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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.مع استمرار تأثير مرض فيروس كورونا 2019 (كوفيد 19) بشكل غير متناسب على الدول ذات الدخل المنخفض والدخل المتوسط، فإن الحاجة تزداد إلى اختبارات تشخيصية بسيطة، ومتواترة، ويسهل الوصول لها. في الأوضاع منخفضة الموارد، غالبًا ما يكون اكتشاف الحالات محدودًا بسبب نقص الاختبارات المتاحة لمتلازمة الجهاز التنفسي الحادة الشديدة فيروس كورونا 2 (SARS-CoV-2). للتعامل مع التفاوتات العالمية في الاختبار، يمكن استخدام أنواع بديلة من العينات لزيادة الوصول إلى الاختبار عن طريق تقليل التكاليف المرتبطة به. اللعاب هو عينة تشخيصية حساسة، قليلة التوغل، وغير مكلفة للكشف عن SARS-CoV-2، وهي مناسبة للمراقبة بدون أعراض، واختبار الأعراض، والتجميع في المنزل. يمكن أن يقلل اختبار اللعاب من تحديين رئيسيين تواجههما الدول ذات الدخل المنخفض والمتوسط، ألا وهما: الموارد المحدودة والعاملين في المجال الصحي المثقلين بالأعباء. يتيح أخذ عينات اللعاب جمعها بشكل ذاتي ومناسب، ويتطلب موارد أقل عن الطرق المعتمدة على المسحة. ومع ذلك، لم يتم تنفيذ اختبار اللعاب لتشخيصات SARS-CoV-2 على نطاق واسع في الدول ذات الدخل المنخفضة والمتوسطة. في حين أن العديد من الدراسات التي تعتمد على هذه الأوضاع قد أثبتت فائدة أخذ عينات اللعاب، إلا أنه لم يكن هناك اهتمام كافٍ بتحسين تنفيذه في الممارسة العملية. نحن نزعم بأن البحث العلمي للتنفيذ ضروري لسد هذه الفجوة بين الدليل والممارسة. تواجه الدول ذات الدخل المنخفض والمتوسطة العديد من العقبات، بينما تواصل جهودها لتوفير اختبار جماعي لفيروس كوفيد 19 في مواجهة التفاوتات الكبيرة في الحصول العالمي على اللقاحات. يجب أن تتطلع المختبرات إلى تكرار الأساليب الناجحة للكشف الدقيق عن فيروس SARS-CoV-2 في اللعاب، بينما يجب على الحكومات العمل لتسهيل الاختبارات الجماعية عن طريق رفع القيود التي تحد من تنفيذ الأساليب المعتمدة على اللعاب.随着新型冠状病毒肺炎 (COVID-19) 持续对中低收入国家造成严重的影响,对简便、易于频繁使用的诊断检测方法的需在不断增长。在资源匮乏地区,因缺乏可用于严重急性呼吸综合征冠状病毒-2 (SARS-CoV-2) 的检测方法,病例检测也通常会受到限制。为解决全球范围内检测方法不足的问题,可使用替代样本类型来降低相关成本,从而增加检测的使用率。对于 SARS-CoV-2 检测,唾液是一种成本低廉的敏感性微创诊断样本,可用于无症状监测、症状性检测和在家自检。唾液检测可缓解中低收入国家面临的两大挑战:资源有限和医护人员的工作负担过重。唾液取样便于开展自检,并且与基于拭子的方法相比,所消耗的资源也更少。然而,在中低收入国家,通过唾液检测进行 SARS-CoV-2 诊断尚未得到大规模的实施。尽管大量在此类地区开展的研究表明唾液样本很有帮助,但是人们对优化其实际实施的关注度仍不足。我们认为需要对其实施开展科学研究,以弥补循证与实践之间的差距。在全球疫苗获取率严重不足的形势下,中低收入国家在继续推进实施大规模 COVID-19 检测方面仍面临许多障碍。各国的实验室应效仿其他地区在采用唾液样本进行敏感性 SARS-CoV-2 检测中的成功方法,同时各国政府应采取行动解除在实施唾液检测方法方面的限制,以促进大规模检测方法的实施。.Поскольку коронавирусная инфекция - 2019 (COVID-19) продолжает в большей степени поражать страны с низким и средним уровнем дохода, растет потребность в проведении простых, доступных и частых диагностических исследований. В условиях нехватки ресурсов выявление случаев заболевания часто ограничивается отсутствием доступных тестов на наличие коронавирусной инфекции 2-го типа, вызывающей развитие тяжелого острого респираторного синдрома (SARS-CoV-2). Для решения проблемы глобального неравенства в отношении тестирования могут быть использованы альтернативные типы образцов для расширения доступа к проведению тестирования за счет снижения сопутствующих расходов. Слюна является чувствительным, минимально инвазивным и недорогим диагностическим образцом для выявления SARS-CoV-2, который подходит для наблюдения за бессимптомной формой, симптоматического тестирования и сбора в домашних условиях. Анализ слюны может способствовать решению двух основных проблем, с которыми сталкиваются страны с низким и средним уровнем дохода: ограниченности ресурсов и перегруженности медицинских работников. Взятие образцов слюны предполагает удобный самостоятельный сбор и требует меньше ресурсов, чем методы на основе мазков. Однако анализ слюны для диагностики SARS-CoV-2 не был внедрен в широких масштабах в странах с низким и средним уровнем дохода. Несмотря на результаты многочисленных исследований, проведенных в этих условиях, которые продемонстрировали целесообразность взятия образцов слюны, вопросам оптимизации их применения на практике уделялось недостаточно внимания. Для преодоления этого разрыва между фактическими данными и применением на практике необходимы научные исследования в области внедрения. Страны с низким и средним уровнем дохода сталкиваются со многими трудностями, прилагая усилия по проведению массового тестирования на COVID-19 в условиях существенного неравенства в доступе к вакцинам на глобальном уровне. Лабораториям следует стремиться повторить успешные методы для диагностики вируса SARS-CoV-2 в слюне, а государствам следует принять меры для содействия массовому тестированию путем снятия ограничений на использование методов на основе слюны.

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.

BackgroundWhile the sensitivity of detection of pneumococcal carriage can be improved by testing respiratory tract samples with qPCR, concerns have been raised regarding the specificity of this approach. We therefore investigated the reliability of the widely-usedlytAqPCR assay when applied to saliva samples from older adults in relation to a more specific qPCR assay (piaB).MethodsDuring the autumn/winter seasons of 2018/2019 and 2019/2020, saliva was collected at multiple timepoints from 103 healthy adults aged 21-40 (n=34) and ≥64 (n=69) years. Following culture-enrichment, extracted DNA was tested using qPCR forpiaBandlytA. By sequencing the variable region ofrpsB(S2-typing), we identified the species of bacteria isolated from samples testinglytA-positive only.ResultsWhile 30/344 (8.7%) saliva samples (16.5% individuals) tested qPCR-positive for bothpiaBandlytA, 52 (15.1%) samples testedlytA-positive only. No samples testedpiaB-positive only. Through extensive re-culture of the 32lytA-positive samples collected in 2018/2019, we isolated 23 strains (from 8 samples, from 5 individuals) that were also qPCR-positive for onlylytA. Sequencing determined thatStreptococcus mitisandStreptococcus infantiswere predominantly responsible for thislytA-positive qPCR signal.ConclusionsWe identified a comparatively large proportion of samples generating positive signals with the widely usedlytA-qPCR and identified non-pneumococcal streptococcal 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 respiratory tract samples.

BackgroundStreptococcus pneumoniae interacts with numerous viral respiratory pathogens in the upper airway. It is unclear whether similar interactions occur with SARS-CoV-2.MethodsWe 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.ResultsAnalyses 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 CT 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).ConclusionsAssociations 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 pointsIn 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.

BackgroundWhether vaccination or natural infection provides greater benefit regarding the development of sustained immunity against SARS-CoV-2 remains unknown. Therefore, the aim of this study was to provide a direct comparison of IgG durability in vaccinated and unvaccinated adults.MethodsThis was a prospective, cross-sectional study of antibody durability in 1087 individuals with a median (IQR) age of 42 (35, 52) years who were unvaccinated and previously infected with SARS-CoV-2 (Arm 1, n=351) or vaccinated against the virus (Arm 2, n=737). Participants self-reported vaccination and infection history and provided self-collected serology samples using mailed collection kits.ResultsAnti-S1 IgG seroprevalence was 15.6% higher in vaccinated versus unvaccinated, previously-infected individuals across intervals ranging from 1 to 12 months and antibody survival was sustained near 100% through 12 months in the vaccinated group.ConclusionsThese findings suggest that vaccination as opposed to natural infection alone provides significant advantages in terms of sustained and effective immunity against prior variants of SARS-CoV-2. Future efforts to characterize SARS-CoV-2 immune responses should address hybrid immunity, booster status and formulation, and protection against (sub)variants of Omicron and future lineages, as well as weigh the potential impact of other immune system mechanisms.

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.

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.

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 participant’s 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.

BackgroundThe Omicron SARS-CoV-2 variant resulted in significant community-based transmission. Numerous occupational settings have employed surveillance testing strategies to minimize occupational exposure and return workers safely to work following isolation.MethodsFrom an occupational COVID-19 testing program, we obtained longitudinal (February 2021-January 2022) saliva-based RT-qPCR results and starting December 27th, 2021, daily on-site molecular over-the-counter (OTC) nasal swab-based isothermal nucleic acid amplification test (molecular OTC; Cue Health COVID-19 test) results. We quantified the fraction of tests with PCR cycle threshold (Ct) values 30, or two negative molecular OTC tests over a 24 hour period, or a single PCR test >30 plus negative molecular OTC test.ResultsFrom the paired testing cohort, 37 (48%) individuals tested positive; all 37 were vaccinated. All individuals tested positive ≤1 day after a previous negative test, and 19 (51.3%) remained PCR-positive with Ct values p=0.49). Peak viral load measured by PCR was 1.97 days from the initial positive test. There were no cases of transmission after returning to work.ConclusionsA large percentage of individuals remain contagious at day 5 post first positive test based on serial PCR testing and can continue until day 12. Early discontinuation of isolation can utilize a two test framework separated by 24 hours. Rapid onsite tests may be useful.

As the second year of the COVID-19 pandemic begins, it remains clear that a massive increase in the ability to test for SARS-CoV-2 infections in a myriad of settings is critical to controlling the pandemic and to preparing for future outbreaks. The current gold standard for molecular diagnostics is the polymerase chain reaction (PCR), but the extraordinary and unmet demand for testing in a variety of environments means that both complementary and supplementary testing solutions are still needed. This review highlights the role that loop-mediated isothermal amplification (LAMP) has had in filling this global testing need, providing a faster and easier means of testing, and what it can do for future applications, pathogens, and the preparation for future outbreaks. This review describes the current state of the art for research of LAMP-based SARS-CoV-2 testing, as well as its implications for other pathogens and testing. The authors represent the global LAMP (gLAMP) Consortium, an international research collective, which has regularly met to share their experiences on LAMP deployment and best practices; sections are devoted to all aspects of LAMP testing, including preanalytic sample processing, target amplification, and amplicon detection, then the hardware and software required for deployment are discussed, and finally, a summary of the current regulatory landscape is provided. Included as well are a series of first-person accounts of LAMP method development and deployment. The final discussion section provides the reader with a distillation of the most validated testing methods and their paths to implementation. This review also aims to provide practical information and insight for a range of audiences: for a research audience, to help accelerate research through sharing of best practices; for an implementation audience, to help get testing up and running quickly; and for a public health, clinical, and policy audience, to help convey the breadth of the effect that LAMP methods have to offer.

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°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.

The performance of Covid-19 diagnostic tests must continue to be reassessed with new variants of concern. The objective of this study was to describe the discordance in saliva SARS-CoV-2 PCR and nasal rapid antigen test results during the early infectious period. We identified a high-risk occupational case cohort of 30 individuals with daily testing during an Omicron outbreak in December 2021. Based on viral load and transmissions confirmed through epidemiological investigation, most Omicron cases were infectious for several days before being detectable by rapid antigen tests.

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/µL, 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.

Background Infections with respiratory viruses [e.g. influenza and respiratory syncytial virus (RSV)] can increase the risk of severe pneumococcal infections. Likewise, pneumococcal coinfection is associated with poorer outcomes in viral respiratory infection. However, there are limited data describing the frequency of pneumococcus and SARS-CoV-2 coinfection and the role of coinfection in influencing COVID-19 severity. We, therefore, investigated the detection of pneumococcus in COVID-19 inpatients during the early pandemic period. Methods The study included patients aged 18 years and older, admitted to the 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). Results Among 148 subjects, the median age was 65 years; 54.7% were male; 50.7% had an ICU stay; 64.9% received antibiotics; and 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. Conclusions Pneumococcal lower respiratory tract infection (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.

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 minutes, 95°C for 5 minutes or 65°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.