SARS-CoV-2 Detection and Culture in Different Biological Specimens from Immunocompetent and Immunosuppressed COVID-19 Patients Infected with Two Different Viral Strains.

Introduction-The dynamics of SARS-CoV-2 shedding and replication in humans remain incompletely understood. Methods-We analyzed SARS-CoV-2 shedding from multiple sites in individuals with an acute COVID-19 infection by weekly sampling for five weeks in 98 immunocompetent and 25 immunosuppressed individuals. Samples and culture supernatants were tested via RT-PCR for SARS-CoV-2 to determine viral clearance rates and in vitro replication. Results-A total of 2447 clinical specimens were evaluated, including 557 nasopharyngeal swabs, 527 saliva samples, 464 urine specimens, 437 anal swabs and 462 blood samples. The SARS-CoV-2 genome sequences at each site were classified as belonging to the B.1.128 (ancestral strain) or Gamma lineage. SARS-CoV-2 detection was highest in nasopharyngeal swabs regardless of the virus strain involved or the immune status of infected individuals. The duration of viral shedding varied between clinical specimens and individual patients. Prolonged shedding of potentially infectious virus varied from 10 days up to 191 days, and primarily occurred in immunosuppressed individuals. Virus was isolated in culture from 18 nasal swab or saliva samples collected 10 or more days after onset of disease. Conclusions-Our findings indicate that persistent SARS-CoV-2 shedding may occur in both competent or immunosuppressed individuals, at multiple clinical sites and in a minority of subjects is capable of in vitro replication.

Shedding of infectious SARS-COV-2 in two asymptomatic children / Excreción de SARS-CoV-2 infeccioso en dos niños asin tomáticos

Abstract Asymptomatic infections with SARS-CoV-2 are associ ated with viral transmission and have a key role in the propagation of the pandemic. Understanding viral shed ding during asymptomatic infections is critical. Unfor tunately, data on asymptomatic SARS-CoV-2 infection in children is extremely limited. To determine the presence of viral viable shedding, we prospectively followed two healthy children of a family where both parents devel oped mild COVID-19 (April 2021). SARS-CoV-2 detection was made by RT-PCR and virus isolation by cell culture from saliva samples. Positive samples were sequenced to identify variants of SARS-CoV-2. Serum samples were evaluated to determine the presence of antibodies using a single enzyme-linked immunosorbent assay (ELISA, COVIDAR IgG). Both children were SARS-CoV-2 positive and asymptomatic. In addition, the virus grew in cell cul ture from saliva samples. Furthermore, one child showed viable SARS-CoV-2 for at least 17 days after the onset symptoms from his father. The recommended isolation period for asymptomatic contacts during the acquisition of data had been established for 10 days; however, this child remained with viable virus beyond that period. The positive samples from both children were consistent with B.1.1.28.1 lineage (Gamma). In both asymptomatic children, anti-Spike IgG was detected. Asymptomatic children may represent a source of infection that should not be underestimated during this pandemic.

Resumen Las infecciones asintomáticas por SARS-CoV-2 están asociadas a la transmisión viral y tienen un papel cla ve en la propagación de la pandemia. Comprender la excreción viral durante las infecciones asintomáticas es fundamental. Desafortunadamente, los datos sobre la infección asintomática por SARS-CoV-2 en niños son extremadamente limitados. Para determinar la presencia de excreción de virus viable, se siguió prospectivamente a dos niños sanos de una familia en la que ambos padres desarrollaron COVID-19 leve (abril 2021). La detección de SARS-CoV-2 se realizó por RT-PCR y el aislamiento del virus por cultivo celular a partir de muestras de saliva. Las muestras positivas se secuenciaron para identificar variantes de SARS-CoV-2. En las muestras de suero se determinó la presencia de anticuerpos utilizando un ensayo de ELISA (COVIDAR IgG). Ambos niños fueron positivos para SARS-CoV-2 y asintomáticos. Además, el virus creció en cultivos celulares a partir de muestras de saliva. Uno de los niños mantuvo SARS-CoV-2 via bles durante al menos 17 días después de la aparición de los síntomas de su padre. El período de aislamiento recomendado para contactos asintomáticos durante la adquisición de datos se había establecido en 10 días, sin embargo, este niño permaneció con virus viable más allá de ese período. Las muestras positivas de estos niños correspondieron al linaje B.1.1.28.1 (Gamma). En ambos niños asintomáticos se detectó anticuerpos IgG anti-Spike. Concluimos que los niños asintomáticos pueden representar una fuente de infección que no debe subestimarse durante esta pandemia.

Shedding of infectious SARS-CoV-2 in two asymptomatic children.

Asymptomatic infections with SARS-CoV-2 are associated with viral transmission and have a key role in the propagation of the pandemic. Understanding viral shedding during asymptomatic infections is critical. Unfortunately, data on asymptomatic SARS-CoV-2 infection in children is extremely limited. To determine the presence of viral viable shedding, we prospectively followed two healthy children of a family where both parents developed mild COVID-19 (April 2021). SARS-CoV-2 detection was made by RT-PCR and virus isolation by cell culture from saliva samples. Positive samples were sequenced to identify variants of SARS-CoV-2. Serum samples were evaluated to determine the presence of antibodies using a single enzyme-linked immunosorbent assay (ELISA, COVIDAR IgG). Both children were SARS-CoV-2 positive and asymptomatic. In addition, the virus grew in cell culture from saliva samples. Furthermore, one child showed viable SARS-CoV-2 for at least 17 days after the onset symptoms from his father. The recommended isolation period for asymptomatic contacts during the acquisition of data had been established for 10 days; however, this child remained with viable virus beyond that period. The positive samples from both children were consistent with B.1.1.28.1 lineage (Gamma). In both asymptomatic children, anti-Spike IgG was detected. Asymptomatic children may represent a source of infection that should not be underestimated during this pandemic.

Las infecciones asintomáticas por SARS-CoV-2 están asociadas a la transmisión viral y tienen un papel clave en la propagación de la pandemia. Comprender la excreción viral durante las infecciones asintomáticas es fundamental. Desafortunadamente, los datos sobre la infección asintomática por SARS-CoV-2 en niños son extremadamente limitados. Para determinar la presencia de excreción de virus viable, se siguió prospectivamente a dos niños sanos de una familia en la que ambos padres desarrollaron COVID-19 leve (abril 2021). La detección de SARS-CoV-2 se realizó por RT-PCR y el aislamiento del virus por cultivo celular a partir de muestras de saliva. Las muestras positivas se secuenciaron para identificar variantes de SARS-CoV-2. En las muestras de suero se determinó la presencia de anticuerpos utilizando un ensayo de ELISA (COVIDAR IgG). Ambos niños fueron positivos para SARS-CoV-2 y asintomáticos. Además, el virus creció en cultivos celulares a partir de muestras de saliva. Uno de los niños mantuvo SARS-CoV-2 viables durante al menos 17 días después de la aparición de los síntomas de su padre. El período de aislamiento recomendado para contactos asintomáticos durante la adquisición de datos se había establecido en 10 días, sin embargo, este niño permaneció con virus viable más allá de ese período. Las muestras positivas de estos niños correspondieron al linaje B.1.1.28.1 (Gamma). En ambos niños asintomáticos se detectó anticuerpos IgG anti-Spike. Concluimos que los niños asintomáticos pueden representar una fuente de infección que no debe subestimarse durante esta pandemia.

SARS-CoV-2 infection-induced immunity and the duration of viral shedding: Results from a Nicaraguan household cohort study.

BACKGROUND: Much of the world's population has been infected with SARS-CoV-2. Thus, immunity from prior infection will play a critical role in future SARS-CoV-2 transmission. We investigated the impact of infection-induced immunity on viral shedding duration and viral load. METHODS: We conducted a household cohort study in Managua, Nicaragua, with an embedded transmission study that closely monitors participants regardless of symptoms. Real-time reverse-transcription polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assays (ELISAs) were used to measure infections and seropositivity, respectively. Blood samples were collected twice annually and surrounding household intensive monitoring periods. We used accelerated failure time models to compare shedding times. Participants vaccinated ≥14 days prior to infection were excluded from primary analyses. RESULTS: There were 600 RT-PCR-confirmed SARS-CoV-2 infections in unvaccinated participants between May 1, 2020, and March 10, 2022, with prior ELISA data. Prior infection was associated with 48% shorter shedding times (event time ratio [ETR] 0.52, 95% CI: 0.39-0.69, mean shedding: 13.7 vs. 26.4 days). A fourfold higher anti-SARS-CoV-2 spike titer was associated with 17% shorter shedding (ETR 0.83, 95% CI: 0.78-0.90). Similarly, maximum viral loads (lowest cycle threshold [CT]) were lower for previously infected individuals (mean CT 29.8 vs. 28.0, p = 4.02 × 10-3 ), for adults and children ≥10 years, but not for children 0-9 years; there was little difference in CT levels for previously infected versus naïve adults aged above 60 years. CONCLUSIONS: Prior infection-induced immunity was associated with shorter viral shedding and lower viral loads, which may be important in the transition from pandemic to endemicity.

¿Cuál es el período de excreción viral e infectividad de la variante Ómicron del virus SARS-CoV-2?: reporte breve de evidencia / What is the period of viral shedding and infectivity of the omicron variant of Sars-cov-2 virus?: brief report of evidence

ANTECEDENTES Y OBJETIVO Han pasado 2 años y medio desde que la Organización Mundial de la Salud decretó emergencia sanitaria por la pandemia ocasionada por SARS-CoV-2. Durante este tiempo, se han presentado una serie de desafíos para evitar su propagación, uno de ellos corresponde a las mutaciones y cambios que ha sufrido el virus. En noviembre de 2021 se notificó la variante Ómicron B.1.1.529, que presenta algunas mutaciones de preocupación, asociándose con mayor transmisibilidad y menor susceptibilidad a los anticuerpos neutralizantes. En este contexto la Jefatura de la División de Planificación Sanitaria solicita este reporte breve de evidencia con el objetivo de conocer el período de excreción viral e infectividad de la variante Ómicron. METODOLOGÍA Se buscaron revisiones sistemáticas que respondieron la pregunta en las bases de datos MEDLINE y EMBASE a través de OVID y en Epistemonikos, con fecha 30 de agosto 2022. Debido a no encontrarse evidencia en los motores de búsqueda seleccionados, se realizó una búsqueda en Google académico, sin embargo, tampoco fue identificada evidencia alguna. Se incluyen revisiones sistemáticas, estudios que describieron o midieran los períodos de infectividad y excreción de la variante Ómicron del virus SARS-CoV-2. Se excluyen períodos de infectividad y excreción de otras variantes del Virus SARS-CoV-2. RESULTADOS -No se encontró evidencia que describiera el periodo de infectividad y excreción del virus en personas contagiadas con SARS-COV-2 variante Ómicron.

SARS-CoV-2 loads in urine, sera and stool specimens in association with clinical features of COVID-19 patients.

Background: COVID-19 pandemic continues to be a priority in public health worldwide, and factors inherent to SARS-CoV-2 pathogenesis and genomic characteristics are under study. Investigations that evaluate possible risk factors for infection, clinical manifestations, and viral shedding in different specimens also need to clarify possible associations with COVID-19 prognosis and disease outcomes. Study design: In this study, we evaluated SARS-CoV-2 positivity and estimated viral loads by real-time RT-PCR in stool, sera, and urine samples from 35 patients, with a positive SARS-CoV-2 RNA molecular test in respiratory sample, attended at a University COVID-19 referral hospital in Goiania, Goias, Brazil. Whole-genome sequencing was also performed in samples with higher viral load. Results: The positivity index was 51.43%, 14.28%, and 5.71% in stool, sera, and urine specimens, respectively. The median viral load was 8.01 × 106 GC/g, 2.03 × 106 GC/mL, and 1.36 × 105 GC/mL in stool, sera, and urine, respectivelly. Of all patients, 88.57% had previous comorbidities, and 48.39% of them had detectable SARS-CoV-2 RNA in at least one type of clinical specimen evaluated by this study (stool, sera or urine). A higher viral load was observed in patients with more than two previous comorbidities and that were classified as severe or critical conditions. Samples with the highest viral loads were sequenced and characterized as B.1.1.33 variant. Conclusion: We conclude that SARS-CoV-2 RNA is present in more than one type of clinical specimen during the infection, and that the most critical patients had detectable viral RNA in more than one clinical specimen at the same time point.

Atypical Prolonged Viral Shedding With Intra-Host SARS-CoV-2 Evolution in a Mildly Affected Symptomatic Patient.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection is caused by a respiratory virus with a wide range of manifestations, varying from asymptomatic to fatal cases, with a generally short outcome. However, some individuals present long-term viral shedding. We monitored 38 individuals who were mildly affected by the SARS-CoV-2 infection. Out of the total studied population, three (7.9%) showed atypical events regarding the duration of positivity for viral RNA detection. In one of these atypical cases, a previously HIV-positive male patient presented a SARS-CoV-2 RNA shedding and subgenomic RNA (sgRNA) detected from the upper respiratory tract, respectively, for 232 and 224 days after the onset of the symptoms. The SARS-CoV-2 B.1.1.28 lineage, one of the most prevalent in Brazil in 2020, was identified in this patient in three serial samples. Interestingly, the genomic analyses performed throughout the infectious process showed an increase in the genetic diversity of the B.1.1.28 lineage within the host itself, with viral clearance occurring naturally, without any intervention measures to control the infection. Contrasting widely spread current knowledge, our results indicate that potentially infectious SARS-CoV-2 virus might be shed by much longer periods by some infected patients. This data call attention to better adapted non-pharmacological measures and clinical discharge of patients aiming at preventing the spread of SARS-CoV-2 to the population.

Clinical practice in COVID-19: The most frequently asked questions to infectious diseases specialists.

Since the emergence of the disease caused by the severe respiratory syndrome coronavirus 2 (SARS-CoV-2) - COVID-19 - in late December 2019, a vast number of publications on the subject appeared in peer-reviewed journals and preprints. Despite the significant amount of available information, infectious disease physicians are requested to solve questions from colleagues, patients, and relatives on a daily basis. Here, we aim to describe the evidence supporting the answers for frequently asked questions, based on a literature review. We created a web-based questionnaire which was distributed to a group of 70 infectious disease specialists and medical residents, asking what questions and issues they most frequently faced. The 10 most frequent questions guided the topics for a narrative review. We provide evidence and consensus-based information on subjects such as infection and transmission, isolation, management of COVID-19 confirmed cases, reinfection, clinical-therapeutic management, vaccination, and antibodies post-infection/vaccination. Correctly clarifying doubts and providing clear information to physicians, patients, and family members helps to better manage COVID-19 in the community and the hospital settings.

Respiratory Viral Shedding in Healthcare Workers Reinfected with SARS-CoV-2, Brazil, 2020.

We documented 4 cases of severe acute respiratory syndrome coronavirus 2 reinfection by non-variant of concern strains among healthcare workers in Campinas, Brazil. We isolated infectious particles from nasopharyngeal secretions during both infection episodes. Improved and continued protection measures are necessary to mitigate the risk for reinfection among healthcare workers.

A household case evidences shorter shedding of SARS-CoV-2 in naturally infected cats compared to their human owners.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been detected in domestic and wild cats. However, little is known about natural viral infections of domestic cats, although their importance for modelling disease spread, informing strategies for managing positive human-animal relationships and disease prevention. Here, we describe the SARS-CoV-2 infection in a household of two human adults and sibling cats (one male and two females) using real-time RT-PCR, an ELISA test, viral sequencing, and virus isolation. On May 5th, 2020, the cat-owners tested positive for SARS-CoV-2. Two days later, the male cat showed mild respiratory symptoms and tested positive. Four days after the male cat, the two female cats became positive, asymptomatically. Also, one human and one cat showed antibodies against SARS-CoV-2. All cats excreted detectable SARS-CoV-2 RNA for a shorter duration than humans and viral sequences analysis confirmed human-to-cat transmission. We could not determine if cat-to-cat transmission also occurred.

No effects of noncytopathic bovine viral diarrhea virus type 2 on the reproductive tract of experimentally inoculated boars.

Bovine viral diarrhea virus (BVDV) infections in pigs may result in transient leukopenia, chronic gastroenteritis, septicemia, and hemorrhagic lesions. Both classical swine fever virus (CSF) and the atypical porcine pestivirus (APPV) are shed in the semen of infected boars. Because these viruses share conserved regions and present antigenic similarity, they may not be the only species belonging to the genus Pestivirus that can be shed in the semen of infected pigs. The objective of this study was to evaluate the testicular and epididymal changes, seminal parameters, and viral shedding in the reproductive tract of boars experimentally inoculated with noncytopathic BVDV-2. Six males were selected, and samples of blood, semen, and preputial swabs were collected every four days until the 52nd day after inoculation. The samples were tested for the presence of viral RNA by RT-PCR. An aliquot of whole blood was used to perform hematological analyses, which showed a significant reduction in monocyte counts and a significant increase in lymphocyte counts when comparing the pre- and postinoculation periods. The neutralizing antibody titers were determined by the virus neutralization test. None of the animals presented clinical signs or worsening of the seminal parameters that were evaluated. Moreover, BVDV-2 shedding by the reproductive route was not observed.

Effect of Oral Oseltamivir on Virological Outcomes in Low-risk Adults With Influenza: A Randomized Clinical Trial.

BACKGROUND: Duration of viral shedding is a determinant of infectivity and transmissibility, but few data exist about oseltamivir's ability to alter viral shedding. METHODS: From January 2012 through October 2017, a randomized, double-blinded multicenter clinical trial was conducted in adults aged 18-64 years at 42 sites in Thailand, the United States, and Argentina. Participants with influenza A or B and without risk factors for complications of influenza were screened for the study. Eligible participants were randomized to receive oseltamivir 75 mg or placebo twice daily for 5 days. The primary endpoint was the percentage of participants with virus detectable by polymerase chain reaction in nasopharyngeal swab at day 3. RESULTS: Of 716 adults screened for the study, 558 were randomized, and 501 were confirmed to have influenza. Forty-six participants in the pilot study were excluded, and 449 of the 455 participants in the population for the primary analysis had day 3 viral shedding results. Ninety-nine (45.0%) of 220 participants in the oseltamivir arm had virus detected at day 3 compared with 131 (57.2%) of 229 participants in the placebo arm (absolute difference of -12.2% [-21.4%, -3.0%], P =; .010). The median time to alleviation of symptoms was 79.0 hours for the oseltamivir arm and 84.0 hours for the placebo arm (P =; .34) in those with confirmed influenza infection. CONCLUSIONS: Oseltamivir decreased viral shedding in this low-risk population. However, in the population enrolled in this study, it did not significantly decrease the time to resolution of clinical symptoms. CLINICAL TRIALS REGISTRATION: NCT01314911.

Intermittent elimination of Porcine Parvovirus 4 (PPV4) in naturally infected swine semen: first report / Primeiro relato de eliminação intermitente de Parvovírus suíno 4 (PPV4) em sêmen de suíno infectado naturalmente

Porcine parvovirus 4 (PPV4) has been reported in several countries and the high rate of concurrent PCV2 infection with PPV4 may trigger the "porcine circovirus disease" (PCVD). This has awakened the interest in how PPV4 virus behaves. The aim of this study was to show that, like other swine viruses, the elimination of PPV4 in semen is intermittent. The study was conducted in the Unidade de Pesquisa e Desenvolvimento de Itapeva da Agência Paulista de Tecnologia dos Agronegócios Regional (Apta Regional). Over a period of six months, four boars were monitored and the semen was collected every 10 days, totalizing 74 semen samples. Through PCR testing, PPV4 was detected in 32/74 (43.2%) semen samples. Three sequences of 284 nt, showed identity varying from 98.2% to 100%, confirming the detection of the virus in all boars. Semen volume and sperm motility did not indicate significant difference between PPV4 positive or negative semen samples (p > 0.05). There was no significant difference between the monthly distributions of positive and negative samples. This is the first study showing intermittent shedding of PPV4 in naturally infected boars. Absence of typical clinical signs and/or influence in semen quantity was also observed.(AU)

O Parvovírus suíno 4 (PPV4) foi descrito em vários países e a alta taxa de infecção simultânea entre PCV2 e PPV4 está associada ao desencadeamento da Circovirose. Isso despertou o interesse em estudar o comportamento do PPV4. O objetivo deste estudo foi mostrar que, assim como outros vírus suínos, a eliminação de PPV4 no sêmen é intermitente. O estudo foi realizado na Unidade de Pesquisa e Desenvolvimento de Itapeva da Agência Paulista de Tecnologia dos Agronegócios Regionais. Durante um período de seis meses, quatro cachaços foram monitorados e o sêmen foi coletado a cada 10 dias, totalizando 74 amostras de sêmen. Através de testes de PCR, o PPV4 foi detectado em 43,2% (32/74) das amostras de sêmen. Três sequências de 284 nt apresentaram 100% de identidade entre si, e quando, comparadas com sequências de PPV4 disponíveis no GenBank, mostraram uma identidade de 98.2% a 100%, confirmando a detecção do PPV4 nas amostras de sêmen dos cachaços. O volume de sêmen e a motilidade espermática não mostraram diferença significativa entre amostras positivas ou negativas de PPV4 (p > 0,05). Não houve diferença significativa entre as distribuições mensais de amostras positivas e negativas. Este é o primeiro estudo que mostra a eliminação intermitente de PPV4 em cachaços infectados naturalmente. Ausência de sinais clínicos típicos e / ou influência na quantidade de sêmen também foi observada.(AU)

Intermittent elimination of Porcine Parvovirus 4 (PPV4) in naturally infected swine semen: first report / Primeiro relato de eliminação intermitente de Parvovírus suíno 4 (PPV4) em sêmen de suíno infectado naturalmente

Porcine parvovirus 4 (PPV4) has been reported in several countries and the high rate of concurrent PCV2 infection with PPV4 may trigger the "porcine circovirus disease" (PCVD). This has awakened the interest in how PPV4 virus behaves. The aim of this study was to show that, like other swine viruses, the elimination of PPV4 in semen is intermittent. The study was conducted in the Unidade de Pesquisa e Desenvolvimento de Itapeva da Agência Paulista de Tecnologia dos Agronegócios Regional (Apta Regional). Over a period of six months, four boars were monitored and the semen was collected every 10 days, totalizing 74 semen samples. Through PCR testing, PPV4 was detected in 32/74 (43.2%) semen samples. Three sequences of 284 nt, showed identity varying from 98.2% to 100%, confirming the detection of the virus in all boars. Semen volume and sperm motility did not indicate significant difference between PPV4 positive or negative semen samples (p > 0.05). There was no significant difference between the monthly distributions of positive and negative samples. This is the first study showing intermittent shedding of PPV4 in naturally infected boars. Absence of typical clinical signs and/or influence in semen quantity was also observed.

O Parvovírus suíno 4 (PPV4) foi descrito em vários países e a alta taxa de infecção simultânea entre PCV2 e PPV4 está associada ao desencadeamento da Circovirose. Isso despertou o interesse em estudar o comportamento do PPV4. O objetivo deste estudo foi mostrar que, assim como outros vírus suínos, a eliminação de PPV4 no sêmen é intermitente. O estudo foi realizado na Unidade de Pesquisa e Desenvolvimento de Itapeva da Agência Paulista de Tecnologia dos Agronegócios Regionais. Durante um período de seis meses, quatro cachaços foram monitorados e o sêmen foi coletado a cada 10 dias, totalizando 74 amostras de sêmen. Através de testes de PCR, o PPV4 foi detectado em 43,2% (32/74) das amostras de sêmen. Três sequências de 284 nt apresentaram 100% de identidade entre si, e quando, comparadas com sequências de PPV4 disponíveis no GenBank, mostraram uma identidade de 98.2% a 100%, confirmando a detecção do PPV4 nas amostras de sêmen dos cachaços. O volume de sêmen e a motilidade espermática não mostraram diferença significativa entre amostras positivas ou negativas de PPV4 (p > 0,05). Não houve diferença significativa entre as distribuições mensais de amostras positivas e negativas. Este é o primeiro estudo que mostra a eliminação intermitente de PPV4 em cachaços infectados naturalmente. Ausência de sinais clínicos típicos e / ou influência na quantidade de sêmen também foi observada.