Open-source real-time quantitative RT-PCR-based on a RNA standard for the assessment of SARS-CoV-2 viral load

BACKGROUND Detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) target genes by molecular methods has been chosen as the main approach to identify individuals with Coronavirus disease 2019 (COVID-19) infection. OBJECTIVES In this study, we developed an open-source RNA standard-based real-time quantitative RT-PCR (RT-qPCR) assay for quantitative diagnostics of SARS-CoV-2 from nasopharynx, oropharynx, saliva and plasma samples. METHODS AND FINDINGS We evaluated three SARS-CoV-2 target genes and selected the RNA-dependent RNA polymerase (RdRp) gene, given its better performance. To improve the efficiency of the assay, a primer gradient containing 25 primers forward and reverse concentration combinations was performed. The forward and reverse primer pairs with 400 nM and 500 nM concentrations, respectively, showed the highest sensitivity. The LOD95% was ~60 copies per reaction. From the four biological matrices tested, none of them interfered with the viral load measurement. Comparison with the AllplexTM 2019-nCoV assay (Seegene) demonstrated that our test presents 90% sensitivity and 100% specificity. MAIN CONCLUSIONS We developed an efficient molecular method able to measure absolute SARS-CoV-2 viral load with high replicability, sensitivity and specificity in different clinical samples.

Avaliação funcional de pacientes internados na Unidade de Terapia Intensiva adulto do Hospital Universitário de Canoas / Evaluación funcional de pacientes internados en la unidad de cuidados intensivos adultos de Hospital Universitario de Canoas / Functional assessment of inpatients in the Intensive Care Unit of the University Hospital of Canoas

RESUMO A sobrevida de pacientes críticos tem aumentado com o tempo. No entanto, a imobilidade e o tempo de internação estão contribuindo para o seu declínio funcional e da sua qualidade de vida. O objetivo do estudo foi avaliar a independência funcional dos pacientes internados na Unidade de Terapia Intensiva (UTI) Adulto do Hospital Universitário de Canoas. Pesquisa de coorte prospectiva executada de fevereiro a dezembro de 2016. Os pacientes foram avaliados quanto à capacidade funcional, força muscular, força de preensão palmar, mobilidade, equilíbrio e marcha. Foram avaliados 90 pacientes com média de idade de 59,6±16,1 anos, com predominância do gênero masculino (51,1%). A mediana do tempo de internação na UTI foi de 5 (3-9) dias, e de internação hospitalar de 13 (10-20) dias. Houve melhora significativa nos resultados de capacidade funcional (p<0,001), mobilidade (p=0,004) e equilíbrio (p=0,009). Os pacientes internados apresentaram um declínio funcional (com relação à normalidade) nos momentos avaliados. Entretanto, houve melhora nos valores até momento da alta hospitalar.

RESUMEN La sobrevida de pacientes críticos ha aumentado con el tiempo. Sin embargo, la inmovilidad y el tiempo de internación están contribuyendo a su declive funcional y su calidad de vida. El objetivo del estudio ha sido evaluar la independencia funcional de los pacientes internados en la Unidad de Cuidados Intensivos (UCI) Adulto de Hospital Universitário de Canoas. Investigación de cohorte prospectiva ejecutada de febrero a diciembre de 2016. Los pacientes han sido evaluados en cuanto a la capacidad funcional, fuerza muscular, fuerza de prensión de mano, movilidad, equilibrio y marcha. Se evaluaron 90 pacientes con una media de edad de 59.6±16.1 años, con predominio del género masculino (51.1%). La mediana del tiempo de internación en la UCI ha sido de 5 (3-9) días, y de internación hospitalaria de 13 (10-20) días. Se observó una mejora significativa en los resultados de capacidad funcional (p<0.001), movilidad (p=0.004) y equilibrio (p=0.009). Los pacientes internados presentaron un declive funcional (con relación a la normalidad) en los momentos evaluados. Sin embargo, hubo mejora en los valores hasta el momento del alta hospitalaria.

ABSTRACT Survival of critically ill patients has increased over time. However, immobility and length of hospital stay contribute to these patient's functional decline and reduction in quality of life. To assess the functional independence of patients admitted to the Intensive Care Unit (ICU) of the University Hospital of Canoas, a prospective cohort study was performed from February to December 2016. Functional capacity, muscle strength, hand grip strength, mobility, balance, and gait were assessed. 90 patients aged on average 59.6±16.1 years old were assessed, with a predominance of male individuals (51.1%). The median of length of stay in the ICU was 5 (3-9) days and the median of hospital stay was 13 (10-20) days. Functional capacity (p<0.001), mobility (p=0.004), and balance (p=0.009) significantly improved. Hospitalized patients showed functional decline (compared to normative values) in all assessments. However, all scores improved after hospital discharge.

Detection of human adenovirus, rotavirus and enterovirus in water samples collected on dairy farms from Tenente Portela, Northwest of Rio Grande do Sul, Brazil

Viral gastroenteritis and other waterborne diseases are a major concern for health in Brazil. A number of studies were conducted about the presence of viruses on water samples from Brazilian areas. However, the knowledge about the occurrence of viral contamination of drinking water sources in rural settings of the country is insufficient. On the present work, 15 samples from 5 dairy farms located at the municipality of Tenente Portela were collected and analysed for the presence of human adenoviruses (HAdV), as well as human enteroviruses (EV) and rotaviruses (RV). HAdV was present on 66.66% of the water samples, and have been found in all samples from artesian wells and springs, which are used as sources of drinking water for the individuals inhabiting those farms. EV and RV found only in one sample each. The detection rates of HAdV on the water from these dairy farms are alarming and point towards a situation of elevated environmental contamination by fecal microorganisms of human origin and poor basic sanitation conditions.

Detecção molecular de vírus da bronquite infecciosa em plantéis de avós, matrizes e frangos de corte no Rio Grande do Sul e Mato Grosso / Molecular detection of infectious bronchitis virus in flocks of grandmothers, mothers and broiler chicken in Rio Grande do Sul and Mato Grosso

O vírus da Bronquite Infecciosa das galinhas (VBI) pertence ao grupo 3 da família Coronaviridae e é o causador de desordens respiratórias e renais em frangos de corte. A vacinação com vacinas vivas é praticada em matrizes e avós e muitas vezes também nos plantéis destinados ao abate. As vacinas utilizadas no Brasil são usualmente do sorogrupo Massachusetts e baseadas nas amostras H120 e H52. É comum que após a vacinação o vírus vacinal seja detectado por isolamento em ovos embrionados ou por métodos moleculares por até 4 semanas. Após essa data, normalmente, não há detecção de vírus e o VBI, quando encontrado, pode representar recirculação do vírus vacinal no plantel ou a introdução de uma nova cepa do vírus. No presente estudo, para avaliar a circulação do vírus em plantéis de frangos e reprodutoras nos estados do Rio Grande do Sul e Mato Grosso do Sul, foram coletadas 240 traqueias e rins de aves de 48 plantéis, sendo (20 exemplares/4 plantéis) de avós, (80 exemplares/16 plantéis) de matrizes e (140 exemplares/28 plantéis) de frangos de corte, as quais foram analisadas em misturas de cinco amostras. Todos os animais eram vacinados e as amostras foram coletadas ao redor de 2 a 48 semanas após a vacinação. A presença de VBI foi determinada com auxílio de uma reação em cadeia da polimerase tipo nested, direcionada ao gene da proteína S1, padronizada neste estudo. Das 48 amostras testadas, 14 resultaram positivas: cinco foram oriundas de aves vacinadas há menos de quatro semanas na data da coleta e nove eram de amostras de aves vacinadas há mais de quatro semanas, o que pode ser devido à recirculação do vírus vacinal ou mesmo introdução de vírus selvagem nos plantéis.

Infectious bronchitis virus (IBV, Avian Coronavirus) from chickens belongs to group 3 of the family Coronaviridae and causes respiratory and renal disorders in broilers. Vaccination using live vaccines is generally performed in mothers and grandmothers, as well as often in flocks for slaughter. The vaccines used in Brazil are usually from serogroup Massachusetts and based on standard samples of the virus at passages H120 and H52. It is common that after vaccination the vaccine virus is detected by isolation in embryonated eggs or by molecular methods for up to four weeks. After, there is usually no virus detection and any IBV found may represent recirculation of the vaccine virus in the flock or the introduction of a new strain. In this study, to evaluate the circulation of the virus in poultry flocks and breeders in the state of Rio Grande do Sul and Mato Grosso do Sul, 240 samples were collected from tracheas and kidneys of birds from 48 flocks, and (20 biological samples / 4 flocks) from grandmothers (80 samples/16 flocks) and mothers (140 samples/28 flocks) from broilers, which were analyzed in pools of five samples. All animals were vaccinated and samples were collected around 2-48 weeks after vaccination. The presence of IBV was determined with the aid of a polymerase chain reaction "nested" gene-directed protein S1, standardized in this study. From the 48 samples tested, 14 were positive: 5 were from birds vaccinated after less than 4 weeks and 9 were from birds vaccinated more than four weeks should be wild viruses or represent the recirculation of the vaccine virus.