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1.
Open Forum Infectious Diseases ; 8(SUPPL 1):S284, 2021.
Article in English | EMBASE | ID: covidwho-1746632

ABSTRACT

Background. Quickly detecting and isolating individuals positive for SARSCoV-2 is essential for limiting virus spread. Policy makers rely on the number of active cases to make decisions, and individuals use this information to evaluate risk should they return to public spaces. Robust testing strategies have been plagued with limited authorized diagnostic assays and high test prices, with large-scale implementation hampered by worldwide supply chain issues. Methods. Having identified its potential early in the pandemic, we simplified saliva-based COVID-19 diagnostic testing by (1) not requiring collection tubes with preservatives, (2) replacing nucleic acid extraction with a simple enzymatic and heating step, and (3) testing specimens for SARS-CoV-2 in dualplex RT-qPCR. Moreover, we validated this approach ("SalivaDirect") with reagents and instruments from multiple vendors to circumvent supply chain disruptions. Results. SalivaDirect's simplified protocol does not compromise on sensitivity. In our hospital cohort, we found a high positive agreement (94%) between saliva tested with SalivaDirect and nasopharyngeal swabs tested with a commercial RT-qPCR kit. With the National Basketball Association we tested 3,779 saliva specimens from healthy individuals and detected low rates of invalid (0.3%) and false-positive (< 0.05%) results. Using comparative assays and sample types, we also demonstrated SalivaDirect to efficiently detect SARS-CoV-2 in asymptomatic individuals. SalivaDirect is a simplified method for SARS-CoV-2 detection (A) Schematic overview of SalivaDirect workflow depicting the main steps of mixing saliva with proteinase K, heat inactivation, and dualplex qRT-PCR testing. Figure created with Biorender.com. (B) SARS-CoV-2 is stable in saliva for at least 7 days at 4C, room temperature (RT;19C), and 30C without addition of stabilizing buffers. Spiked-in saliva samples of low virus concentrations (12, 25, and 50 SARSCoV-2 copies/mL) were kept at the indicated temperature for 7 days and then tested with SalivaDirect. N1 cycle threshold (Ct) values were lower when kept for 7 days at 30C as compared to fresh specimens (Kruskal-Wallis;p = 0.03). Horizontal bars indicate the median. (C) Comparing Ct values for saliva treated with proteinase K and heat as compared to nucleic extraction yields higher N1 Ct values without extraction (Wilcoxon;p < 0.01). (D) Testing extracted nucleic acid from saliva with the N1 primer-probe set (singleplex) as compared to a multiplex assay showed stronger N1 detection in multiplex (Wilcoxon;p < 0.01). The dotted line in (B)-(D) indicates the limit of detection. Conclusion. Saliva is a valid alternative to swabs for SARS-CoV-2 screening. Importantly, SalivaDirect enables labs to utilize existing infrastructure, improving test implementation time and requiring limited investment to scale-up to meet mass testing needs. With the safe and reliable self-collection of saliva, our vision is to help provide accessible and equitable testing solutions, especially in low-resource and remote settings.

2.
Open Forum Infectious Diseases ; 8(SUPPL 1):S749, 2021.
Article in English | EMBASE | ID: covidwho-1746303

ABSTRACT

Background. Despite the widespread use of pneumococcal conjugate vaccines, particularly in children, an important burden of pneumococcal disease remains in older adults. The acquisition and transmission rates of pneumococcus between older adults have not been well characterized. Methods. Between October 2020-June 2021, couples living in the Greater New Haven Area were enrolled if both individuals were over the age of 60 years and did not have any individuals under the age of 60 years living in the household. Saliva samples and questionnaires regarding social patterns 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 qPCR Ct-values for piaB +/- lytA were less than 40. Results. To date, we have collected 495 saliva samples from 95 individuals (48 households). Of 495 saliva samples, 31 (5.9%) have tested positive for pneumococcus by either piaB only (n=9) or both lytA and piaB (n=22). Of 95 individuals, 16 (16.8%) (representing 13, or 27.1% households) have tested positive at least once. Six of the 16 (37.5%) carriers tested positive at multiple timepoints, though none were colonized at all 6 time points over the course of the 10 weeks of study enrolment. For 3 of the 48 (6.3%) households, both members of the couple were identified as carriers, though not necessarily at the same sampling moment. Conclusion. The preliminary findings of this longitudinal transmission model demonstrate evidence of pneumococcal acquisition among older adults measured by molecular tools. These transmission patterns and high rates of pneumococcal carriage in adults were observed during a period when the COVID-19 pandemic led to numerous preventative public health measures that may have reduced pneumococcal transmission (e.g., social distancing, mask wearing, bans on mass gatherings, restaurant closures, travel restrictions).

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