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The global urgency to uncover medical countermeasures to combat the COVID-19 pandemic caused by the severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2) has revealed an unmet need for robust tissue culture models that faithfully recapitulate key features of human tissues and disease. Infection of the nose is considered the dominant initial site for SARS-CoV-2 infection and models that replicate this entry portal offer the greatest potential for examining and demonstrating the effectiveness of countermeasures designed to prevent or manage this highly communicable disease. Here, we test an air-liquid-interface (ALI) differentiated human nasal epithelium (HNE) culture system as a model of authentic SARS-CoV-2 infection. Progenitor cells (basal cells) were isolated from nasal turbinate brushings, expanded under conditionally reprogrammed cell (CRC) culture conditions and differentiated at ALI. Differentiated cells were inoculated with different SARS-CoV-2 clinical isolates. Infectious virus release into apical washes was determined by TCID50, while infected cells were visualized by immunofluorescence and confocal microscopy. We demonstrate robust, reproducible SARS-CoV-2 infection of ALI-HNE established from different donors. Viral entry and release occurred from the apical surface, and infection was primarily observed in ciliated cells. In contrast to the ancestral clinical isolate, the Delta variant caused considerable cell damage. Successful establishment of ALI-HNE is donor dependent. ALI-HNE recapitulate key features of human SARS-CoV-2 infection of the nose and can serve as a pre-clinical model without the need for invasive collection of human respiratory tissue samples.
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COVID-19/virologia , Mucosa Nasal/citologia , Mucosa Nasal/virologia , Técnicas de Cultura de Tecidos/métodos , Adolescente , Adulto , Enzima de Conversão de Angiotensina 2/metabolismo , Técnicas de Cultura de Células , Diferenciação Celular , Células Epiteliais/citologia , Células Epiteliais/virologia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Modelos Biológicos , SARS-CoV-2 , Internalização do VírusRESUMO
BACKGROUND: On 31 December 2019, the World Health Organization recognised clusters of pneumonia-like cases due to a novel coronavirus disease (COVID-19). COVID-19 became a pandemic 71 days later. AIM: To report the clinical and epidemiological features, laboratory data and outcomes of the first group of 11 returned travellers with COVID-19 in Australia. METHODS: This is a retrospective, multi-centre case series. All patients with confirmed COVID-19 infection were admitted to tertiary referral hospitals in New South Wales, Queensland, Victoria and South Australia. RESULTS: The median age of the patient cohort was 42 years (interquartile range (IQR), 24-53 years) with six men and five women. Eight (72.7%) patients had returned from Wuhan, one from Shenzhen, one from Japan and one from Europe. Possible human-to-human transmission from close family contacts in gatherings overseas occurred in two cases. Symptoms on admission were fever, cough and sore throat (n = 9, 81.8%). Co-morbidities included hypertension (n = 3, 27.3%) and hypercholesterolaemia (n = 2, 18.2%). No patients developed severe acute respiratory distress nor required intensive care unit admission or mechanical ventilation. After a median hospital stay of 14.5 days (IQR, 6.75-21), all patients were discharged. CONCLUSIONS: This is a historical record of the first COVID-19 cases in Australia during the early biocontainment phase of the national response. These findings were invaluable for establishing early inpatient and outpatient COVID-19 models of care and informing the management of COVID-19 over time as the outbreak evolved. Future research should extend this Australian case series to examine global epidemiological variation of this novel infection.
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COVID-19/epidemiologia , Adulto , Austrália/epidemiologia , COVID-19/terapia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Alta do Paciente , Estudos Retrospectivos , Centros de Atenção Terciária , Adulto JovemRESUMO
BACKGROUND: Robust serological assays are essential for long-term control of the COVID-19 pandemic. Many recently released point-of-care (PoCT) serological assays have been distributed with little premarket validation. METHODS: Performance characteristics for 5 PoCT lateral flow devices approved for use in Australia were compared to a commercial enzyme immunoassay (ELISA) and a recently described novel surrogate virus neutralization test (sVNT). RESULTS: Sensitivities for PoCT ranged from 51.8% (95% confidence interval [CI], 43.1%-60.4%) to 67.9% (95% CI, 59.4%-75.6%), and specificities from 95.6% (95% CI, 89.2%-98.8%) to 100.0% (95% CI, 96.1%-100.0%). ELISA sensitivity for IgA or IgG detection was 67.9% (95% CI, 59.4%-75.6%), increasing to 93.8% (95% CI, 85.0%-98.3%) for samples >14 days post symptom onset. sVNT sensitivity was 60.9% (95% CI, 53.2%-68.4%), rising to 91.2% (95% CI, 81.8%-96.7%) for samples >14 days post symptom onset, with specificity 94.4% (95% CI, 89.2%-97.5%). CONCLUSIONS: Performance characteristics for COVID-19 serological assays were generally lower than those reported by manufacturers. Timing of specimen collection relative to onset of illness or infection is crucial in reporting of performance characteristics for COVID-19 serological assays. The optimal algorithm for implementing serological testing for COVID-19 remains to be determined, particularly in low-prevalence settings.
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Infecções por Coronavirus/sangue , Pneumonia Viral/sangue , Algoritmos , Anticorpos Antivirais/sangue , Austrália/epidemiologia , Betacoronavirus/isolamento & purificação , COVID-19 , Teste para COVID-19 , Técnicas de Laboratório Clínico , Infecções por Coronavirus/diagnóstico , Infecções por Coronavirus/epidemiologia , Ensaio de Imunoadsorção Enzimática , Humanos , Imunoglobulina A/sangue , Imunoglobulina G/sangue , Testes de Neutralização/métodos , Pandemias , Pneumonia Viral/diagnóstico , Pneumonia Viral/epidemiologia , Prevalência , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , SARS-CoV-2 , Testes Sorológicos/métodos , Testes Sorológicos/normasRESUMO
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has resulted in an unprecedented need for diagnostic testing that is critical in controlling the spread of COVID-19. We propose a portable infrared spectrometer with purpose-built transflection accessory for rapid point-of-care detection of COVID-19 markers in saliva. Initially, purified virion particles were characterized with Raman spectroscopy, synchrotron infrared (IR) and AFM-IR. A data set comprising 171 transflection infrared spectra from 29 subjects testing positive for SARS-CoV-2 by RT-qPCR and 28 testing negative, was modeled using Monte Carlo Double Cross Validation with 50 randomized test and model sets. The testing sensitivity was 93 % (27/29) with a specificity of 82 % (23/28) that included positive samples on the limit of detection for RT-qPCR. Herein, we demonstrate a proof-of-concept high throughput infrared COVID-19 test that is rapid, inexpensive, portable and utilizes sample self-collection thus minimizing the risk to healthcare workers and ideally suited to mass screening.
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Teste para COVID-19/métodos , COVID-19/diagnóstico , Saliva/química , Animais , Chlorocebus aethiops , Estudos de Coortes , Análise Discriminante , Humanos , Análise dos Mínimos Quadrados , Método de Monte Carlo , Testes Imediatos , Estudo de Prova de Conceito , SARS-CoV-2 , Sensibilidade e Especificidade , Manejo de Espécimes , Espectrofotometria Infravermelho , Células VeroRESUMO
OBJECTIVES: To describe the first isolation and sequencing of SARS-CoV-2 in Australia and rapid sharing of the isolate. SETTING: SARS-CoV-2 was isolated from a 58-year-old man from Wuhan, China who arrived in Melbourne on 19 January 2020 and was admitted to the Monash Medical Centre, Melbourne from the emergency department on 24 January 2020 with fever, cough, and progressive dyspnoea. MAJOR OUTCOMES: Clinical course and laboratory features of the first reported case of COVID-19 (the illness caused by SARS-CoV-2) in Australia; isolation, whole genome sequencing, imaging, and rapid sharing of virus from the patient. RESULTS: A nasopharyngeal swab and sputum collected when the patient presented to hospital were each positive for SARS-CoV-2 (reverse transcription polymerase chain reaction). Inoculation of Vero/hSLAM cells with material from the nasopharyngeal swab led to the isolation of SARS-CoV-2 virus in culture. Electron microscopy of the supernatant confirmed the presence of virus particles with morphology characteristic of viruses of the family Coronaviridae. Whole genome sequencing of the viral isolate and phylogenetic analysis indicated the isolate exhibited greater than 99.99% sequence identity with other publicly available SARS-CoV-2 genomes. Within 24 hours of isolation, the first Australian SARS-CoV-2 isolate was shared with local and overseas reference laboratories and major North American and European culture collections. CONCLUSIONS: The ability to rapidly identify, propagate, and internationally share our SARS-CoV-2 isolate is an important step in collaborative scientific efforts to deal effectively with this international public health emergency by developing better diagnostic procedures, vaccine candidates, and antiviral agents.
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Betacoronavirus/genética , Infecções por Coronavirus/genética , Disseminação de Informação/métodos , Isolamento de Pacientes/métodos , Pneumonia Viral/genética , Austrália , COVID-19 , Infecções por Coronavirus/diagnóstico , Humanos , Masculino , Pessoa de Meia-Idade , Pandemias , Pneumonia Viral/diagnóstico , SARS-CoV-2 , Sequenciamento Completo do GenomaRESUMO
OBJECTIVES: To design and evaluate 3D-printed nasal swabs for collection of samples for SARS-CoV-2 testing. DESIGN: An iterative design process was employed. Laboratory evaluation included in vitro assessment of mock nasopharyngeal samples spiked with two different concentrations of gamma-irradiated SARS-CoV-2. A prospective clinical study compared SARS-CoV-2 and human cellular material recovery by 3D-printed swabs and standard nasopharyngeal swabs. SETTING, PARTICIPANTS: Royal Melbourne Hospital, May 2020. Participants in the clinical evaluation were 50 hospital staff members attending a COVID-19 screening clinic and two inpatients with laboratory-confirmed COVID-19. INTERVENTION: In the clinical evaluation, a flocked nasopharyngeal swab sample was collected with the Copan ESwab and a mid-nasal sample from the other nostril was collected with the 3D-printed swab. RESULTS: In the laboratory evaluation, qualitative agreement with regard to SARS-CoV-2 detection in mock samples collected with 3D-printed swabs and two standard swabs was complete. In the clinical evaluation, qualitative agreement with regard to RNase P detection (a surrogate measure of adequate collection of human cellular material) in samples collected from 50 hospital staff members with standard and 3D-printed swabs was complete. Qualitative agreement with regard to SARS-CoV-2 detection in three pairs of 3D-printed mid-nasal and standard swab samples from two inpatients with laboratory-confirmed SARS-CoV-2 was also complete. CONCLUSIONS: Using 3D-printed swabs to collect nasal samples for SARS-CoV-2 testing is feasible, acceptable to patients and health carers, and convenient.
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Técnicas de Laboratório Clínico/instrumentação , Infecções por Coronavirus/diagnóstico , Técnicas de Diagnóstico do Sistema Respiratório/instrumentação , Aceitação pelo Paciente de Cuidados de Saúde/estatística & dados numéricos , Pneumonia Viral/diagnóstico , Impressão Tridimensional , Adulto , Betacoronavirus/isolamento & purificação , COVID-19 , Teste para COVID-19 , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Nasofaringe/virologia , Pandemias , SARS-CoV-2RESUMO
The infectious etiology of myocarditis often remains unidentified. We report a case of myocarditis associated with human parechovirus (HPeV) infection in an adult. HPeV is an emerging pathogen that can cause serious illness, including myocarditis, in adults. Testing for HPeV should be considered in differential diagnosis of myocarditis.
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Miocardite/diagnóstico , Parechovirus/genética , Infecções por Picornaviridae/diagnóstico , RNA Viral/genética , Adulto , Diagnóstico Diferencial , Humanos , Masculino , Miocardite/etiologia , Miocardite/fisiopatologia , Miocardite/virologia , Parechovirus/isolamento & purificação , Infecções por Picornaviridae/complicações , Infecções por Picornaviridae/fisiopatologia , Infecções por Picornaviridae/virologiaRESUMO
The Gram-positive bacterium, Staphylococcus aureus (S. aureus), is a major pathogen responsible for a variety of infectious diseases ranging from cellulitis to more serious conditions such as septic arthritis and septicaemia. Timely treatment with appropriate antibiotic therapy is essential to ensure clinical defervescence and to prevent further complications such as infective endocarditis or organ impairment due to septic shock. To date, initial antibiotic choice is empirical, using a "best guess" of likely organism and sensitivity- an approach adopted due to the lack of rapid identification methods for bacteria. Current culture based methods take up to 5 days to identify the causative bacterial pathogen and its antibiotic sensitivity. This paper provides proof of concept for a biosensor, based on interdigitated electrodes, to detect the presence of S. aureus and ascertain its sensitivity to flucloxacillin rapidly (within 2 hours) in a cost effective manner. The proposed method is label-free and uses non-faradic measurements. This is the first study to successfully employ interdigitated electrodes for the rapid detection of antibiotic resistance. The method described has important potential outcomes of faster definitive antibiotic treatment and more rapid clinical response to treatment.
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Técnicas Biossensoriais/instrumentação , Resistência Microbiana a Medicamentos , Dispositivos Lab-On-A-Chip , Staphylococcus aureus Resistente à Meticilina/isolamento & purificação , Aderência Bacteriana , Eletrodos , Interações Hidrofóbicas e Hidrofílicas , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos , Fatores de TempoRESUMO
Serological diagnostic assays are essential tools for determining an individual's protection against viruses like SARS-CoV-2, tracking the spread of the virus in the community, and evaluating population immunity. To assess the diversity and quality of the anti-SARS-CoV-2 antibody response, we have compared the antibody profiles of people with mild, moderate, and severe COVID-19 using a dot blot assay. The test targeted the four major structural proteins of SARS-CoV-2, namely the nucleocapsid (N), spike (S) protein domains S1 and S2, and receptor-binding domain (RBD). Serum samples were collected from 63 participants at various time points for up to 300 days after disease onset. The dot blot assay revealed patient-specific differences in the anti-SARS-CoV-2 antibody profiles. Out of the 63 participants with confirmed SARS-CoV-2 infections and clinical COVID-19, 35/63 participants exhibited diverse and robust responses against the tested antigens, while 14/63 participants displayed either limited responses to a subset of antigens or no detectable antibody response to any of the antigens. Anti-N-specific antibody levels decreased within 300 days after disease onset, whereas anti-S-specific antibodies persisted. The dynamics of the antibody response did not change during the test period, indicating stable antibody profiles. Among the participants, 28/63 patients with restricted anti-S antibody profiles or undetectable anti-S antibody levels in the dot blot assay also exhibited weak neutralization activity, as measured by a surrogate virus neutralization test (sVNT) and a microneutralization test. These results indicate that in some cases, natural infections do not lead to the production of neutralizing antibodies. Furthermore, the study revealed significant serological variability among patients, regardless of the severity of their COVID-19 illness. These differences need to be carefully considered when evaluating the protective antibody status of individuals who have experienced primary SARS-CoV-2 infections.
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Murray Valley encephalitis virus (MVEV) is a mosquito-borne virus that is found across Australia, Papua New Guinea and Irian Jaya. MVEV is endemic to northern Australia and causes occasional outbreaks across south-eastern Australia. 2011 saw a dramatic increase in MVEV activity in endemic regions and the re-emergence of MVEV in south-eastern Australia. This followed significant regional flooding and increased numbers of the main mosquito vector, Culex annulirostris, and was evident from the widespread seroconversion of sentinel chickens, fatalities among horses and several cases in humans, resulting in at least three deaths. The last major outbreak in Australia was in 1974, during which 58 cases were identified and the mortality rate was about 20%. With the potential for a further outbreak of MVEV in the 2011-2012 summer and following autumn, we highlight the importance of this disease, its clinical characteristics and radiological and laboratory features. We present a suspected but unproven case of MVEV infection to illustrate some of the challenges in clinical management. It remains difficult to establish an early diagnosis of MVEV infection, and there is a lack of proven therapeutic options.
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Vírus da Encefalite do Vale de Murray/isolamento & purificação , Encefalite por Arbovirus , Corticosteroides/uso terapêutico , Idoso , Antivirais/uso terapêutico , Encefalite por Arbovirus/diagnóstico , Encefalite por Arbovirus/tratamento farmacológico , Encefalite por Arbovirus/prevenção & controle , Evolução Fatal , Humanos , Imunoglobulinas Intravenosas/uso terapêutico , Fatores Imunológicos/uso terapêutico , Imageamento por Ressonância Magnética , MasculinoRESUMO
BACKGROUND: During 2001-2008, the Victorian Infectious Diseases Reference Laboratory (VIDRL) prepared and provided a measles and rubella proficiency test panel for distribution to the World Health Organization (WHO) measles and rubella network laboratories as part of their annual laboratory accreditation assessment. METHODS: Panel test results were forwarded to VIDRL, and results from 8 consecutive years were analyzed. We assessed the type of assays used and results achieved on the basis of the positive and negative interpretation of submitted results, by year and WHO region, for measles and rubella. RESULTS: Over time, there has been a noticeable increase in laboratory and WHO regional participation. For all panels, the proportion of laboratories in all WHO regions using the WHO-validated Dade Behring assay for measles and rubella-specific IgM antibodies ranged from 35% to 100% and 59% to 100%, respectively. For all regions and years, the proportion of laboratories obtaining a pass score ranged from 87% to 100% for measles and 93% to 100% for rubella. CONCLUSIONS: During 2001-2008, a large proportion of laboratories worldwide achieved and maintained a pass score for both measles and rubella. Measles and rubella proficiency testing is regarded as a major achievement for the WHO measles and rubella laboratory program.
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Saúde Global , Sarampo/diagnóstico , Garantia da Qualidade dos Cuidados de Saúde , Rubéola (Sarampo Alemão)/diagnóstico , Organização Mundial da Saúde/organização & administração , Técnicas de Laboratório Clínico/normas , Controle de Doenças Transmissíveis/métodos , Coleta de Dados , Cooperação Internacional , Sarampo/epidemiologia , Vigilância da População , Avaliação de Programas e Projetos de Saúde , Garantia da Qualidade dos Cuidados de Saúde/organização & administração , Garantia da Qualidade dos Cuidados de Saúde/normas , Rubéola (Sarampo Alemão)/epidemiologia , Inquéritos e Questionários , Fatores de TempoRESUMO
Background: Regular repeat surveillance testing is a strategy to identify asymptomatic individuals with SARS-CoV-2 infections in high-risk work settings to prevent onward community transmission. Saliva sampling is less invasive compared to nasal/oropharyngeal sampling, thus making it suitable for regular testing. In this multi-centre evaluation, we aimed to validate RT-PCR using salivary swab testing of SARS-CoV-2 for large-scale surveillance testing and assess implementation amongst staff working in the hotel quarantine system in Victoria, Australia. Methods: A multi-centre laboratory evaluation study was conducted to systematically validate the in vitro and clinical performance of salivary swab RT-PCR for implementation of SARS-CoV-2 surveillance testing. Analytical sensitivity for multiple RT-PCR platforms was assessed using a dilution series of known SARS-CoV-2 viral loads, and assay specificity was examined using a panel of viral pathogens other than SARS-CoV-2. In addition, we tested capacity for large-scale saliva testing using a four-sample pooling approach, where positive pools were subsequently decoupled and retested. Regular, frequent self-collected saliva swab RT-PCR testing was implemented for staff across fourteen quarantine hotels. Samples were tested at three diagnostic laboratories validated in this study, and results were provided back to staff in real-time. Findings: The agreement of self-collected saliva swabs for RT-PCR was 84.5% (95% CI 68.6 to 93.8) compared to RT-PCR using nasal/oropharyngeal swab samples collected by a healthcare practitioner, when saliva samples were collected within seven days of symptom onset. Between 7th December 2020 and 17th December 2021, almost 500,000 RT-PCR tests were performed on saliva swabs self-collected by 102 staff working in quarantine hotels in Melbourne. Of these, 20 positive saliva swabs were produced by 13 staff (0.004%). The majority of staff that tested positive occurred during periods of community transmission of the SARS-CoV-2 Delta variant. Interpretation: Salivary RT-PCR had an acceptable level of agreement compared to standard nasal/oropharyngeal swab RT-PCR within early symptom onset. The scalability, tolerability and ease of self-collection highlights utility for frequent or repeated testing in high-risk settings, such as quarantine or healthcare environments where regular monitoring of staff is critical for public health, and protection of vulnerable populations. Funding: This work was funded by the Victorian Department of Health.
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BACKGROUND: Three patients who received visceral-organ transplants from a single donor on the same day died of a febrile illness 4 to 6 weeks after transplantation. Culture, polymerase-chain-reaction (PCR) and serologic assays, and oligonucleotide microarray analysis for a wide range of infectious agents were not informative. METHODS: We evaluated RNA obtained from the liver and kidney transplant recipients. Unbiased high-throughput sequencing was used to identify microbial sequences not found by means of other methods. The specificity of sequences for a new candidate pathogen was confirmed by means of culture and by means of PCR, immunohistochemical, and serologic analyses. RESULTS: High-throughput sequencing yielded 103,632 sequences, of which 14 represented an Old World arenavirus. Additional sequence analysis showed that this new arenavirus was related to lymphocytic choriomeningitis viruses. Specific PCR assays based on a unique sequence confirmed the presence of the virus in the kidneys, liver, blood, and cerebrospinal fluid of the recipients. Immunohistochemical analysis revealed arenavirus antigen in the liver and kidney transplants in the recipients. IgM and IgG antiviral antibodies were detected in the serum of the donor. Seroconversion was evident in serum specimens obtained from one recipient at two time points. CONCLUSIONS: Unbiased high-throughput sequencing is a powerful tool for the discovery of pathogens. The use of this method during an outbreak of disease facilitated the identification of a new arenavirus transmitted through solid-organ transplantation.
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Infecções por Arenaviridae/virologia , Arenavirus/classificação , Transplante de Rim/efeitos adversos , Transplante de Fígado/efeitos adversos , Análise de Sequência de DNA/métodos , Adulto , Anticorpos Antivirais/sangue , Infecções por Arenaviridae/transmissão , Arenavirus/genética , Arenavirus/isolamento & purificação , Biologia Computacional , Transmissão de Doença Infecciosa , Feminino , Humanos , Imuno-Histoquímica , Rim/ultraestrutura , Rim/virologia , Pessoa de Meia-Idade , Análise de Sequência com Séries de Oligonucleotídeos , Reação em Cadeia da Polimerase/métodos , RNA Viral/análiseRESUMO
The COVID-19 pandemic has placed unprecedented global demand on laboratory supplies required for testing. Sample pooling has been investigated by laboratories as a strategy to preserve testing capacity. We evaluate the performance of Cepheid Xpert® Xpress SARS-CoV-2 RT-PCR assay for testing samples in pools of 4 and 6. Clinical samples containing SARS-CoV-2, and confirmed negative clinical samples were used to create sample pools. Clinical samples had 'neat' Xpert® E gene cycle threshold values ranging between 20 and 28 and all were detected qualitatively when contained in pools of 4 or 6 samples. For these samples, pooling had a median change in cycle threshold value of 2.0 in pools of 4, and of 2.9 in pools of 6. With the use of Cepheid Xpert® Xpress SARS-CoV-2 RT-PCR assay, pooling of 4 or 6 samples may be an effective strategy to increase testing capacity.
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Teste para COVID-19/métodos , COVID-19/diagnóstico , Técnicas de Laboratório Clínico/métodos , Técnicas de Diagnóstico Molecular/métodos , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Humanos , Nasofaringe/virologia , SARS-CoV-2/genética , Sensibilidade e EspecificidadeRESUMO
BACKGROUND: Serological testing for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) complements nucleic acid tests for patient diagnosis and enables monitoring of population susceptibility to inform the coronavirus disease 2019 (COVID-19) pandemic response. It is important to understand the reliability of assays with different antigen or antibody targets to detect humoral immunity after SARS-CoV-2 infection and to understand how antibody (Ab) binding assays compare to those detecting neutralizing antibody (nAb), particularly as we move into the era of vaccines. METHODS: We evaluated the performance of 6 commercially available enzyme-linked immunosorbent assays (ELISAs), including a surrogate virus neutralization test (sVNT), for detection of SARS-CoV-2 immunoglobulins (IgA, IgM, IgG), total or nAb. A result subset was compared with a cell culture-based microneutralization (MN) assay. We tested sera from patients with prior reverse transcription polymerase chain reaction-confirmed SARS-CoV-2 infection, prepandemic sera, and potential cross-reactive sera from patients with other non-COVID-19 acute infections. RESULTS: For sera collected >14 days post-symptom onset, the assay achieving the highest sensitivity was the Wantai total Ab at 100% (95% CI, 94.6%-100%), followed by 93.1% for Euroimmun NCP-IgG, 93.1% for GenScript sVNT, 90.3% for Euroimmun S1-IgG, 88.9% for Euroimmun S1-IgA, and 83.3% for Wantai IgM. Specificity for the best-performing assay was 99.5% for the Wantai total Ab, and for the lowest-performing assay it was 97.1% for sVNT (as per the Instructions for Use [IFU]). The Wantai Total Ab had the best agreement with MN at 98% followed by Euroimmun S1-IgA, Euro NCP-IgG, and sVNT (as per IFU) with 97%, 97% and 95%, respectively; Wantai IgM had the poorest agreement at 93%. CONCLUSIONS: Performance characteristics of the SARS-CoV-2 serology assays detecting different antibody types are consistent with those found in previously published reports. Evaluation of the surrogate virus neutralization test in comparison to the Ab binding assays and a cell culture-based neutralization assay showed good result correlation between all assays. However, correlation between the cell-based neutralization test and some assays detecting Ab's not specifically involved in neutralization was higher than with the sVNT. This study demonstrates the reliability of different assays to detect the humoral immune response following SARS-CoV-2 infection, which can be used to optimize serological test algorithms for assessing antibody responses post-SARS-CoV-2 infection or vaccination.
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Emerging testing technologies for detection of SARS-CoV-2 include those that are rapid and can be used at point-of-care (POC), and those facilitating high throughput laboratory-based testing. Tests designed to be performed at POC (such as antigen tests and molecular assays) have the potential to expedite isolation of infectious patients and their contacts, but most are less sensitive than standard-of-care reverse transcription polymerase chain reaction (RT-PCR). Data on clinical performance of the majority of emerging assays are limited with most evaluations performed on contrived or stored laboratory samples. Further evaluations of these assays are required, particularly when performed at POC on symptomatic and asymptomatic patients and at various time-points after symptom onset. A few studies have so far shown several of these assays have high specificity. However, large prospective evaluations are needed to confirm specificity, particularly before the assays are implemented in low prevalence settings or asymptomatic populations. High throughput laboratory-based testing includes the use of new sample types (e.g., saliva to increase acceptability) or innovative uses of existing technology (e.g., sample pooling). Information detailing population-wide testing strategies for SARS-COV-2 is largely missing from peer-reviewed literature. Logistics and supply chains are key considerations in any plan to 'scale up' testing in the Australian context. The strategic use of novel assays will help strike the balance between achieving adequate test numbers without overwhelming laboratory capacity. To protect testing of high-risk populations, the aims of testing with respect to the phase of the pandemic must be considered.
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Teste para COVID-19/métodos , COVID-19/diagnóstico , Austrália , Humanos , SARS-CoV-2RESUMO
Saliva has recently been proposed as a suitable specimen for the diagnosis of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Use of saliva as a diagnostic specimen may present opportunities for SARS-CoV-2 reverse transcription polymerase chain reaction (RT-PCR) testing in remote and low-resource settings. Determining the stability of SARS-CoV-2 RNA in saliva over time is an important step in determining optimal storage and transport times. We undertook an in vitro study to assess whether SARS-CoV-2 could be detected in contrived saliva samples. The contrived saliva samples comprised 10 ml pooled saliva spiked with gamma-irradiated SARS-CoV-2 to achieve a concentration of 2.58×104 copies ml SARS-CoV-2, which was subsequently divided into 2 ml aliquots comprising: (i) neat saliva; and a 1â:â1 dilution with (ii) normal saline; (iii) viral transport media, and (iv) liquid Amies medium. Contrived samples were made in quadruplicate, with two samples of each stored at either: (i) room temperature or (ii) 4 °C. SARS-CoV-2 was detected in all SARS-CoV-2 spiked samples at time point 0, day 1, 3 and 7 at both storage temperatures using the N gene RT-PCR assay and time point 0, day 1 and day 7 using the Xpert Xpress SARS-CoV-2 (Cepheid, Sunnyvale, USA) RT-PCR assay. The ability to detect SARS-CoV-2 in saliva over a 1 week period is an important finding that presents further opportunities for saliva testing as a diagnostic specimen for the diagnosis of SARS-CoV-2.
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Teste para COVID-19/métodos , COVID-19/diagnóstico , RNA Viral/análise , SARS-CoV-2/genética , Saliva/virologia , Humanos , Técnicas de Diagnóstico Molecular , Reação em Cadeia da Polimerase Via Transcriptase Reversa , SARS-CoV-2/isolamento & purificação , Manejo de EspécimesRESUMO
We describe severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-specific immune responses in a patient with lymphoma and recent programmed death 1 (PD-1) inhibitor therapy with late onset of severe coronavirus disease 2019 disease and prolonged SARS-CoV-2 replication, in comparison to age-matched and immunocompromised controls. High levels of HLA-DR+/CD38+ activation, interleukin 6, and interleukin 18 in the absence of B cells and PD-1 expression was observed. SARS-CoV-2-specific antibody responses were absent and SARS-CoV-2-specific T cells were minimally detected. This case highlights challenges in managing immunocompromised hosts who may fail to mount effective virus-specific immune responses.
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BACKGROUND: A cornerstone of Australia's ability to control COVID-19 has been effective border control with an extensive supervised quarantine programme. However, a rapid recrudescence of COVID-19 was observed in the state of Victoria in June, 2020. We aim to describe the genomic findings that located the source of this second wave and show the role of genomic epidemiology in the successful elimination of COVID-19 for a second time in Australia. METHODS: In this observational, genomic epidemiological study, we did genomic sequencing of all laboratory-confirmed cases of COVID-19 diagnosed in Victoria, Australia between Jan 25, 2020, and Jan 31, 2021. We did phylogenetic analyses, genomic cluster discovery, and integrated results with epidemiological data (detailed information on demographics, risk factors, and exposure) collected via interview by the Victorian Government Department of Health. Genomic transmission networks were used to group multiple genomic clusters when epidemiological and genomic data suggested they arose from a single importation event and diversified within Victoria. To identify transmission of emergent lineages between Victoria and other states or territories in Australia, all publicly available SARS-CoV-2 sequences uploaded before Feb 11, 2021, were obtained from the national sequence sharing programme AusTrakka, and epidemiological data were obtained from the submitting laboratories. We did phylodynamic analyses to estimate the growth rate, doubling time, and number of days from the first local infection to the collection of the first sequenced genome for the dominant local cluster, and compared our growth estimates to previously published estimates from a similar growth phase of lineage B.1.1.7 (also known as the Alpha variant) in the UK. FINDINGS: Between Jan 25, 2020, and Jan 31, 2021, there were 20 451 laboratory-confirmed cases of COVID-19 in Victoria, Australia, of which 15 431 were submitted for sequencing, and 11 711 met all quality control metrics and were included in our analysis. We identified 595 genomic clusters, with a median of five cases per cluster (IQR 2-11). Overall, samples from 11 503 (98·2%) of 11 711 cases clustered with another sample in Victoria, either within a genomic cluster or transmission network. Genomic analysis revealed that 10 426 cases, including 10 416 (98·4%) of 10 584 locally acquired cases, diagnosed during the second wave (between June and October, 2020) were derived from a single incursion from hotel quarantine, with the outbreak lineage (transmission network G, lineage D.2) rapidly detected in other Australian states and territories. Phylodynamic analyses indicated that the epidemic growth rate of the outbreak lineage in Victoria during the initial growth phase (samples collected between June 4 and July 9, 2020; 47·4 putative transmission events, per branch, per year [1/years; 95% credible interval 26·0-85·0]), was similar to that of other reported variants, such as B.1.1.7 in the UK (mean approximately 71·5 1/years). Strict interventions were implemented, and the outbreak lineage has not been detected in Australia since Oct 29, 2020. Subsequent cases represented independent international or interstate introductions, with limited local spread. INTERPRETATION: Our study highlights how rapid escalation of clonal outbreaks can occur from a single incursion. However, strict quarantine measures and decisive public health responses to emergent cases are effective, even with high epidemic growth rates. Real-time genomic surveillance can alter the way in which public health agencies view and respond to COVID-19 outbreaks. FUNDING: The Victorian Government, the National Health and Medical Research Council Australia, and the Medical Research Future Fund.