Detection of pneumococcus during hospitalization for SARS-CoV-2

BackgroundInfections with respiratory viruses (e.g., influenza, RSV) can increase the risk of severe pneumococcal infections. Likewise, pneumococcal co-infection is associated with poorer outcomes in viral respiratory infection. However, there are limited data describing the frequency of pneumococcus and SARS-CoV-2 co-infection and the role of co-infection in influencing COVID-19 severity. MethodsThe study included patients admitted to Yale-New Haven Hospital who were symptomatic for respiratory infection and tested positive for SARS-CoV-2 during March-August 2020. Patients were tested for pneumococcus through culture-enrichment of saliva followed by RT-qPCR (to identify carriage) and serotype-specific urine antigen detection (UAD) assays (to identify presumed lower respiratory tract pneumococcal disease). ResultsAmong 148 subjects, the median age was 65 years; 54.7% were male; 50.7% had an ICU stay; 64.9% received antibiotics; 14.9% died while admitted. Pneumococcal carriage was detected in 3/96 (3.1%) individuals tested by saliva RT-qPCR. Additionally, pneumococcus was detected in 14/127 (11.0%) individuals tested by UAD, and more commonly in severe than moderate COVID-19 (OR: 2.20; 95% CI: [0.72, 7.48]); however, the numbers were small with a high degree of uncertainty. None of the UAD-positive individuals died. ConclusionsPneumococcal LRTI, as detected by positive UAD, occurred in patients hospitalized with COVID-19. Moreover, pneumococcal LRTI was more common in those with more serious COVID-19 outcomes. Future studies should assess how pneumococcus and SARS-CoV-2 interact to influence COVID-19 severity in hospitalized patients. One Sentence SummaryPneumococcal lower respiratory tract infection, as detected by positive UAD, occurred in patients hospitalized with COVID-19 at rates similar to those reported prepandemic.

Persistence of pneumococcal carriage among older adults in the community despite COVID-19 mitigation measures

BackgroundReported 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. MethodsBetween October 2020-August 2021, couples living in the Greater New Haven Area 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 less than 40. ResultsWe collected 567 saliva samples from 95 individuals aged 60 years and above (47 household pairs and one singleton). Of those, 7.1% of samples tested positive for pneumococcus by either piaB only (n=6) or both piaB and lytA (n=34), representing 22/95 (23.2%) individuals and 16/48 (33.3%) households over the course of the 10-week study period. 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., 2-9 year olds) had a higher prevalence of carriage (15.9% vs 5.4%). ConclusionsDespite COVID-19-related disruptions, a large proportion of older adults carried pneumococcus at least once during the 10-week study period. Prevalence was particularly high among those who had contact with school-aged children, but carriage was not limited to this group.

Routine Saliva Testing for SARS-CoV-2 in Children: Partnering with Childcare Centers in the Greater New Haven Community

BackgroundWhile 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, 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. MethodsWe enrolled children (aged 6 months to 7 years) and staff at 8 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 for RT-PCR testing using SalivaDirect and results were uploaded to each participants online patient portal within 24 hours. ResultsThis study fostered 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 were overcome by working with parents/guardians to conceptualize new collection strategies and by offering parents/guardians continued virtual guidance and support. ConclusionSARS-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 the childcare age group, especially as a routine surveillance tool, an at-home saliva collection method provides an attractive alternative. 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.

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

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 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 assay. A pool size of five, with or without heat inactivation at 65{degrees}C for 15 minutes 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.

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{degrees}C for 30 minutes, 95{degrees}C for 5 minutes or 65{degrees}C for 15 minutes) 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.

Evaluation of the Liberty16 mobile real time PCR device for use with the SalivaDirect assay for SARS-CoV-2 testing

The COVID-19 pandemic has highlighted the need and benefits for all communities to be permitted timely access to on-demand screening for infectious respiratory diseases. This can be achieved with simplified testing approaches and affordable access to core resources. While RT-qPCR-based tests remain the gold standard for SARS-CoV-2 detection due to their high sensitivity, implementation of testing requires high upfront costs to obtain the necessary instrumentation. This is particularly restrictive in low-resource settings. The Ubiquitome Liberty16 system was developed as an inexpensive, portable, battery-operated single-channel RT-qPCR device with an associated iPhone app to simplify assay set-up and data reporting. When coupled with the SalivaDirect protocol for testing saliva samples for SARS-CoV-2, the Liberty16 device yielded a limit of detection (LOD) of 12 SARS-CoV-2 RNA copies/{micro}L, comparable to the upper end of the LOD range for the standard SalivaDirect protocol when performed on larger RT-qPCR instruments. While further optimization may deliver even greater sensitivity and assay speed, findings from this study indicate that small portable devices such as the Liberty16 can deliver reliable results and provide the opportunity to further increase access to gold standard SARS-CoV-2 testing.

Usability of saliva collection devices for SARS-CoV-2 diagnostics

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. In this study, we observed thirty 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. 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 RNase P detection. Here, we demonstrate inexpensive, generic, buffer free collection devices suitable for unsupervised and home saliva self-collection.