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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.

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.

Cache Valley virus (CVV) is a prevalent emerging pathogen of significant importance to agricultural and human health in North America. Emergence in livestock can result in substantial agroeconomic losses resulting from the severe embryonic lethality associated with infection during pregnancy. Although CVV pathogenesis has been well described in ruminants, small animal models are still unavailable, which limits our ability to study its pathogenesis and perform preclinical testing of therapeutics. Herein, we explored CVV pathogenesis, tissue tropism, and disease outcomes in a variety of murine models, including immune -competent and -compromised animals. Our results show that development of CVV disease in mice is dependent on innate immune responses, and type I interferon signalling is essential for preventing infection in mice. IFN-αβR-/- mice infected with CVV present with significant disease and lethal infections, with minimal differences in age-dependent pathogenesis, suggesting this model is appropriate for pathogenesis-related, and short- and long-term therapeutic studies. We also developed a novel CVV in utero transmission model that showed high rates of transmission, spontaneous abortions, and congenital malformations during infection. CVV infection presents a wide tissue tropism, with significant amplification in liver, spleen, and placenta tissues. Immune-competent mice are generally resistant to infection, and only show disease in an age dependent manner. Given the high seropositivity rates in regions of North America, and the continuing geographic expansion of competent mosquito vectors, the risk of epidemic and epizootic emergence of CVV is high, and interventions are needed for this important pathogen.

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.

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.

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.

The agents of equine piroplasmosis, Theileria equi and Babesia caballi, are endemic in Trinidad, West Indies. While transmission is mainly by ixodid ticks, transplacental transmission of T. equi has also been reported. This disease has contributed to foetal losses as well as morbidity and mortality of neonatal foals and adult horses. Previous 18S rRNA-based phylogenetic studies indicated a noticeable degree of variation within and among B. caballi and T. equi isolates from different geographical regions. The objective of this study was to evaluate the diversity of T. equi and B. caballi obtained from horses in Trinidad by amplifying a region of the 18S rRNA gene. The phylogenetic trees for T. equi sequences obtained from horses in 2006 and 2011-2013 revealed that Trinidad sequences were of genotype A. Additionally, all of the B. caballi sequences from Trinidad were grouped together with other B. caballi sequences of genotype A. However, T. equi sequences from horses in Saint Kitts and Nevis clustered with sequences of genotype C. This study also identified two genotypes of T. equi in the equine population of Brazil. All of the T. equi and B. caballi sequences obtained from horses in Trinidad belong to genotype A and were similar to T. equi and B. caballi sequences of the same genotype that were submitted to GenBank™ databases. Countries in close proximity to Trinidad have T. equi sequences belonging to genotype C; therefore, movement of horses between these countries can introduce a new genotype of T. equi into the equid population of Trinidad.

SalivaDirect™ is an RNA-extraction free, dual-plexed RT-qPCR method for SARS-CoV-2 detection. It can be broadly implemented as it (1) does not require saliva collection tubes containing preservatives, (2) does not require specialized equipment for RNA extraction, and (3) is validated for use with products from multiple vendors. Thus, the simplicity and flexibility of SalivaDirect™ means that it is not as affected by supply chain bottlenecks as some other assays. Our method is RNA-extraction free which enables testing of low volume and minimally processed saliva in dual-plexed RT-qPCR for SARS-CoV-2 detection. Saliva will be treated with proteinase K followed by a heat inactivation step, and is then directly used as input in the dual-plexed RT-qPCR test. Our aim was not to design new primers and probes for RT-qPCR testing, but rather to use validated primer and probe sets (N1 and RP) developed by the US CDC. The human Ribonuclease P (RP) probe was modified with a different fluorophore so that the primer/probe set could be combined in a dualplex assay, reducing the number of tests to 1 assay with 2 sets. Version 2 includes: Optimized thermocycler conditions Locally validated alternative options for Proteinase K, RT-qPCR master mix, and thermocyclers Use of 8-strip tubes for sample processing step, due to contamination issues in 96-well plates. Version 3 has been updated to remove steps for sample self-collection. Version 4 has updated Ct thresholds for the ABI 7500 Fast Dx. Version 5 has an updated description for use, additional RP probe with ATTO647 fluorophore, and a detailed table with catalog numbers. Version 6 includes: Introducing multiple workflows for sample processing including heat Pre-treatment as an alternative to Proteinase K Updated list of RT-qPCR reagents, theromcyclers and protocol for 384 well format.

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.

Dengue viruses (DENVs) are classified into four serotypes, each of which contains multiple genotypes. DENV genotypes introduced into the Americas over the past five decades have exhibited different rates and patterns of spatial dispersal. In order to understand factors underlying these patterns, we utilized a statistical framework that allows for the integration of ecological, socioeconomic, and air transport mobility data as predictors of viral diffusion while inferring the phylogeographic history. Predictors describing spatial diffusion based on several covariates were compared using a generalized linear model approach, where the support for each scenario and its contribution is estimated simultaneously from the data set. Although different predictors were identified for different serotypes, our analysis suggests that overall diffusion of DENV-1, -2, and -3 in the Americas was associated with airline traffic. The other significant predictors included human population size, the geographical distance between countries and between urban centers and the density of people living in urban environments. ispartof: VIRUS EVOLUTION vol:6 issue:2 ispartof: location:England status: published

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). Disclosures Anne Wyllie, PhD, Global Diagnostic Systems (Consultant)Pfizer (Advisor or Review Panel member, Research Grant or Support)PPS Health (Consultant)Tempus Labs, Inc (Research Grant or Support) Ronika Alexander-Parrish, RN, MAEd, Pfizer (Employee, Shareholder) Adriano Arguedas, MD, Pfizer (Employee) Bradford D. Gessner, MD, MPH, Pfizer Inc. (Employee) Daniel Weinberger, PhD, Affinivax (Consultant)Merck (Consultant, Grant/Research Support)Pfizer (Consultant, Grant/Research Support)

Local transmission of chikungunya virus (CHIKV) was first detected in the Americas in December 2013, after which it spread rapidly throughout the Caribbean islands and American mainland, causing a major chikungunya fever epidemic. Previous phylogenetic analysis of CHIKV from a limited number of countries in the Americas suggests that an Asian genotype strain was responsible, except in Brazil where both Asian and East/Central/South African (ECSA) lineage strains were detected. In this study, we sequenced thirty-three complete CHIKV genomes from viruses isolated in 2014 from fourteen Caribbean islands, the Bahamas and two mainland countries in the Americas. Phylogenetic analyses confirmed that they all belonged to the Asian genotype and clustered together with other Caribbean and mainland sequences isolated during the American outbreak, forming an 'Asian/American' lineage defined by two amino acid substitutions, E2 V368A and 6K L20M, and divided into two well-supported clades. This lineage is estimated to be evolving at a mean rate of 5 x 10-4 substitutions per site per year (95% higher probability density, 2.9-7.9 x 10-4) and to have arisen from an ancestor introduced to the Caribbean (most likely from Oceania) in about March 2013, 9 months prior to the first report of CHIKV in the Americas. Estimation of evolutionary rates for individual gene regions and selection analyses indicate that (in contrast to the Indian Ocean Lineage that emerged from the ECSA genotype followed by adaptive evolution and with a significantly higher substitution rate) the evolutionary dynamics of the Asian/American lineage are very similar to the rest of the Asian genotype and natural selection does not appear to have played a major role in its emergence. However, several codon sites with evidence of positive selection were identified within the non-structural regions of Asian genotype sequences outside of the Asian/American lineage.

Local transmission of Chikungunya virus (CHIKV) was first documented in Trinidad and Tobago (T&T) in July 2014 preceding a large epidemic. At initial presentation, it is difficult to distinguish chikungunya fever (CHIKF) from other acute undifferentiated febrile illnesses (AUFIs), including life-threatening dengue disease. We characterised and compared dengue virus (DENV) and CHIKV infections in 158 patients presenting with suspected dengue fever (DF) and CHIKF at a major hospital in T&T, and performed phylogenetic analyses on CHIKV genomic sequences recovered from 8 individuals. The characteristics of patients with and without PCR-confirmed CHIKV were compared using Pearson’s χ2 and student’s t-tests, and adjusted odds ratios (aORs) and 95% confidence intervals (CIs) were determined using logistic regression. We then compared signs and symptoms of people with RT-qPCR-confirmed CHIKV and DENV infections using the Mann-Whitney U, Pearson’s χ2 and Fisher’s exact tests. Among the 158 persons there were 8 (6%) RT-qPCR-confirmed DENV and 30 (22%) RT-qPCR-confirmed CHIKV infections. Phylogenetic analyses showed that the CHIKV strains belonged to the Asian genotype and were most closely related to a British Virgin Islands strain isolated at the beginning of the 2013/14 outbreak in the Americas. Compared to persons who were RT-qPCR-negative for CHIKV, RT-qPCR-positive individuals were significantly more likely to have joint pain (aOR: 4.52 [95% CI: 1.28–16.00]), less likely to be interviewed at a later stage of illness (days post onset of fever—aOR: 0.56 [0.40–0.78]) and had a lower white blood cell count (aOR: 0.83 [0.71–0.96]). Among the 38 patients with RT-qPCR-confirmed CHIKV or DENV, there were no significant differences in symptomatic presentation. However when individuals with serological evidence of recent DENV or CHIKV infection were included in the analyses, there were key differences in clinical presentation between CHIKF and other AUFIs including DF, which can be used to triage patients for appropriate care in the clinical setting., Author Summary Chikungunya virus (CHIKV) recently emerged in the Americas and caused a major epidemic of chikungunya fever (CHIKF). While not usually life threatening, CHIKF is a debilitating and often chronic illness resulting in major morbidity and economic losses. It is difficult to distinguish CHIKF from other viral illnesses that cause acute fevers including dengue fever (DF), an important consideration since DF can be life-threatening and early identification and treatment of cases is key to reducing mortality. In this study we investigated individuals presenting to a major hospital in Trinidad and Tobago (T&T) with suspected DF or CHIKF, and identified signs and symptoms that distinguish these two illnesses. We also recovered complete genome sequences for CHIKV and show that the etiologic strain in T&T is a closely related descendent of the strain first isolated from the British Virgin Islands at the beginning of the outbreak in the Americas.

Changes in Dengue virus (DENV) disease patterns in the Americas over recent decades have been attributed, at least in part, to repeated introduction of DENV strains from other regions, resulting in a shift from hypoendemicity to hyperendemicity. Using newly sequenced DENV-1 and DENV-3 envelope (E) gene isolates from 11 Caribbean countries, along with sequences available on GenBank, we sought to document the population genetic and spatiotemporal transmission histories of the four main invading DENV genotypes within the Americas and investigate factors that influence the rate and intensity of DENV transmission. For all genotypes, there was an initial invasion phase characterized by rapid increases in genetic diversity, which coincided with the first confirmed cases of each genotype in the region. Rapid geographic dispersal occurred upon each genotype's introduction, after which individual lineages were locally maintained, and gene flow was primarily observed among neighboring and nearby countries. There were, however, centers of viral diversity (Barbados, Puerto Rico, Colombia, Suriname, Venezuela, and Brazil) that were repeatedly involved in gene flow with more distant locations. For DENV-1 and DENV-2, we found that a "distance-informed" model, which posits that the intensity of virus movement between locations is inversely proportional to the distance between them, provided a better fit than a model assuming equal rates of movement between all pairs of countries. However, for DENV-3 and DENV-4, the more stochastic "equal rates" model was preferred.

Bat rabies is an emerging disease of public health significance in the Americas. The Caribbean island of Trinidad experiences periodic outbreaks within the livestock population. We performed molecular characterisation of Trinidad rabies virus (RABV) and used a Bayesian phylogeographic approach to investigate the extent to which outbreaks are a result of in situ evolution versus importation of virus from the nearby South American mainland. Trinidadian RABV sequences were confirmed as bat variant and clustered with Desmodus rotundus (vampire bat) related sequences. They fell into two largely temporally defined lineages designated Trinidad I and II. The Trinidad I lineage which included sequences from 1997–2000 (all but two of which were from the northeast of the island) was most closely related to RABV from Ecuador (2005, 2007), French Guiana (1990) and Venezuela (1993, 1994). Trinidad II comprised sequences from the southwest of the island, which clustered into two groups: Trinidad IIa, which included one sequence each from 2000 and 2007, and Trinidad IIb including all 2010 sequences. The Trinidad II sequences were most closely related to sequences from Brazil (1999, 2004) and Uruguay (2007, 2008). Phylogeographic analyses support three separate RABV introductions from the mainland from which each of the three Trinidadian lineages arose. The estimated dates for the introductions and subsequent lineage expansions suggest periods of in situ evolution within Trinidad following each introduction. These data also indicate co-circulation of Trinidad lineage I and IIa during 2000. In light of these findings and the likely vampire bat origin of Trinidadian RABV, further studies should be conducted to investigate the relationship between RABV spatiotemporal dynamics and vampire bat population ecology, in particular any movement between the mainland and Trinidad., Author Summary The Caribbean island of Trinidad experiences periodic rabies virus (RABV) outbreaks within the livestock population. In this study, we inferred the evolutionary history of RABV in the Americas and reconstructed past patterns of RABV geographic spread in order to address the question of whether Trinidadian outbreaks arise from locally maintained RABV or are the result of virus importation from the mainland (presumably via infected bats). Our results provide statistical support for three importation events that gave rise to each of three Trinidadian vampire bat-associated lineages identified in the study. They also indicate limited periods of in situ evolution within Trinidad following each of these introductions. The results also support Mexico and Brazil as major epicenters for the expansion of RABV associated with vampire bats throughout the Americas and consequently to Trinidad. The findings of our study are particularly relevant to local RABV monitoring and control. In addition to justifying vampire bats as the main target for active rabies surveillance and control activities in Trinidad, they suggest that more intense surveillance of regions that lie close to the mainland may be warranted. Finally, in light of these findings, further studies should be conducted to investigate the relationship between RABV spatiotemporal dynamics and vampire bat population ecology.