnant diagnostic material, typically nasopharyngeal (NP) swabs, as input into whole-genome SARS-CoV-2 next-generation sequencing pipelines. Saliva has proven to be a safe and stable specimen for the detection of SARS-CoV-2 RNA via traditional diagnostic assays; however, saliva is not commonly used for SARS-CoV-2 sequencing. Using the ARTIC Network amplicon-generation approach with sequencing on the Oxford Nanopore MinION, we demonstrate that sequencing SARS-CoV-2 from saliva produces genomes comparable to those from NP swabs, and that RNA extraction is necessary to generate complete genomes from saliva. In this study, we show that saliva is a useful specimen type for genomic studies of SARS-CoV-2.

Evaluation of saliva self-collection devices for SARS-CoV-2 diagnostics.

BACKGROUND: There is an urgent need to expand testing for SARS-CoV-2 and other respiratory pathogens as the global community struggles to control the COVID-19 pandemic. Current diagnostic methods can be affected by supply chain bottlenecks and require the assistance of medical professionals, impeding the implementation of large-scale testing. Self-collection of saliva may solve these problems, as it can be completed without specialized training and uses generic materials. METHODS: We observed 30 individuals who self-collected saliva using four different collection devices and analyzed their feedback. Two of these devices, a funnel and bulb pipette, were used to evaluate at-home saliva collection by 60 individuals. SARS-CoV-2-spiked saliva samples were subjected to temperature cycles designed to simulate the conditions the samples might be exposed to during the summer and winter seasons and sensitivity of detection was evaluated. RESULTS: All devices enabled the safe, unsupervised self-collection of saliva. The quantity and quality of the samples received were acceptable for SARS-CoV-2 diagnostic testing, as determined by human RNase P detection. There was no significant difference in SARS-CoV-2 nucleocapsid gene (N1) detection between the freshly spiked samples and those incubated with the summer and winter profiles. CONCLUSION: We demonstrate inexpensive, generic, buffer free collection devices suitable for unsupervised and home saliva self-collection.

Single-cell multi-omics reveals dyssynchrony of the innate and adaptive immune system in progressive COVID-19.

Dysregulated immune responses against the SARS-CoV-2 virus are instrumental in severe COVID-19. However, the immune signatures associated with immunopathology are poorly understood. Here we use multi-omics single-cell analysis to probe the dynamic immune responses in hospitalized patients with stable or progressive course of COVID-19, explore V(D)J repertoires, and assess the cellular effects of tocilizumab. Coordinated profiling of gene expression and cell lineage protein markers shows that S100Ahi/HLA-DRlo classical monocytes and activated LAG-3hi T cells are hallmarks of progressive disease and highlights the abnormal MHC-II/LAG-3 interaction on myeloid and T cells, respectively. We also find skewed T cell receptor repertories in expanded effector CD8+ clones, unmutated IGHG+ B cell clones, and mutated B cell clones with stable somatic hypermutation frequency over time. In conclusion, our in-depth immune profiling reveals dyssynchrony of the innate and adaptive immune interaction in progressive COVID-19.