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1.
Sci Rep ; 12(1): 14438, 2022 08 24.
Article in English | MEDLINE | ID: covidwho-2000930

ABSTRACT

The lack of therapeutic options to fight Covid-19 has contributed to the current global pandemic. Despite the emergence of effective vaccines, development of broad-spectrum antiviral treatment remains a significant challenge, in which antimicrobial photodynamic therapy (aPDT) may play a role, especially at early stages of infection. aPDT of the nares with methylene blue (MB) and non-thermal light has been successfully utilized to inactivate both bacterial and viral pathogens in the perioperative setting. Here, we investigated the effect of MB-aPDT to inactivate human betacoronavirus OC43 and SARS-CoV-2 in vitro and in a proof-of-principle COVID-19 clinical trial to test, in a variety of settings, the practicality, technical feasibility, and short-term efficacy of the method. aPDT yielded inactivation of up to 6-Logs in vitro, as measured by RT-qPCR and infectivity assay. From a photo-physics perspective, the in vitro results suggest that the response is not dependent on the virus itself, motivating potential use of aPDT for local destruction of SARS-CoV-2 and its variants. In the clinical trial we observed variable effects on viral RNA in nasal-swab samples as assessed by RT-qPCR attributed to aPDT-induced RNA fragmentation causing falsely-elevated counts. However, the viral infectivity in clinical nares swabs was reduced in 90% of samples and undetectable in 70% of samples. This is the first demonstration based on quantitative clinical viral infectivity measurements that MB-aPDT is a safe, easily delivered and effective front-line technique that can reduce local SARS-CoV-2 viral load.


Subject(s)
COVID-19 , Disinfection , Nose , Photochemotherapy , Anti-Infective Agents/adverse effects , Anti-Infective Agents/pharmacology , COVID-19/drug therapy , Disinfection/methods , Feasibility Studies , Humans , Methylene Blue/adverse effects , Methylene Blue/pharmacology , Nose/virology , Pandemics , RNA, Viral/analysis , SARS-CoV-2/drug effects , SARS-CoV-2/genetics , SARS-CoV-2/isolation & purification , Treatment Outcome , Viral Load/drug effects
2.
PLoS One ; 17(2): e0264130, 2022.
Article in English | MEDLINE | ID: covidwho-1910538

ABSTRACT

The global COVID-19 pandemic has highlighted the need for rapid, accurate and accessible nucleic acid tests to enable timely identification of infected individuals. We optimized a sample-to-answer nucleic acid test for SARS-CoV-2 that provides results in <1 hour using inexpensive and readily available reagents. The test workflow includes a simple lysis and viral inactivation protocol followed by direct isothermal amplification of viral RNA using RT-LAMP. The assay was validated using two different instruments, a portable isothermal fluorimeter and a standard thermocycler. Results of the RT-LAMP assay were compared to traditional RT-qPCR for nasopharyngeal swabs, nasal swabs, and saliva collected from a cohort of patients hospitalized due to COVID-19. For all three sample types, positive agreement with RT-LAMP performed using the isothermal fluorimeter was 100% for samples with Ct <30 and 69-91% for samples with Ct <40. Following validation, the test was successfully scaled to test the saliva of up to 400 asymptomatic individuals per day as part of the campus surveillance program at Rice University. Successful development, validation, and scaling of this sample-to-answer, extraction-free real-time RT-LAMP test for SARS-CoV-2 adds a highly adaptable tool to efforts to control the COVID-19 pandemic, and can inform test development strategies for future infectious disease threats.


Subject(s)
COVID-19 Testing , COVID-19/diagnosis , Nasopharynx/virology , Nose/virology , Population Surveillance/methods , SARS-CoV-2/isolation & purification , Saliva/virology , COVID-19/virology , Humans , Molecular Diagnostic Techniques/methods , Nucleic Acid Amplification Techniques/methods , RNA, Viral/genetics , Sensitivity and Specificity
4.
Microbiol Spectr ; 10(1): e0245521, 2022 02 23.
Article in English | MEDLINE | ID: covidwho-1691400

ABSTRACT

Containment measures employed during the COVID-19 pandemic included prompt recognition of cases, isolation, and contact tracing. Bilateral nasal (NA) swabs applied to a commercial antigen-based rapid diagnostic test (Ag-RDT) offer a simpler and more comfortable alternative to nasopharyngeal (NP) collection; however, little is known about the sensitivity of this method in an asymptomatic population. Participants in community-based asymptomatic testing sites were screened for SARS-CoV-2 using an Ag-RDT with NP sampling. Positive individuals returned for confirmatory molecular testing and consented to repeating the Ag-RDT using a bilateral NA swab for comparison. Residual test buffer (RTB) from Ag-RDTs was subjected to real-time reverse transcription-PCR (RT-PCR). Of 123,617 asymptomatic individuals, 197 NP Ag-RDT-positive participants were included, with 175 confirmed positive by RT-PCR. Of these cases, 154 were identified from the NA swab collection with Ag-RDT, with a sensitivity of 88.0% compared to the NP swab collection. Stratifying results by RT-PCR cycle threshold demonstrated that sensitivity of the nasal collection method varied based on the cycle threshold (CT) value of the paired RT-PCR sample. RT-PCR testing on the RTB from the Ag-RDT using NP and NA swab collections resulted in 100.0% and 98.7% sensitivity, respectively. NA swabs provide an adequate alternative to NP swab collection for use with Ag-RDT, with the recognition that the test is most sensitive in specimens with high viral loads. With the high sensitivity of RT-PCR testing on RTB from Ag-RDT, a more streamlined approach to confirmatory testing is possible without recollection or use of paired collections strategies. IMPORTANCE Nasal swabbing for SARS-CoV-2 (COVID-19) comes with many benefits but is slightly less sensitive than traditional nasopharyngeal swabbing; however, confirmatory lab-based testing could be performed directly from the residual buffer from either sample type.


Subject(s)
Antigens, Viral/analysis , COVID-19/virology , Carrier State/virology , Nasopharynx/virology , Nose/virology , SARS-CoV-2/isolation & purification , Specimen Handling/methods , Antigens, Viral/genetics , Antigens, Viral/immunology , Asymptomatic Diseases , COVID-19/diagnosis , COVID-19 Serological Testing , Humans , Reverse Transcriptase Polymerase Chain Reaction , SARS-CoV-2/classification , SARS-CoV-2/genetics , SARS-CoV-2/immunology , Sensitivity and Specificity
5.
Viruses ; 14(2)2022 02 05.
Article in English | MEDLINE | ID: covidwho-1674824

ABSTRACT

SARS-CoV-2 can efficiently infect both children and adults, albeit with morbidity and mortality positively associated with increasing host age and presence of co-morbidities. SARS-CoV-2 continues to adapt to the human population, resulting in several variants of concern (VOC) with novel properties, such as Alpha and Delta. However, factors driving SARS-CoV-2 fitness and evolution in paediatric cohorts remain poorly explored. Here, we provide evidence that both viral and host factors co-operate to shape SARS-CoV-2 genotypic and phenotypic change in primary airway cell cultures derived from children. Through viral whole-genome sequencing, we explored changes in genetic diversity over time of two pre-VOC clinical isolates of SARS-CoV-2 during passage in paediatric well-differentiated primary nasal epithelial cell (WD-PNEC) cultures and in parallel, in unmodified Vero-derived cell lines. We identified a consistent, rich genetic diversity arising in vitro, variants of which could rapidly rise to near fixation within two passages. Within isolates, SARS-CoV-2 evolution was dependent on host cells, with paediatric WD-PNECs showing a reduced diversity compared to Vero (E6) cells. However, mutations were not shared between strains. Furthermore, comparison of both Vero-grown isolates on WD-PNECs disclosed marked growth attenuation mapping to the loss of the polybasic cleavage site (PBCS) in Spike, while the strain with mutations in Nsp12 (T293I), Spike (P812R) and a truncation of Orf7a remained viable in WD-PNECs. Altogether, our work demonstrates that pre-VOC SARS-CoV-2 efficiently infects paediatric respiratory epithelial cells, and its evolution is restrained compared to Vero (E6) cells, similar to the case of adult cells. We highlight the significant genetic plasticity of SARS-CoV-2 while uncovering an influential role for collaboration between viral and host cell factors in shaping viral evolution and ultimately fitness in human respiratory epithelium.


Subject(s)
Evolution, Molecular , Respiratory Mucosa/virology , SARS-CoV-2/genetics , Animals , Cells, Cultured , Child , Chlorocebus aethiops , Genotype , Humans , Mutation , Nose/cytology , Nose/virology , Phenotype , SARS-CoV-2/classification , SARS-CoV-2/growth & development , Vero Cells , Whole Genome Sequencing
6.
Microbiol Spectr ; 10(1): e0202921, 2022 02 23.
Article in English | MEDLINE | ID: covidwho-1673365

ABSTRACT

The objective of our study was to evaluate the sensitivity and specificity of rapid antigen detection tests versus those of reverse transcriptase PCR (RT-PCR) using oral, anterior nasal, and nasopharyngeal swabs. The underlying prospective, diagnostic case-control-type accuracy study included 87 hospitalized and nonhospitalized participants in a positive and a negative sample cohort between 16 March and 14 May 2021 in two hospitals in Vienna. SARS-CoV-2 infection status was confirmed by RT-PCR. Participants self-performed one oral and one anterior nasal swab for the rapid antigen test, immediately followed by two nasopharyngeal swabs for the rapid antigen test and RT-PCR by the investigator. Test results were read after 15 min, and participants completed a questionnaire in the meantime. Test parameters were calculated based on the evaluation of 87 participants. The overall sensitivity of rapid antigen detection tests versus that of RT-PCR with oral, anterior nasal, and nasopharyngeal samples was 18.18% (95% confidence interval [CI] 8.19% to 32.71%), 63.04% (95% CI 47.55% to 76.79%), and 73.33% (95% CI 58.06% to 85.4%), respectively. All sampling methods had a test specificity of 100% regardless of the cycle threshold (CT) value. Rapid antigen detection tests using self-collected anterior nasal swabs proved to be as sensitive as and more tolerable than professionally collected nasopharyngeal swabs for CT values up to 30 determined by RT-PCR. This finding illustrates the reliability of tests obtained by adequate self-collected anterior nasal specimen. Sensitivity was dependent upon the CT value for each sampling method. While the main advantage of rapid antigen detection tests is the immediate availability of results, PCR should be preferred in crucial settings wherever possible. IMPORTANCE Rapid antigen detection devices for SARS-CoV-2 represent a valuable tool for monitoring the spread of infection. However, the reliability of the tests depends largely on the test performance and the respective sampling method. Nasopharyngeal swabs mark the gold standard for sample collection in suspected respiratory tract infections but are unsuitable for widespread application, as they must be performed by medically trained personnel. With the underlying study, the head-to-head test performance and the usability of self-collected samples for SARS-CoV-2 detection using rapid antigen detection devices were evaluated. The results confirm similar sensitivity of self-collected anterior nasal swabs to that of professionally collected nasopharyngeal swabs for patients with a CT of < 30 determined by RT-PCR.


Subject(s)
COVID-19 Serological Testing/methods , COVID-19/diagnosis , Mouth/virology , Nasopharynx/virology , Nose/virology , SARS-CoV-2/isolation & purification , Adult , Aged , Aged, 80 and over , Antigens, Viral/analysis , COVID-19/virology , COVID-19 Nucleic Acid Testing , Female , Humans , Male , Middle Aged , Polymerase Chain Reaction , Prospective Studies , SARS-CoV-2/genetics , SARS-CoV-2/immunology , Sensitivity and Specificity , Young Adult
7.
Microbiol Spectr ; 9(3): e0016421, 2021 12 22.
Article in English | MEDLINE | ID: covidwho-1599285

ABSTRACT

Respiratory tract infections (RTIs) are ubiquitous among children in the community. A prospective observational study was performed to evaluate the diagnostic performance and quality of at-home parent-collected (PC) nasal and saliva swab samples, compared to nurse-collected (NC) swab samples, from children with RTI symptoms. Children with RTI symptoms were swabbed at home on the same day by a parent and a nurse. We compared the performance of PC swab samples as the test with NC swab samples as the reference for the detection of respiratory pathogen gene targets by reverse transcriptase PCR, with quality assessment using a human gene. PC and NC paired nasal and saliva swab samples were collected from 91 and 92 children, respectively. Performance and interrater agreement (Cohen's κ) of PC versus NC nasal swab samples for viruses combined showed sensitivity of 91.6% (95% confidence interval [CI], 85.47 to 95.73%) and κ of 0.84 (95% CI, 0.79 to 0.88), respectively; the respective values for bacteria combined were 91.4% (95% CI, 86.85 to 94.87%) and κ of 0.85 (95% CI, 0.80 to 0.89). In saliva samples, viral and bacterial sensitivities were lower at 69.0% (95% CI, 57.47 to 79.76%) and 78.1% (95% CI, 71.60 to 83.76%), as were κ values at 0.64 (95% CI, 0.53 to 0.72) and 0.70 (95% CI, 0.65 to 0.76), respectively. Quality assessment for human biological material (18S rRNA) indicated perfect interrater agreement. At-home PC nasal swab samples performed comparably to NC swab samples, whereas PC saliva swab samples lacked sensitivity for the detection of respiratory microbes. IMPORTANCE RTIs are ubiquitous among children. Diagnosis involves a swab sample being taken by a health professional, which places a considerable burden on community health care systems, given the number of cases involved. The coronavirus disease 2019 (COVID-19) pandemic has seen an increase in the at-home self-collection of upper respiratory tract swab samples without the involvement of health professionals. It is advised that parents conduct or supervise swabbing of children. Surprisingly, few studies have addressed the quality of PC swab samples for subsequent identification of respiratory pathogens. We compared NC and PC nasal and saliva swab samples taken from the same child with RTI symptoms, for detection of respiratory pathogens. The PC nasal swab samples performed comparably to NC samples, whereas saliva swab samples lacked sensitivity for the detection of respiratory microbes. Collection of swab samples by parents would greatly reduce the burden on community nurses without reducing the effectiveness of diagnoses.


Subject(s)
Respiratory Tract Infections/diagnosis , Specimen Handling/methods , Adult , Bacteria/genetics , Bacteria/isolation & purification , Child, Preschool , Female , Health Personnel , Humans , Infant , Male , Middle Aged , Nose/microbiology , Nose/virology , Parents , Prospective Studies , Respiratory Tract Infections/microbiology , Respiratory Tract Infections/virology , Saliva , Specimen Handling/standards , Viruses/genetics , Viruses/isolation & purification , Young Adult
8.
Nat Immunol ; 23(1): 23-32, 2022 01.
Article in English | MEDLINE | ID: covidwho-1585822

ABSTRACT

Systemic immune cell dynamics during coronavirus disease 2019 (COVID-19) are extensively documented, but these are less well studied in the (upper) respiratory tract, where severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) replicates1-6. Here, we characterized nasal and systemic immune cells in individuals with COVID-19 who were hospitalized or convalescent and compared the immune cells to those seen in healthy donors. We observed increased nasal granulocytes, monocytes, CD11c+ natural killer (NK) cells and CD4+ T effector cells during acute COVID-19. The mucosal proinflammatory populations positively associated with peripheral blood human leukocyte antigen (HLA)-DRlow monocytes, CD38+PD1+CD4+ T effector (Teff) cells and plasmablasts. However, there was no general lymphopenia in nasal mucosa, unlike in peripheral blood. Moreover, nasal neutrophils negatively associated with oxygen saturation levels in blood. Following convalescence, nasal immune cells mostly normalized, except for CD127+ granulocytes and CD38+CD8+ tissue-resident memory T cells (TRM). SARS-CoV-2-specific CD8+ T cells persisted at least 2 months after viral clearance in the nasal mucosa, indicating that COVID-19 has both transient and long-term effects on upper respiratory tract immune responses.


Subject(s)
CD4-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/immunology , Nasopharynx/immunology , Nose/cytology , Respiratory Mucosa/immunology , SARS-CoV-2/immunology , Antibodies, Viral/blood , COVID-19/immunology , COVID-19/pathology , Granulocytes/immunology , HLA-DR Antigens/metabolism , Humans , Killer Cells, Natural/immunology , Monocytes/immunology , Nasopharynx/cytology , Nasopharynx/virology , Neutrophils/immunology , Nose/immunology , Nose/virology , Prospective Studies , Respiratory Mucosa/cytology , Respiratory Mucosa/virology
10.
Viruses ; 13(11)2021 11 11.
Article in English | MEDLINE | ID: covidwho-1512701

ABSTRACT

Small animal models are of crucial importance for assessing COVID-19 countermeasures. Common laboratory mice would be well-suited for this purpose but are not susceptible to infection with wild-type SARS-CoV-2. However, the development of mouse-adapted virus strains has revealed key mutations in the SARS-CoV-2 spike protein that increase infectivity, and interestingly, many of these mutations are also present in naturally occurring SARS-CoV-2 variants of concern. This suggests that these variants might have the ability to infect common laboratory mice. Herein we show that the SARS-CoV-2 beta variant attains infectibility to BALB/c mice and causes pulmonary changes within 2-3 days post infection, consistent with results seen in other murine models of COVID-19, at a reasonable virus dose (2 × 105 PFU). The findings suggest that common laboratory mice can serve as the animal model of choice for testing the effectiveness of antiviral drugs and vaccines against SARS-CoV-2.


Subject(s)
COVID-19/virology , Disease Models, Animal , SARS-CoV-2 , Animals , Brain/virology , COVID-19/pathology , Female , Inflammation , Lung/pathology , Lung/virology , Male , Mice, Inbred BALB C , Nose/virology , Pulmonary Alveoli/pathology , SARS-CoV-2/isolation & purification
11.
APMIS ; 130(2): 95-100, 2022 Feb.
Article in English | MEDLINE | ID: covidwho-1511282

ABSTRACT

The aim was to determine the accuracy of anterior nasal swab in rapid antigen (Ag) tests in a low SARS-CoV-2 prevalence and massive screened community. Individuals, aged 18 years or older, who self-booked an appointment for real-time reverse transcriptase-polymerase chain reaction (RT-PCR) test in March 2021 at a public test center in Copenhagen, Denmark were included. An oropharyngeal swab was collected for RT-PCR testing, followed by a swab from the anterior parts of the nose examined by Ag test (SD Biosensor). Accuracy of the Ag test was calculated with RT-PCR as reference. We included 7074 paired conclusive tests (n = 3461, female: 50.7%). The median age was 48 years (IQR: 36-57 years). The prevalence was 0.9%, that is, 66 tests were positive on RT-PCR. Thirty-two had a paired positive Ag test. The sensitivity was 48.5% and the specificity was 100%. This study conducted in a low prevalence setting in a massive screening set-up showed that the Ag test had a sensitivity of 48.5% and a specificity of 100%, that is, no false positive tests. The lower sensitivity is a challenge especially if Ag testing is not repeated frequently allowing this scalable test to be a robust supplement to RT-PCR testing in an ambitious public SARS-CoV-2 screening.


Subject(s)
COVID-19 Serological Testing/methods , COVID-19/diagnosis , Nose/virology , Reverse Transcriptase Polymerase Chain Reaction/methods , SARS-CoV-2/isolation & purification , Adult , Antigens, Viral/analysis , Antigens, Viral/immunology , COVID-19/virology , Female , Humans , Male , Middle Aged , Reverse Transcriptase Polymerase Chain Reaction/standards , SARS-CoV-2/genetics , SARS-CoV-2/immunology , Sensitivity and Specificity
12.
Science ; 374(6573): 1343-1353, 2021 Dec 10.
Article in English | MEDLINE | ID: covidwho-1483979

ABSTRACT

Neutralizing antibody responses gradually wane against several variants of concern (VOCs) after vaccination with the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccine messenger RNA-1273 (mRNA-1273). We evaluated the immune responses in nonhuman primates that received a primary vaccination series of mRNA-1273 and were boosted about 6 months later with either homologous mRNA-1273 or heterologous mRNA-1273.ß, which encompasses the spike sequence of the B.1.351 Beta variant. After boost, animals had increased neutralizing antibody responses across all VOCs, which was sustained for at least 8 weeks after boost. Nine weeks after boost, animals were challenged with the SARS-CoV-2 Beta variant. Viral replication was low to undetectable in bronchoalveolar lavage and significantly reduced in nasal swabs in all boosted animals, suggesting that booster vaccinations may be required to sustain immunity and protection.


Subject(s)
/immunology , COVID-19 Vaccines/immunology , COVID-19/prevention & control , Immunogenicity, Vaccine , SARS-CoV-2/immunology , /administration & dosage , Animals , Antibodies, Neutralizing/blood , Antibodies, Viral/analysis , Antibodies, Viral/blood , Bronchoalveolar Lavage Fluid/immunology , Bronchoalveolar Lavage Fluid/virology , COVID-19/immunology , COVID-19/virology , COVID-19 Vaccines/administration & dosage , Immunity, Mucosal , Immunization, Secondary , Macaca mulatta , Nose/immunology , Nose/virology , RNA, Viral/analysis , SARS-CoV-2/genetics , SARS-CoV-2/isolation & purification , SARS-CoV-2/physiology , T Follicular Helper Cells/immunology , Th1 Cells/immunology , Virus Replication
13.
Nat Commun ; 12(1): 5877, 2021 10 07.
Article in English | MEDLINE | ID: covidwho-1462003

ABSTRACT

Several COVID-19 vaccines have recently gained authorization for emergency use. Limited knowledge on duration of immunity and efficacy of these vaccines is currently available. Data on other coronaviruses after natural infection suggest that immunity to SARS-CoV-2 might be short-lived, and preliminary evidence indicates waning antibody titers following SARS-CoV-2 infection. In this work, we model the relationship between immunogenicity and protective efficacy of a series of Ad26 vectors encoding stabilized variants of the SARS-CoV-2 Spike protein in rhesus macaques and validate the analyses by challenging macaques 6 months after immunization with the Ad26.COV2.S vaccine candidate that has been selected for clinical development. We show that Ad26.COV2.S confers durable protection against replication of SARS-CoV-2 in the lungs that is predicted by the levels of Spike-binding and neutralizing antibodies, indicating that Ad26.COV2.S could confer durable protection in humans and immunological correlates of protection may enable the prediction of durability of protection.


Subject(s)
Antibodies, Neutralizing/immunology , Antibodies, Viral/immunology , COVID-19 Vaccines/immunology , COVID-19/immunology , COVID-19/virology , SARS-CoV-2/immunology , Spike Glycoprotein, Coronavirus/immunology , Vaccination , Animals , Female , HEK293 Cells , Humans , Immunity, Humoral , Logistic Models , Lung/immunology , Lung/pathology , Lung/virology , Macaca mulatta , Male , Nose/immunology , Nose/virology , SARS-CoV-2/physiology , Virus Replication/physiology
15.
Front Immunol ; 12: 729837, 2021.
Article in English | MEDLINE | ID: covidwho-1450810

ABSTRACT

We have developed a dual-antigen COVID-19 vaccine incorporating genes for a modified SARS-CoV-2 spike protein (S-Fusion) and the viral nucleocapsid (N) protein with an Enhanced T-cell Stimulation Domain (N-ETSD) to increase the potential for MHC class II responses. The vaccine antigens are delivered by a human adenovirus serotype 5 platform, hAd5 [E1-, E2b-, E3-], previously demonstrated to be effective in the presence of Ad immunity. Vaccination of rhesus macaques with the hAd5 S-Fusion + N-ETSD vaccine by subcutaneous prime injection followed by two oral boosts elicited neutralizing anti-S IgG and T helper cell 1-biased T-cell responses to both S and N that protected the upper and lower respiratory tracts from high titer (1 x 106 TCID50) SARS-CoV-2 challenge. Notably, viral replication was inhibited within 24 hours of challenge in both lung and nasal passages, becoming undetectable within 7 days post-challenge.


Subject(s)
COVID-19 Vaccines/immunology , COVID-19/prevention & control , Coronavirus Nucleocapsid Proteins/immunology , SARS-CoV-2/immunology , Spike Glycoprotein, Coronavirus/immunology , Adenoviruses, Human/genetics , Adenoviruses, Human/immunology , Adenoviruses, Human/metabolism , Administration, Oral , Animals , Antibodies, Neutralizing/blood , Antibodies, Viral/blood , COVID-19 Vaccines/administration & dosage , Cytokines/blood , Immunization, Secondary/methods , Immunoglobulin G/blood , Lung/virology , Macaca mulatta , Nose/virology , Phosphoproteins/immunology , Protein Domains/immunology , T-Lymphocytes, Helper-Inducer/immunology , Vaccination , Virus Replication/immunology
16.
Int J Mol Sci ; 22(19)2021 Sep 30.
Article in English | MEDLINE | ID: covidwho-1444232

ABSTRACT

Natural or experimental infection of domestic cats and virus transmission from humans to captive predatory cats suggest that felids are highly susceptible to SARS-CoV-2 infection. However, it is unclear which cells and compartments of the respiratory tract are infected. To address this question, primary cell cultures derived from the nose, trachea, and lungs of cat and lion were inoculated with SARS-CoV-2. Strong viral replication was observed for nasal mucosa explants and tracheal air-liquid interface cultures, whereas replication in lung slices was less efficient. Infection was mainly restricted to epithelial cells and did not cause major pathological changes. Detection of high ACE2 levels in the nose and trachea but not lung further suggests that susceptibility of feline tissues to SARS-CoV-2 correlates with ACE2 expression. Collectively, this study demonstrates that SARS-CoV-2 can efficiently replicate in the feline upper respiratory tract ex vivo and thus highlights the risk of SARS-CoV-2 spillover from humans to felids.


Subject(s)
COVID-19/veterinary , Cats/virology , Lions/virology , Angiotensin-Converting Enzyme 2/analysis , Animals , COVID-19/transmission , COVID-19/virology , Cat Diseases/transmission , Cat Diseases/virology , Cells, Cultured , Disease Susceptibility , Humans , Lung/cytology , Lung/virology , Nose/cytology , Nose/virology , SARS-CoV-2/isolation & purification , Trachea/cytology , Trachea/virology
17.
PLoS One ; 16(9): e0257350, 2021.
Article in English | MEDLINE | ID: covidwho-1435609

ABSTRACT

SARS-CoV-2 has spread worldwide and has become a global health problem. As a result, the demand for inputs for diagnostic tests rose dramatically, as did the cost. Countries with inadequate infrastructure experience difficulties in expanding their qPCR testing capacity. Therefore, the development of sensitive and specific alternative methods is essential. This study aimed to develop, standardize, optimize, and validate conventional RT-PCR targeting the N gene of SARS-CoV-2 in naso-oropharyngeal swab samples compared to qPCR. Using bioinformatics tools, specific primers were determined, with a product expected to be 519 bp. The reaction conditions were optimized using a commercial positive control, and the detection limit was determined to be 100 fragments. To validate conventional RT-PCR, we determined a representative sampling of 346 samples from patients with suspected infection whose diagnosis was made in parallel with qPCR. A sensitivity of 92.1% and specificity of 100% were verified, with an accuracy of 95.66% and correlation coefficient of 0.913. Under current Brazilian conditions, this method generates approximately 60% savings compared to qPCR costs. Conventional RT-PCR, validated herein, showed sufficient results for the detection of SARS-CoV-2 and can be used as an alternative for epidemiological studies and interspecies correlations.


Subject(s)
COVID-19 Nucleic Acid Testing/methods , COVID-19/diagnosis , Nose/virology , Nucleocapsid Proteins/genetics , Oropharynx/virology , Real-Time Polymerase Chain Reaction/methods , SARS-CoV-2/genetics , Adolescent , Brazil , COVID-19/virology , DNA Primers/genetics , Female , Humans , Male , Molecular Diagnostic Techniques/methods , RNA, Viral/genetics , Reference Standards , Sensitivity and Specificity , Specimen Handling/methods
18.
J Med Microbiol ; 70(9)2021 Sep.
Article in English | MEDLINE | ID: covidwho-1393561

ABSTRACT

Introduction. Non-invasive sample collection and viral sterilizing buffers have independently enabled workflows for more widespread COVID-19 testing by reverse-transcriptase polymerase chain reaction (RT-PCR).Gap statement. The combined use of sterilizing buffers across non-invasive sample types to optimize sensitive, accessible, and biosafe sampling methods has not been directly and systematically compared.Aim. We aimed to evaluate diagnostic yield across different non-invasive samples with standard viral transport media (VTM) versus a sterilizing buffer eNAT- (Copan diagnostics Murrieta, CA) in a point-of-care diagnostic assay system.Methods. We prospectively collected 84 sets of nasal swabs, oral swabs, and saliva, from 52 COVID-19 RT-PCR-confirmed patients, and nasopharyngeal (NP) swabs from 37 patients. Nasal swabs, oral swabs, and saliva were placed in either VTM or eNAT, prior to testing with the Xpert Xpress SARS-CoV-2 (Xpert). The sensitivity of each sampling strategy was compared using a composite positive standard.Results. Swab specimens collected in eNAT showed an overall superior sensitivity compared to swabs in VTM (70 % vs 57 %, P=0.0022). Direct saliva 90.5 %, (95 % CI: 82 %, 95 %), followed by NP swabs in VTM and saliva in eNAT, was significantly more sensitive than nasal swabs in VTM (50 %, P<0.001) or eNAT (67.8 %, P=0.0012) and oral swabs in VTM (50 %, P<0.0001) or eNAT (58 %, P<0.0001). Saliva and use of eNAT buffer each increased detection of SARS-CoV-2 with the Xpert; however, no single sample matrix identified all positive cases.Conclusion. Saliva and eNAT sterilizing buffer can enhance safe and sensitive detection of COVID-19 using point-of-care GeneXpert instruments.


Subject(s)
COVID-19 Nucleic Acid Testing , Specimen Handling/methods , Adult , Aged , COVID-19/diagnosis , Containment of Biohazards , Culture Media , Female , Humans , Male , Middle Aged , Mouth/virology , Nasopharynx/virology , Nose/virology , Point-of-Care Testing , SARS-CoV-2/genetics , SARS-CoV-2/isolation & purification , Saliva/virology , Sensitivity and Specificity , Specimen Handling/standards
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