Use of saliva and RT-PCR screening for SARS-CoV-2 variants of concern: Surveillance and monitoring.

Genomic surveillance has been applied since the beginning of the COVID-19 pandemic to track the spread of the virus, leading to the characterization of multiple SARS-CoV-2 variants, including variants of concern (VOC). Although sequencing is the standard method, a rapid molecular test for screening and surveillance of VOC is considered for detection. Furthermore, using alternative saliva as specimen collection facilitates the implementation of a less invasive, self-collected sample. In this study, we applied a combinatory strategy of saliva collection and reverse transcription polymerase chain reaction (RT-PCR) for SARS-CoV-2 VOC detection. Saliva samples from patients attending a tertiary hospital with suspected COVID-19 were collected and SARS-CoV-2 RNA was detected using SARS-CoV-2 RT-qPCR reagent kit (PerkinElmer). Positive saliva samples were screened for SARS-CoV-2 VOC with previously described RT-PCR for Alpha, Beta, and Gamma variants. Saliva samples were positive in 171 (53%) of 324 tested. A total of 108 (74%) from positive samples were also positive for VOC by RT-PCR screening. Those samples were found between January and August 2021. This approach allowed us to successfully use an alternative and complementary tool to genomic surveillance to monitor the circulation of SARS-CoV-2 VOC in the studied population.

Typical epidemiology of respiratory virus infections in a Brazilian slum.

Host population size, density, immune status, age structure, and contact rates are critical elements of virus epidemiology. Slum populations stand out from other settings and may present differences in the epidemiology of acute viral infections. We collected nasopharyngeal specimens from 282 children aged ≤5 years with acute respiratory tract infection (ARI) during 2005 to 2006 in one of the largest Brazilian slums. We conducted real-time reverse transcription-polymerase chain reaction (RT-PCR) for 16 respiratory viruses, nested RT-PCR-based typing of rhinoviruses (HRVs), and collected clinical symptoms. Viruses were common causes of respiratory disease; with ≥1 virus being detected in 65.2% of patients. We detected 15 different viruses during 1 year with a predominance of HRV (33.0%) and human respiratory syncytial virus (hRSV, 12.1%) infections, and a high rate of viral coinfections (28.3%). We observed seasonality of hRSV, HRV and human coronavirus infections, more severe symptoms in hRSV and influenza virus (FLU) infections and prolonged circulation of seven HRV clusters likely representing distinct serotypes according to genomic sequence distances. Potentially unusual findings included the absence of human metapneumovirus detections and lack of typical FLU seasonal patterns, which may be linked to the population size and density of the slum. Nonetheless, most epidemiological patterns were similar to other studies globally, suggesting surprising similarities of virus-associated ARI across highly diverse settings and a complex impact of population characteristics on respiratory virus epidemiology.

Evidence for an Ancestral Association of Human Coronavirus 229E with Bats.

UNLABELLED: We previously showed that close relatives of human coronavirus 229E (HCoV-229E) exist in African bats. The small sample and limited genomic characterizations have prevented further analyses so far. Here, we tested 2,087 fecal specimens from 11 bat species sampled in Ghana for HCoV-229E-related viruses by reverse transcription-PCR (RT-PCR). Only hipposiderid bats tested positive. To compare the genetic diversity of bat viruses and HCoV-229E, we tested historical isolates and diagnostic specimens sampled globally over 10 years. Bat viruses were 5- and 6-fold more diversified than HCoV-229E in the RNA-dependent RNA polymerase (RdRp) and spike genes. In phylogenetic analyses, HCoV-229E strains were monophyletic and not intermixed with animal viruses. Bat viruses formed three large clades in close and more distant sister relationships. A recently described 229E-related alpaca virus occupied an intermediate phylogenetic position between bat and human viruses. According to taxonomic criteria, human, alpaca, and bat viruses form a single CoV species showing evidence for multiple recombination events. HCoV-229E and the alpaca virus showed a major deletion in the spike S1 region compared to all bat viruses. Analyses of four full genomes from 229E-related bat CoVs revealed an eighth open reading frame (ORF8) located at the genomic 3' end. ORF8 also existed in the 229E-related alpaca virus. Reanalysis of HCoV-229E sequences showed a conserved transcription regulatory sequence preceding remnants of this ORF, suggesting its loss after acquisition of a 229E-related CoV by humans. These data suggested an evolutionary origin of 229E-related CoVs in hipposiderid bats, hypothetically with camelids as intermediate hosts preceding the establishment of HCoV-229E. IMPORTANCE: The ancestral origins of major human coronaviruses (HCoVs) likely involve bat hosts. Here, we provide conclusive genetic evidence for an evolutionary origin of the common cold virus HCoV-229E in hipposiderid bats by analyzing a large sample of African bats and characterizing several bat viruses on a full-genome level. Our evolutionary analyses show that animal and human viruses are genetically closely related, can exchange genetic material, and form a single viral species. We show that the putative host switches leading to the formation of HCoV-229E were accompanied by major genomic changes, including deletions in the viral spike glycoprotein gene and loss of an open reading frame. We reanalyze a previously described genetically related alpaca virus and discuss the role of camelids as potential intermediate hosts between bat and human viruses. The evolutionary history of HCoV-229E likely shares important characteristics with that of the recently emerged highly pathogenic Middle East respiratory syndrome (MERS) coronavirus.

Prevalence of human herpesvirus in plasma and saliva of cirrhotic patients: A pilot study.

AIMS: The objective of this study was to identify the presence of human herpesvirus (HHV) in the plasma and saliva of hepatic-cirrhosis patients and correlate it with clinical data and laboratory tests. This is a pilot, observational, and cross-sectional study. METHODS AND RESULTS: Specimens of plasma and saliva from 72 cirrhotic individuals were analyzed by means of polymerase chain reaction. The patient population had a mean age of 54.84 years old (SD ± 10) and was 70% males (51/72). Approximately 47% (n = 34) of the patients had leukopenia and HHV was not identified in the plasma specimens. The main species of HHV identified in the saliva were HHV-7 (n = 42, 62%) and Epstein-Barr virus (EBV) (n = 30, 41%). Moreover, there was a significant decrease in the total number of leukocytes and lymphocytes in saliva containing EBV (P = .038 and P = .047, respectively). CONCLUSION: The results show that the presence of EBV in the saliva of cirrhotic patients was correlated with their circulating immune status. It may be possible that the immune dysfunction displayed by the cirrhotic patients plays a role in the shedding of EBV into saliva.

Assessing the risks for stillbirth in São Paulo, Brazil: protocol for a multidisciplinary case-control study - FetRisks.

Stillbirth is a fundamental component of childhood mortality, but its causes are still insufficiently understood. This study aims to explore stillbirth risk factors by using a multidisciplinary approach to stimulate public policies and protocols to prevent stillbirth, improve maternal care and support bereaved families. METHODS AND ANALYSIS: In this case-control study with stillbirths and live births in 14 public hospitals in São Paulo, mothers are interviewed at hospitals after delivery, and hospital records and prenatal care registries are reviewed. Maternal and umbilical cord blood samples and placentas are collected to analyse angiogenesis and infection biomarkers, and the placenta's anatomopathological exam. Air pollutant exposure is estimated through the participant's residence and work addresses. Traditional and non-invasive autopsies by image-guided histopathology are conducted in a subset of stillbirths. Subsample mothers of cases are interviewed at home 2 months after delivery on how they were dealing with grief. Information contained in the official prenatal care registries of cases and controls is being compiled. Hospital managers are interviewed about the care offered to stillbirth mothers. Data analysis will identify the main risk factors for stillbirth, investigate their interrelations, and evaluate health services care and support for bereaved families. We hope this project will contribute to the understanding of stillbirth's risk factors and related health services in Brazil, providing new knowledge about this central public health problem, contributing to the improvement of public policies and prenatal and puerperal care, helping to prevent stillbirths and improve the healthcare and support for bereaved families. ETHICS AND DISSEMINATION: This study protocol was approved by the Ethics Committee of the Municipal Health Secretary (process no 16509319.0.3012.5551) and of the Hospital das Clínicas, Faculdade de Medicina, Universidade de São Paulo (process no 16509319.0.0000.0068). Results will be communicated to the study participants, policy-makers and the scientific community.

Investigation of Oral Shedding of Torquetenovirus (TTV) in Moderate-to-Severe COVID-19 Hospitalised Patients.

BACKGROUND: Torquetenovirus (TTV) is a small DNA virus constituting the human virome. High levels of TTV-DNA have been shown to be associated with immunosuppression and inflammatory chronic disorders. AIM: To assess the possible association between the salivary viral load of TTV-DNA in patients hospitalised due to COVID-19 and disease severity. METHODS: Saliva samples collected from 176 patients infected with SARS-CoV-2 were used to investigate the presence of SARS-CoV-2 and TTV-DNA by use of real-time RT-PCR. RESULTS: The majority of patients were male with severe COVID-19. Presence of SARS-CoV-2 was observed in the saliva of 64.77% of patients, showing TTV-DNA in 55.68% of them. Patients with impaired clinical conditions (p < 0.001), which evolved to death (p = 0.003), showed a higher prevalence of TTV-DNA. The median viral load in patients with severe condition was 4.99 log10 copies/mL, in which those who were discharged and those evolving to death had values of 3.96 log10 copies/mL and 6.27 log10 copies/mL, respectively. A statistically significant association was found between the distribution of TTV-DNA viral load in saliva samples and severity of COVID-19 (p = 0.004) and disease outcomes (p < 0.001). CONCLUSIONS: These results indicate that TTV-DNA in saliva could be a useful biomarker of COVID-19 severity and prognosis.

The differential clinical impact of human coronavirus species in children with cystic fibrosis.

We investigated the clinical impact of human coronaviruses (HCoV) OC43, 229E, HKU1 and NL63 in pediatric patients with cystic fibrosis (CF) during routine and exacerbation visits. A total of 408 nasopharyngeal aspirate samples were obtained from 103 patients over a 1-year period. Samples positive for HCoV were submitted for nucleotide sequencing to determine the species. Nineteen samples (4.65%) were positive for HCoV, of which 8 were positive for NL63, 6 for OC43, 4 for HKU1, and 1 for 229E. Identification of HCoV was not associated with an increased rate of respiratory exacerbations, but NL63-positive patients had higher exacerbation rates than patients who were positive for other HCoV species.

SARS-CoV-2 in saliva, viremia and seroprevalence for COVID-19 surveillance at a single hematopoietic stem cell transplantation center: a prospective cohort study.

This prospective cohort study aims to analyze the surveillance of COVID-19 at a single hematopoietic stem cell transplantation (HSCT) center in Brazil, in 29 patients undergoing allogeneic HSCT and 57 healthcare workers (nurses and dentists), through viral shedding of SARS-CoV-2 in saliva and plasma and seroprevalence of anti-SARS-CoV-2 IgG. In addition, we report two cases with prolonged persistent detection of SARS-CoV-2 without seroconversion. The sample collection was performed seven times for patients and five times for healthcare workers. Only two patients tested positive for SARS-CoV-2 in their saliva and plasma samples (6.9%) without seroconversion. All healthcare workers were asymptomatic and none tested positive. Two patients (6.9%) and four nurses (8%) had positive serology. No dentists had positive viral detection or positive serology. Our results reflect a low prevalence of positive RT-PCR and seroprevalence of SARS-CoV-2 in patients and healthcare workers at a single HSCT center. Results have also corroborated how the rigorous protocols adopted in transplant centers were even more strengthened in this pandemic scenario.

Quantification of torque teno virus (TTV) DNA in saliva and plasma samples in patients at short time before and after kidney transplantation.

BACKGROUND: Several reports have proposed that the viral load of torque teno virus (TTV) in plasma is a biomarker of immune function in solid organ transplantation (SOT) and in allogeneic hematopoietic stem cell transplantation. Additionally, for the latter one, TTV-DNA quantification in saliva has also been suggested. AIM: to investigate the correlation between the TTV viral load and immune function in paired saliva and plasma samples in patients on kidney transplantation. MATERIALS AND METHODS: TTV-DNA viral load was quantified in paired samples of saliva and plasma from 71 patients before and a short-time after renal-transplantation by real-time PCR. RESULTS: The data obtained from 213 paired samples showed a slight consistency in the comparison between saliva and plasma, with prevalence of TTV-DNA being 58%, 52% and 60% in saliva samples and 60%, 73% and 90% in plasma samples before and at 15-20 and 45-60 days after transplantation, respectively. Additionally, a high TTV viral load was observed in plasma at 15-20 and 45-60 days after transplantation compared to that observed in saliva at the same time. CONCLUSIONS: Overall, monitoring TTV-DNA in saliva samples could be an additional fast non-invasive option to assess the immune functionality in SOT populations.

Micro-Fourier-transform infrared reflectance spectroscopy as tool for probing IgG glycosylation in COVID-19 patients.

It has been reported that patients diagnosed with COVID-19 become critically ill primarily around the time of activation of the adaptive immune response. However the role of antibodies in the worsening of disease is not obvious. Higher titers of anti-spike immunoglobulin IgG1 associated with low fucosylation of the antibody Fc tail have been associated to excessive inflammatory response. In contrast it has been also reported that NP-, S-, RBD- specific IgA, IgG, and IgM are not associated with SARS-CoV-2 viral load, indicating that there is no obvious correlation between antibody response and viral antigen detection. In the present work the micro-Fourier-transform infrared reflectance spectroscopy (micro-FTIR) was employed to investigate blood serum samples of healthy and COVID-19-ill (mild or oligosymptomatic) individuals (82 healthcare workers volunteers in "Instituto de Infectologia Emilio Ribas", São Paulo, Brazil). The molecular-level-sensitive, multiplexing quantitative and qualitative FTIR data probed on 1 µL of dried biofluid was compared to signal-to-cutoff index of chemiluminescent immunoassays CLIA and ELISA (IgG antibodies against SARS-CoV-2). Our main result indicated that 1702-1785 [Formula: see text] spectral window (carbonyl C=O vibration) is a spectral marker of the degree of IgG glycosylation, allowing to probe distinctive sub-populations of COVID-19 patients, depending on their degree of severity. The specificity was 87.5 % while the detection rate of true positive was 100%. The computed area under the receiver operating curve was equivalent to CLIA, ELISA and other ATR-FTIR methods ([Formula: see text]). In summary, overall discrimination of healthy and COVID-19 individuals and severity prediction as well could be potentially implemented using micro-FTIR reflectance spectroscopy on blood serum samples. Considering the minimal and reagent-free sample preparation procedures combined to fast (few minutes) outcome of FTIR we can state that this technology is suitable for fast screening of immune response of individuals with COVID-19. It would be an important tool in prospective studies, helping investigate the physiology of the asymptomatic, oligosymptomatic, or severe individuals and measure the extension of infection dissemination in patients.

Amplification of emerging viruses in a bat colony.

Bats host noteworthy viral pathogens, including coronaviruses, astroviruses, and adenoviruses. Knowledge on the ecology of reservoir-borne viruses is critical for preventive approaches against zoonotic epidemics. We studied a maternity colony of Myotis myotis bats in the attic of a private house in a suburban neighborhood in Rhineland-Palatinate, Germany, during 2008, 2009, and 2010. One coronavirus, 6 astroviruses, and 1 novel adenovirus were identified and monitored quantitatively. Strong and specific amplification of RNA viruses, but not of DNA viruses, occurred during colony formation and after parturition. The breeding success of the colony was significantly better in 2010 than in 2008, in spite of stronger amplification of coronaviruses and astroviruses in 2010, suggesting that these viruses had little pathogenic influence on bats. However, the general correlation of virus and bat population dynamics suggests that bats control infections similar to other mammals and that they may well experience epidemics of viruses under certain circumstances.

SARS-CoV-2 in saliva, viremia and seroprevalence for COVID-19 surveillance at a single hematopoietic stem cell transplantation center: a prospective cohort study

ABSTRACT This prospective cohort study aims to analyze the surveillance of COVID-19 at a single hematopoietic stem cell transplantation (HSCT) center in Brazil, in 29 patients undergoing allogeneic HSCT and 57 healthcare workers (nurses and dentists), through viral shedding of SARS-CoV-2 in saliva and plasma and seroprevalence of anti-SARS-CoV-2 IgG. In addition, we report two cases with prolonged persistent detection of SARS-CoV-2 without seroconversion. The sample collection was performed seven times for patients and five times for healthcare workers. Only two patients tested positive for SARS-CoV-2 in their saliva and plasma samples (6.9%) without seroconversion. All healthcare workers were asymptomatic and none tested positive. Two patients (6.9%) and four nurses (8%) had positive serology. No dentists had positive viral detection or positive serology. Our results reflect a low prevalence of positive RT-PCR and seroprevalence of SARS-CoV-2 in patients and healthcare workers at a single HSCT center. Results have also corroborated how the rigorous protocols adopted in transplant centers were even more strengthened in this pandemic scenario.