Ultrarapid detection of SARS-CoV-2 RNA using a reverse transcription-free exponential amplification reaction, RTF-EXPAR.

A rapid isothermal method for detecting severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the virus responsible for COVID-19, is reported. The procedure uses an unprecedented reverse transcription-free (RTF) approach for converting genomic RNA into DNA. This involves the formation of an RNA/DNA heteroduplex whose selective cleavage generates a short DNA trigger strand, which is then rapidly amplified using the exponential amplification reaction (EXPAR). Deploying the RNA-to-DNA conversion and amplification stages of the RTF-EXPAR assay in a single step results in the detection, via a fluorescence read-out, of single figure copy numbers per microliter of SARS-CoV-2 RNA in under 10 min. In direct three-way comparison studies, the assay has been found to be faster than both RT-qPCR and reverse transcription loop-mediated isothermal amplification (RT-LAMP), while being just as sensitive. The assay protocol involves the use of standard laboratory equipment and is readily adaptable for the detection of other RNA-based pathogens.

Spray Pattern and Plume Geometry Testing and Methodology: An IPAC-RS Working Group Overview.

Plume characterization for orally inhaled and nasal drug products (OINDP) provides valuable information during OINDP development. Spray pattern and plume geometry techniques, methods, and technology have evolved over the past 20 years since the publication of the original 1998 FDA MDI DPI draft guidance. The International Pharmaceutical Aerosol Consortium on Regulation and Science (IPAC-RS) discusses the historical context and background to plume geometry and spray pattern characterization studies; provides an analysis of the current regulatory context; addresses results from its industry surveys on application and value of such testing; and presents case studies and best practices-seeking to provide insights to regulatory bodies and other stakeholders. Assessment and consideration of published studies and industry experience note the value of plume geometry and spray pattern in development, and that further data is needed regarding their use in assessing formulation characteristics. Continued dialogue between industry and regulatory bodies is needed to establish the optimum use of these techniques.

Cell-based Culture Informs Infectivity and Safe De-Isolation Assessments in Patients with Coronavirus Disease 2019.

BACKGROUND: The detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA by reverse-transcription polymerase chain reaction (PCR) does not necessarily indicate shedding of infective virions. There are limited data on the correlation between the isolation of SARS-CoV-2, which likely indicates infectivity, and PCR. METHODS: A total of 195 patients with Coronavirus disease 2019 were tested (outpatients, n = 178; inpatients, n = 12; and critically unwell patients admitted to the intensive care unit [ICU] patients, n = 5). SARS-CoV-2 PCR-positive samples were cultured in Vero C1008 cells and inspected daily for cytopathic effect (CPE). SARS-CoV-2-induced CPE was confirmed by PCR of culture supernatant. Where no CPE was observed, PCR was performed on day 4 to confirm absence of virus replication. The cycle thresholds (Cts) of the day 4 PCR (Ctculture) and the PCR of the original clinical sample (Ctsample) were compared, and positive cultures were defined where Ctsample - Ctculture was ≥3. RESULTS: Of 234 samples collected, 228 (97%) were from the upper respiratory tract. SARS-CoV-2 was isolated from 56 (24%), including in 28 of 181 (15%), 19 of 42 (45%), and 9 of 11 samples (82%) collected from outpatients, inpatients, and ICU patients, respectively. All 56 samples had Ctsample ≤32; CPE was observed in 46 (20%). The mean duration from symptom onset to culture positivity was 4.5 days (range, 0-18). SARS-CoV-2 was significantly more likely to be isolated from samples collected from inpatients (P < .001) and ICU patients (P < .0001) compared with outpatients, and in samples with lower Ctsample. CONCLUSIONS: SARS-CoV-2 culture may be used as a surrogate marker for infectivity and inform de-isolation protocols.

COVID-19 in Australia: our national response to the first cases of SARS-CoV-2 infection during the early biocontainment phase.

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.

Molecular diagnosis of the myeloproliferative neoplasms: UK guidelines for the detection of JAK2 V617F and other relevant mutations.

Molecular genetic assays for the detection of the JAK2 V617F (c.1849G>T) and other pathogenetic mutations within JAK2 exon 12 and MPL exon 10 are part of the routine diagnostic workup for patients presenting with erythrocytosis, thrombocytosis or otherwise suspected to have a myeloproliferative neoplasm. A wide choice of techniques are available for the detection of these mutations, leading to potential difficulties for clinical laboratories in deciding upon the most appropriate assay, which can lead to problems with inter-laboratory standardization. Here, we discuss the most important issues for a clinical diagnostic laboratory in choosing a technique, particularly for detection of the JAK2 V617F mutation at diagnosis. The JAK2 V617F detection assay should be both specific and sensitive enough to detect a mutant allele burden as low as 1-3%. Indeed, the use of sensitive assays increases the detection rate of the JAK2 V617F mutation within myeloproliferative neoplasms. Given their diagnostic relevance, it is also beneficial and relatively straightforward to screen JAK2 V617F negative patients for JAK2 exon 12 mutations (in the case of erythrocytosis) or MPL exon 10 mutations (thrombocytosis or myelofibrosis) using appropriate assays. Molecular results should be considered in the context of clinical findings and other haematological or laboratory results.

An Amplicon-Based Approach for the Whole-Genome Sequencing of Human Metapneumovirus.

Human metapneumovirus (HMPV) is an important cause of upper and lower respiratory tract disease in individuals of all ages. It is estimated that most individuals will be infected by HMPV by the age of five years old. Despite this burden of disease, there remain caveats in our knowledge of global genetic diversity due to a lack of HMPV sequencing, particularly at the whole-genome scale. The purpose of this study was to create a simple and robust approach for HMPV whole-genome sequencing to be used for genomic epidemiological studies. To design our assay, all available HMPV full-length genome sequences were downloaded from the National Center for Biotechnology Information (NCBI) GenBank database and used to design four primer sets to amplify long, overlapping amplicons spanning the viral genome and, importantly, specific to all known HMPV subtypes. These amplicons were then pooled and sequenced on an Illumina iSeq 100 (Illumina, San Diego, CA, USA); however, the approach is suitable to other common sequencing platforms. We demonstrate the utility of this method using a representative subset of clinical samples and examine these sequences using a phylogenetic approach. Here we present an amplicon-based method for the whole-genome sequencing of HMPV from clinical extracts that can be used to better inform genomic studies of HMPV epidemiology and evolution.

The spatial-temporal dynamics of respiratory syncytial virus infections across the east-west coasts of Australia during 2016-17.

Respiratory syncytial virus (RSV) is an important human respiratory pathogen. In temperate regions, a distinct seasonality is observed, where peaks of infections typically occur in early winter, often preceding the annual influenza season. Infections are associated with high rates of morbidity and mortality and in some populations exceed that of influenza. Two subtypes, RSV-A and RSV-B, have been described, and molecular epidemiological studies have shown that both viruses mostly co-circulate. This trend also appears to be the case for Australia; however, previous genomic studies have been limited to cases from one Eastern state-New South Wales. As such, the broader spatial patterns and viral traffic networks across the continent are not known. Here, we conducted a whole-genome study of RSV comparing strains across eastern and Western Australia during the period January 2016 to June 2017. In total, 96 new RSV genomes were sequenced, compiled with previously generated data, and examined using a phylodynamic approach. This analysis revealed that both RSV-A and RSV-B strains were circulating, and each subtype was dominated by a single genotype, RSV-A ON1-like and RSV-B BA10-like viruses. Some geographical clustering was evident in strains from both states with multiple distinct sub-lineages observed and relatively low mixing across jurisdictions, suggesting that endemic transmission was likely seeded from imported, unsampled locations. Overall, the RSV phylogenies reflected a complex pattern of interactions across multiple epidemiological scales from fluid virus traffic across global and regional networks to fine-scale local transmission events.

Evaluation of a worldwide EQA scheme for complex clonality analysis of clinical lymphoproliferative cases demonstrates a learning effect.

Clonality analysis of immunoglobulin (IG) or T-cell receptor (TR) gene rearrangements is routine practice to assist diagnosis of lymphoid malignancies. Participation in external quality assessment (EQA) aids laboratories in identifying systematic shortcomings. The aim of this study was to evaluate laboratories' improvement in IG/TR analysis and interpretation during five EQA rounds between 2014 and 2018. Each year, participants received a total of five cases for IG and five cases for TR testing. Paper-based cases were included for analysis of the final molecular conclusion that should be interpreted based on the integration of the individual PCR results. Wet cases were distributed for analysis of their routine protocol as well as evaluation of the final molecular conclusion. In total, 94.9% (506/533) of wet tests and 97.9% (829/847) of paper tests were correctly analyzed for IG, and 96.8% (507/524) wet tests and 93.2% (765/821) paper tests were correctly analyzed for TR. Analysis scores significantly improved when laboratories participated to more EQA rounds (p=0.001). Overall performance was significantly lower (p=0.008) for non-EuroClonality laboratories (95% for IG and 93% for TR) compared to EuroClonality laboratories (99% for IG and 97% for TR). The difference was not related to the EQA scheme year, anatomic origin of the sample, or final clinical diagnosis. This evaluation showed that repeated EQA participation helps to reduce performance differences between laboratories (EuroClonality versus non-EuroClonality) and between sample types (paper versus wet). The difficulties in interpreting oligoclonal cases highlighted the need for continued education by meetings and EQA schemes.

Ongoing evolution of Chlamydia trachomatis lymphogranuloma venereum: exploring the genomic diversity of circulating strains.

Lymphogranuloma venereum (LGV), the invasive infection of the sexually transmissible infection (STI) Chlamydia trachomatis, is caused by strains from the LGV biovar, most commonly represented by ompA-genotypes L2b and L2. We investigated the diversity in LGV samples across an international collection over seven years using typing and genome sequencing. LGV-positive samples (n=321) from eight countries collected between 2011 and 2017 (Spain n=97, Netherlands n=67, Switzerland n=64, Australia n=53, Sweden n=37, Hungary n=31, Czechia n=30, Slovenia n=10) were genotyped for pmpH and ompA variants. All were found to contain the 9 bp insertion in the pmpH gene, previously associated with ompA-genotype L2b. However, analysis of the ompA gene shows ompA-genotype L2b (n=83), ompA-genotype L2 (n=180) and several variants of these (n=52; 12 variant types), as well as other/mixed ompA-genotypes (n=6). To elucidate the genomic diversity, whole genome sequencing (WGS) was performed from selected samples using SureSelect target enrichment, resulting in 42 genomes, covering a diversity of ompA-genotypes and representing most of the countries sampled. A phylogeny of these data clearly shows that these ompA-genotypes derive from an ompA-genotype L2b ancestor, carrying up to eight SNPs per isolate. SNPs within ompA are overrepresented among genomic changes in these samples, each of which results in an amino acid change in the variable domains of OmpA (major outer membrane protein, MOMP). A reversion to ompA-genotype L2 with the L2b genomic backbone is commonly seen. The wide diversity of ompA-genotypes found in these recent LGV samples indicates that this gene is under immunological selection. Our results suggest that the ompA-genotype L2b genomic backbone is the dominant strain circulating and evolving particularly in men who have sex with men (MSM) populations.

Impact of a GP cooperative on lower acuity emergency department attendances.

BACKGROUND: In 1998 'Dubdoc', Ireland's first out-of-hours general practice emergency service, opened in an outpatient suite in St James's Hospital with a separate entrance 300 m from the emergency department (ED). Dubdoc was established with the aim of providing an easy access out-of-hours service for ambulatory patients of those doctors supplying the service. AIM: To determine whether ED attendances for patients in the lower acuity triage categories 4 and 5 have changed since the establishment of 'Dubdoc'. METHODS: A retrospective review of all attendances at the 'Dubdoc' service was compared with attendances at the ED for triage categories 4 and 5 of the same hospital over a 9-year period (1999-2007 inclusive) for equivalent times of day. RESULTS: ED attendances during 'Dubdoc' hours have decreased as a proportion of all attendances for triage categories 4 and 5. ED attendances for triage categories 4 and 5 fell substantially during the study period. CONCLUSIONS: Although the presence of the 'Dubdoc' service has resulted in a decrease in ED attendances for triage categories 4 and 5, this is a minor proportion of the overall decrease in attendances in this group of patients.

The Antibody Response to SARS-CoV-2 Infection.

BACKGROUND: Testing for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-specific antibodies has become an important tool, complementing nucleic acid tests (NATs) for diagnosis and for determining the prevalence of coronavirus disease 2019 (COVID-19) in population serosurveys. The magnitude and persistence of antibody responses are critical for assessing the duration of immunity. METHODS: A SARS-CoV-2-specific immunofluorescent antibody (IFA) assay for immunoglobulin G (IgG), immunoglobulin A (IgA), and immunoglobulin M (IgM) was developed and prospectively evaluated by comparison to the reference standard of NAT on respiratory tract samples from individuals with suspected COVID-19. Neutralizing antibody responses were measured in a subset of samples using a standard microneutralization assay. RESULTS: A total of 2753 individuals were eligible for the study (126 NAT-positive; prevalence, 4.6%). The median "window period" from illness onset to appearance of antibodies (range) was 10.2 (5.8-14.4) days. The sensitivity and specificity of either SARS-CoV-2 IgG, IgA, or IgM when collected ≥14 days after symptom onset were 91.3% (95% CI, 84.9%-95.6%) and 98.9% (95% CI, 98.4%-99.3%), respectively. The negative predictive value was 99.6% (95% CI, 99.3%-99.8%). The positive predictive value of detecting any antibody class was 79.9% (95% CI, 73.3%-85.1%); this increased to 96.8% (95% CI, 90.7%-99.0%) for the combination of IgG and IgA. CONCLUSIONS: Measurement of SARS-CoV-2-specific antibody by IFA is an accurate method to diagnose COVID-19. Serological testing should be incorporated into diagnostic algorithms for SARS-CoV-2 infection to identify additional cases where NAT was not performed and resolve cases where false-negative and false-positive NATs are suspected. The majority of individuals develop robust antibody responses following infection, but the duration of these responses and implications for immunity remain to be established.

An emergent clade of SARS-CoV-2 linked to returned travellers from Iran.

The SARS-CoV-2 epidemic has rapidly spread outside China with major outbreaks occurring in Italy, South Korea, and Iran. Phylogenetic analyses of whole-genome sequencing data identified a distinct SARS-CoV-2 clade linked to travellers returning from Iran to Australia and New Zealand. This study highlights potential viral diversity driving the epidemic in Iran, and underscores the power of rapid genome sequencing and public data sharing to improve the detection and management of emerging infectious diseases.

Revealing COVID-19 transmission in Australia by SARS-CoV-2 genome sequencing and agent-based modeling.

In January 2020, a novel betacoronavirus (family Coronaviridae), named severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), was identified as the etiological agent of a cluster of pneumonia cases occurring in Wuhan City, Hubei Province, China1,2. The disease arising from SARS-CoV-2 infection, coronavirus disease 2019 (COVID-19), subsequently spread rapidly causing a worldwide pandemic. Here we examine the added value of near real-time genome sequencing of SARS-CoV-2 in a subpopulation of infected patients during the first 10 weeks of COVID-19 containment in Australia and compare findings from genomic surveillance with predictions of a computational agent-based model (ABM). Using the Australian census data, the ABM generates over 24 million software agents representing the population of Australia, each with demographic attributes of an anonymous individual. It then simulates transmission of the disease over time, spreading from specific infection sources, using contact rates of individuals within different social contexts. We report that the prospective sequencing of SARS-CoV-2 clarified the probable source of infection in cases where epidemiological links could not be determined, significantly decreased the proportion of COVID-19 cases with contentious links, documented genomically similar cases associated with concurrent transmission in several institutions and identified previously unsuspected links. Only a quarter of sequenced cases appeared to be locally acquired and were concordant with predictions from the ABM. These high-resolution genomic data are crucial to track cases with locally acquired COVID-19 and for timely recognition of independent importations once border restrictions are lifted and trade and travel resume.

Evolution of Human Respiratory Syncytial Virus (RSV) over Multiple Seasons in New South Wales, Australia.

There is an ongoing global pandemic of human respiratory syncytial virus (RSV) infection that results in substantial annual morbidity and mortality. In Australia, RSV is a major cause of acute lower respiratory tract infections (ALRI). Nevertheless, little is known about the extent and origins of the genetic diversity of RSV in Australia, nor the factors that shape this diversity. We have conducted a genome-scale analysis of RSV infections in New South Wales (NSW). RSV genomes were successfully sequenced for 144 specimens collected between 2010⁻2016. Of these, 64 belonged to the RSVA and 80 to the RSVB subtype. Phylogenetic analysis revealed a wide diversity of RSV lineages within NSW and that both subtypes evolved rapidly in a strongly clock-like manner, with mean rates of approximately 6⁻8 × 10-4 nucleotide substitutions per site per year. There was only weak evidence for geographic clustering of sequences, indicative of fluid patterns of transmission within the infected population and no evidence of any clustering by patient age such that viruses in the same lineages circulate through the entire host population. Importantly, we show that both subtypes circulated concurrently in NSW with multiple introductions into the Australian population in each year and only limited evidence for multi-year persistence.

Simultaneous co-detection of wild-type and vaccine strain measles virus using the BD MAX system.

Despite the reported elimination of measles virus in Australia, importation of cases from endemic countries continues to lead to secondary local transmission and outbreaks. Rapid laboratory confirmation of measles is paramount for individual patient management and outbreak responses. Further, it is important to rapidly distinguish infection from wild-type virus or vaccine strains to guide public health responses. We developed a high throughput, TaqMan-based multiplex reverse-transcription-polymerase chain reaction (PCR) assay using the BD MAX platform (Becton Dickinson) that simultaneously detects measles virus and differentiates between wild-type and vaccine strains without the need for sequencing.

The Role of Animal Translocations in Conserving British Wildlife: An Overview of Recent Work and Prospects for the Future.

We provide an overview of terrestrial animal translocations carried out for conservation purposes in Britain, summarising what has been achieved in recent decades and discussing the issues raised by this approach to conservation. In the last 40 years, at least nine species have been reintroduced following extinction in Britain (or at least one country within Britain), including five birds, one mammal, one amphibian and two invertebrates. Many more species have been translocated within Britain to establish additional populations in order to improve conservation status. We discuss the guidelines and protocols used to assess translocation projects in Britain, notably the IUCN guidelines, most recently revised in 2013. We also discuss the likely use of species translocations in future and suggest that, in our increasingly fragmented landscapes, they will have an important role to play in conservation restoration, especially for animals with limited mobility. Moving species around is a complex undertaking and our understanding of the inherent risks involved, including the risks from disease, has improved significantly in recent years. Conservation translocations should be considered in the context of species recovery targets and high standards should be maintained so that disease risks and other potentially negative impacts are minimised.

Planning for pandemic influenza surveillance in NSW.

Early detection of a novel strain (genotype) of influenza virus in the NSW population is the key to controlling a pandemic. If this occurs, ongoing surveillance will help determine the epidemiology and risk factors of the virus as well as its impact on essential services. Important components of surveillance preparedness in NSW include: border surveillance; hospital-based screening for suspected cases; protocols for efficient transport and testing of viral specimens; flexible, robust electronic tools for rapid surveillance data collection; management and reporting; and creation of surveillance surge capacity.

Single-center Series of Bone Marrow Biopsy-Defined Large Granular Lymphocyte Leukemia: High Rates of Sustained Response to Oral Methotrexate.

INTRODUCTION: Large granular lymphocyte (LGL) leukemia is a rare chronic lymphoproliferative disorder, with few large series reported to date. Series using stringent diagnostic criteria incorporating bone marrow biopsy (BMB), immunophenotyping, and T-cell receptor rearrangements are even scarcer. PATIENTS AND METHODS: The present study was a single-center series of 39 patients with LGL leukemia diagnosed using immunohistochemical analysis of BMB samples and flow cytometric and molecular data. RESULTS: With a median follow-up of 3.2 years (range, 1.0-15.1 years), 15 patients (38%) never required treatment. Of the remaining 24 patients requiring treatment, 13 were initially treated with prednisolone, for an overall response rate (ORR) of 84.6% and a median duration of response (DOR) of 13.5 months (range, 5.7-70.3 months). Of the 24 patients, 9 received oral low-dose weekly methotrexate as first-line therapy, with 8 (89%) achieving a hematologic response and a median DOR of 132.7 months (range, 6.7-180.5 months). Another 5 patients received methotrexate after prednisolone failure; all 5 responded, with a median DOR of 14 months (range, 4-96 months). Only 2 patients developed progression during methotrexate therapy, and 4 patients experienced responses lasting ≥ 5 years. CONCLUSION: Single-agent oral methotrexate appears to be highly efficacious, resulting in long response durations and minimal toxicity.

Indirect fluorescent antibody testing of nasopharyngeal swabs for influenza diagnosis in lung transplant recipients.

BACKGROUND: Rapid and reliable diagnosis of respiratory viral infections (RVI) in lung transplant recipients is essential to direct therapy of acute graft dysfunction and identify epidemic trends. Traditional techniques of serology and viral culture are limited by the lack of antibody response and delay in diagnosis. METHODS: We examined the clinical utility of indirect fluorescent antibody (IFA) testing in adult lung transplant patients with suspected RVI, compared with serology and culture. Nasopharyngeal and throat swabs (NT) were obtained to sample epithelial cells, followed by application of monoclonal antibody to respiratory syncytial virus, adenovirus, parainfluenza 1-3 and influenza A and B. The Bartels Respiratory Viral Detection kit was used with IFA results available within 24 hours. RESULTS: Nine of 18 patients tested positive for RVI with influenza A (n = 8) and influenza B (n = 1) detected. The sensitivity of IFA (67%) was higher than that of cell culture (45%). With intensive supportive therapy, infection was self-limiting in bronchiolitis obliterans syndrome (BOS) Grade 0-2 patients. However, patients with BOS Grade 3 manifested an acute exacerbation of airflow obstruction, which proved to be irreversible. CONCLUSIONS: Lung transplant patients with "flu-like" symptoms should proceed to IFA testing of NT swab specimens for early diagnosis. Samples collected within 7 days of symptom onset have high sensitivity as compared with serology and viral culture techniques.