Isolation and rapid sharing of the 2019 novel coronavirus (SARS-CoV-2) from the first patient diagnosed with COVID-19 in Australia.

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.

Defective Severe Acute Respiratory Syndrome Coronavirus 2 Immune Responses in an Immunocompromised Individual With Prolonged Viral Replication.

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.

The FDA-approved drug ivermectin inhibits the replication of SARS-CoV-2 in vitro.

Although several clinical trials are now underway to test possible therapies, the worldwide response to the COVID-19 outbreak has been largely limited to monitoring/containment. We report here that Ivermectin, an FDA-approved anti-parasitic previously shown to have broad-spectrum anti-viral activity in vitro, is an inhibitor of the causative virus (SARS-CoV-2), with a single addition to Vero-hSLAM cells 2 h post infection with SARS-CoV-2 able to effect ~5000-fold reduction in viral RNA at 48 h. Ivermectin therefore warrants further investigation for possible benefits in humans.

Validation of a single-step, single-tube reverse transcription loop-mediated isothermal amplification assay for rapid detection of SARS-CoV-2 RNA.

Introduction. The SARS-CoV-2 pandemic of 2020 has resulted in unparalleled requirements for RNA extraction kits and enzymes required for virus detection, leading to global shortages. This has necessitated the exploration of alternative diagnostic options to alleviate supply chain issues.Aim. To establish and validate a reverse transcription loop-mediated isothermal amplification (RT- LAMP) assay for the detection of SARS-CoV-2 from nasopharyngeal swabs.Methodology. We used a commercial RT-LAMP mastermix from OptiGene in combination with a primer set designed to detect the CDC N1 region of the SARS-CoV-2 nucleocapsid (N) gene. A single-tube, single-step fluorescence assay was implemented whereby 1 µl of universal transport medium (UTM) directly from a nasopharyngeal swab could be used as template, bypassing the requirement for RNA purification. Amplification and detection could be conducted in any thermocycler capable of holding 65 °C for 30 min and measure fluorescence in the FAM channel at 1 min intervals.Results. Assay evaluation by assessment of 157 clinical specimens previously screened by E-gene RT-qPCR revealed assay sensitivity and specificity of 87 and 100%, respectively. Results were fast, with an average time-to-positive (Tp) for 93 clinical samples of 14 min (sd±7 min). Using dilutions of SARS-CoV-2 virus spiked into UTM, we also evaluated assay performance against FDA guidelines for implementation of emergency-use diagnostics and established a limit-of-detection of 54 Tissue Culture Infectious Dose 50 per ml (TCID50 ml-1), with satisfactory assay sensitivity and specificity. A comparison of 20 clinical specimens between four laboratories showed excellent interlaboratory concordance; performing equally well on three different, commonly used thermocyclers, pointing to the robustness of the assay.Conclusion. With a simplified workflow, The N1 gene Single Tube Optigene LAMP assay (N1-STOP-LAMP) is a powerful, scalable option for specific and rapid detection of SARS-CoV-2 and an additional resource in the diagnostic armamentarium against COVID-19.

Applicability of oral fluid collected onto filter paper for detection and genetic characterization of measles virus strains.

Expansion of measles molecular surveillance to developing countries where measles is endemic will help facilitate measles control. Limited infrastructure in these areas is a barrier to referral of specimens suitable for measles virus (MV) genotyping. In this study, we demonstrate that oral fluid dried onto filter paper can be used for the detection and characterization of MV strains. Using this approach, an MV-positive sample by reverse transcriptase PCR could be obtained from 67% of serologically confirmed acute measles cases. Mimicking certain environmental conditions and duration of transportation established that MV RNA remained detectable and suitable for nucleic acid sequencing in oral fluid spots for at least 1 week. In the context of a measles outbreak in a remote region of the world where infrastructure is poor, oral fluid samples dried onto filter paper and sent to a specialized laboratory for testing will aid in the identification and characterization of the causative MV strain.

Improving HIV surveillance in Victoria: the role of the "detuned" enzyme immunoassay.

Between 1999 and 2000, new diagnoses of HIV in Victoria (Australia) rose by 41%, from 140 to 197. In this time period, sera from new HIV diagnoses were tested using the Organon Teknika "detuned" enzyme immunoassay (EIA). We compared the results of the detuned EIA with incident infections defined by surveillance (on the basis of a previous negative or indeterminate HIV test and/or a seroconversion illness within the 12 months preceding HIV diagnosis). Of 317 specimens, 97 (31%) incident infections and 114 (36%) recent infections were detected using surveillance and detuned EIA, respectively. The detuned assay misclassified 11 cases with AIDS and 2 cases with CD4 counts < or = 200 micro3 (probable long-standing infections) as recent infections and was unable to identify 31 (32%) of 97 cases previously classified as incident cases by surveillance. The assay detected an extra 35 recent infections that were previously classified as nonincident by surveillance. By combining the detuned assay and surveillance, 132 (42%) incident infections were identified from 317 specimens, 36% more than surveillance alone. We recommend that a detuned assay or similar test become part of the routine strategy to identify incident infections in Victoria. Incident infections provide important information for targeting prevention strategies and the opportunity to interrupt ongoing viral transmission.

Detection of measles virus-specific immunoglobulin M in dried venous blood samples by using a commercial enzyme immunoassay.

The optical densities (ODs) of 216 dried venous blood (DVB) samples submitted to the Victorian Infectious Diseases Reference Laboratory as part of enhanced measles surveillance were compared to the ODs of the corresponding serum samples collected at the same time. DVB samples, stored for up to 24 months at 4 degrees C, were tested by the Dade Behring Enzygnost Anti-Measles-Virus/IgM immunoassay. Elution and testing conditions were optimized with the use of spiked DVB samples. The assay showed an overall sensitivity of 90.2% and a specificity of 98.8% for DVB samples compared to the results for serum. When the results were analyzed according to the length of time that the DVB sample had been stored, the assay was 100% sensitive and 97% specific according to the ODs for those samples stored for less than 6 months compared to the results for the corresponding serum samples, with 97.7% agreement between the results for the two sample types. These results demonstrate the potential for the use of DVB samples for the diagnosis of measles in routine diagnostic laboratories.