Background Torque teno virus (TTV) viral loads may reflect patients' net state of immunosuppression and identify patients at risk of infections and rejection early after solid organ transplantation. However, its role in long-term kidney recipients (KTRs) is less clear. Methods This single-centre prospective cohort study evaluated the association between TTV viral load at recruitment and severe infections requiring hospitalisation within 12 months as the primary outcome in kidney recipients > 1-year post-transplant and on stable immunosuppression for > 3 months. Participants were followed up for 12 months, until change of immunosuppression, graft loss, or death. Pre-specified, exploratory secondary outcomes, including opportunistic infections, malignancy, composite outcomes were also analysed. Results Median time after transplant amongst the 171 included participants was 10.3 years (interquartile range (IQR) 4.9-16.0). Thirty-one developed severe infections 105 days (IQR 65-204) after baseline TTV measurement. Higher baseline log10-transformed TTV viral load (logTTV) was associated with severe infections within 12 months (odds ratio (OR) 1.37, 95% confidence interval (CI) 1.01-1.86, p = 0.045) with a modest area under receiver operating characteristics curve of 0.60 (95% CI 0.50-0.71). Compared to logTTV < 4.6 copies/mL, logTTV > 6.6 copies/mL (hazard ratio (HR) 3.45, 95% CI 1.10-10.9, p = 0.04) was associated with time to hospitalisations for infections but not logTTV 4.6-6.6 copies/mL (HR 2.30, 95% CI 0.97-5.47, p = 0.06). The ORs for the association between logTTV and malignancy, opportunistic infection and biopsy-proven rejection were 1.86 (95% CI 1.09-3.16), 1.21 (95% CI 0.69-2.13) and 1.09 (95% CI 0.70-1.69) respectively. Conclusion Elevated TTV viral loads were associated with severe infections amongst our cohort of long-term KTRs and may be a useful biomarker in this population.

BACKGROUND:The emerging fungal pathogen Candida auris poses a serious threat to global public health due to its worldwide distribution, multidrug resistance, high transmissibility, propensity to cause outbreaks, and high mortality. We aimed to characterise three unusual C auris isolates detected in Singapore, and to determine whether they constitute a novel clade distinct from all previously known C auris clades (I-V). METHODS:In this genotypic and phenotypic study, we characterised three C auris clinical isolates, which were cultured from epidemiologically unlinked inpatients at a large tertiary hospital in Singapore. The index isolate was detected in April, 2023. We performed whole-genome sequencing (WGS) and obtained hybrid assemblies of these C auris isolates. The complete genomes were compared with representative genomes of all known C auris clades. To provide a global context, 3651 international WGS data from the National Center for Biotechnology Information (NCBI) database were included in a high-resolution single nucleotide polymorphism (SNP) analysis. Antifungal susceptibility testing was done and antifungal resistance genes, mating-type locus, and chromosomal rearrangements were characterised from the WGS data of the three investigated isolates. We further implemented Bayesian logistic regression models to classify isolates into known clades and simulate the automatic detection of isolates belonging to novel clades as their WGS data became available. FINDINGS:The three investigated isolates were separated by at least 37 000 SNPs (range 37 000-236 900) from all existing C auris clades. These isolates had opposite mating-type allele and different chromosomal rearrangements when compared with their closest clade IV relatives. The isolates were susceptible to all tested antifungals. Therefore, we propose that these isolates represent a new clade of C auris, clade VI. Furthermore, an independent WGS dataset from Bangladesh, accessed via the NCBI Sequence Read Archive, was found to belong to this new clade. As a proof-of-concept, our Bayesian logistic regression model was able to flag these outlier genomes as a potential new clade. INTERPRETATION:The discovery of a new C auris clade in Singapore and Bangladesh in the Indomalayan zone, showing a close relationship to clade IV members most commonly found in South America, highlights the unknown genetic diversity and origin of C auris, particularly in under-resourced regions. Active surveillance in clinical settings, along with effective sequencing strategies and downstream analysis, will be essential in the identification of novel strains, tracking of transmission, and containment of adverse clinical effects of C auris infections. FUNDING:Duke-NUS Academic Medical Center Nurturing Clinician Researcher Scheme, and the Genedant-GIS Innovation Program.

Good syndrome (GS) is a rare acquired immunodeficiency disease characterized by the presence of thymoma with combined B and T cell immunodeficiency in adults. Recurrent bacterial infections, particularly sinopulmonary infections caused by encapsulated bacteria, remain the most common infective presentation of GS; however, relapsing viral infections have also been reported, likely due to impaired T cell-mediated immunity. Relapsing COVID-19 infection, however, has not been previously reported as a manifestation of GS. We present two cases of relapsing COVID-19 infection in patients with GS; in one case, relapsing COVID-19 was the first manifestation of newly diagnosed GS.

Summary Background The emerging fungal pathogen Candida auris poses a serious threat to global public health due to its worldwide distribution, multidrug-resistance, high transmissibility, propensity to cause outbreaks and high mortality rates. We report three C. auris isolates detected in Singapore, which are genetically distinct from all known clades (I-V) and represent a new clade (Clade VI). Methods Three epidemiologically unlinked clinical isolates belonging to the potential new C. auris clade were whole-genome sequenced and phenotypically characterized. The complete genomes of these isolates were compared to representative genomes of all known clades. To provide a global context, 3,651 international whole-genome sequences (WGS) from the NCBI database were included in the high-resolution single nucleotide polymorphism (SNP) analysis. Antifungal resistance genes, mating type locus, and chromosomal rearrangements were characterized from the WGS data of the Clade VI isolates. We further implemented Bayesian logistic regression models to simulate the automatic detection of Clade V and VI as their WGS data became available. Findings The three Clade VI isolates were separated by >36,000 SNPs from all existing C. auris clades. These isolates had opposite mating type allele and different chromosomal rearrangements when compared to their closest Clade IV relatives. As a proof-of-concept, our classification model was able to flag these outlier genomes as a potential new clade. Furthermore, an independent WGS submission from Bangladesh was found to belong to this new clade. Interpretation The discovery of a new C. auris clade in Singapore and Bangladesh, showing close relationship to Clade IV members in South America, highlights the unknown genetic diversity and origin of C. auris , particularly in under-resourced regions. Active surveillance in clinical settings, along with effective sequencing strategies and downstream analysis, will be essential in the identification of novel strains, tracking of transmission, and containment of adverse clinical impacts caused by C. auris infections. Funding This work was supported by the Singapore National Medical Research Council (NMRC) research training fellowship (MOH-FLWSHP19may-0005), the NCRS Duke-NUS Academic Medical Center Academic Clinical Program grant (09/FY2022/P1/17-A32, GRDUKP003401), and the Genedant-GIS Innovation Program grant. Research in context Evidence before this study We searched PubMed using the search terms “ Candida auris ” AND “clade”, for papers published between Jan 1, 2009, and July 1, 2023. This search retrieved 115 publications. 60 relevant publications were identified. 28 studies analyzed and discussed the molecular epidemiology of C. auris , including the description of C. auris clades, either in outbreak or surveillance settings. There were 11 case reports of C. auris clinical cases that included clade determination. Two studies focused on the detection and clade determination of C. auris from non-healthcare environments. Clade-specific characteristics were described or analyzed in 14 studies. One study applied machine learning to C. auris drug resistance analysis, but not for clade determination. Four studies focused on the description of potentially new C. auris lineages, subclades, or clades. All publications described isolates that belong to one of the five known C. auris clades (I-V). All publications found that strains from different clades differed by more than 35,000 SNPs, and that there are clade-specific differences in geographical distribution, phenotypic characteristics, antifungal susceptibility profile, outbreak potential, and clinical manifestations. The NCBI Pathogen Detection system contained 4,506 C. auris genomes on July 1, 2023. There were ten (0·22%) submissions from Southeast Asian countries and 92 (2·04%) submissions from South Asia and the Indian subcontinent, which are parts of the Indomalayan biogeographic realm. Added value of this study To the best of our knowledge, we are the first group to perform hybrid assemblies on three representative isolates in a new C. auris clade, which is separated from all other existing clades (I-V) by >36,000 SNPs. Whole-genome SNP analysis and phenotypic characterization of these epidemiologically unlinked isolates detected in Singapore suggest that they represent a previously unreported sixth major clade. High-resolution SNP analysis of 3,651 international whole-genome sequences from the NCBI database, which generated the final dataset consisting of more than 6.6 million genome pairs, revealed six distinct genetic clusters representing the five known clades and the new sixth clade (Indomalayan). In addition, we demonstrate that a machine learning approach can be used to flag these outlier genomes for further investigations as soon as they become available, thus providing the earliest possible alert for potential new public health threats. Implications of all the available evidence Despite the high antimicrobial resistance burden in Southeast Asia and South Asia, these regions are disproportionately underrepresented in terms of genomic surveillance of C. auris , a multidrug-resistant fungal pathogen. The detection of three epidemiologically unlinked C. auris isolates in Singapore belonging to a new C. auris clade suggests that yet-to-be-reported strains may be circulating in the region. Given the propensity for multidrug resistance, healthcare-associated infection outbreaks, and the associated high mortality, active surveillance and continued vigilance is necessary.

Background The emerging fungal pathogen Candida auris poses a serious threat to global public health due to its worldwide distribution, multidrug-resistance, high transmissibility, propensity to cause outbreaks and high mortality rates. We report three C. auris isolates detected in Singapore, which are genetically distinct from all known clades (I-V) and represent a new clade (Clade VI). Methods Three epidemiologically unlinked clinical isolates belonging to the potential new C. auris clade were whole-genome sequenced and phenotypically characterized. The complete genomes of these isolates were compared to representative genomes of all known clades. To provide a global context, 3,651 international whole-genome sequences (WGS) from the NCBI database were included in the high-resolution single nucleotide polymorphism (SNP) analysis. Antifungal resistance genes, mating type locus, and chromosomal rearrangements were characterized from the WGS data of the Clade VI isolates. We further implemented Bayesian logistic regression models to simulate the automatic detection of Clade V and VI as their WGS data became available. Findings The three Clade VI isolates were separated by >36,000 SNPs from all existing C. auris clades. These isolates had opposite mating type allele and different chromosomal rearrangements when compared to their closest Clade IV relatives. As a proof-of-concept, our classification model was able to flag these outlier genomes as a potential new clade. Furthermore, an independent WGS submission from Bangladesh was found to belong to this new clade. Interpretation The discovery of a new C. auris clade in Singapore and Bangladesh, showing close relationship to Clade IV members in South America, highlights the unknown genetic diversity and origin of C. auris , particularly in under-resourced regions. Active surveillance in clinical settings, along with effective sequencing strategies and downstream analysis, will be essential in the identification of novel strains, tracking of transmission, and containment of adverse clinical impacts caused by C. auris infections. Funding This work was supported by the Singapore National Medical Research Council (NMRC) research training fellowship (MOH-FLWSHP19may-0005), the NCRS Duke-NUS Academic Medical Center Academic Clinical Program grant (09/FY2022/P1/17-A32, GRDUKP003401), and the Genedant-GIS Innovation Program grant. Evidence before this study We searched PubMed using the search terms “ Candida auris ” AND “clade”, for papers published between Jan 1, 2009, and July 1, 2023. This search retrieved 115 publications. 60 relevant publications were identified. 28 studies analyzed and discussed the molecular epidemiology of C. auris , including the description of C. auris clades, either in outbreak or surveillance settings. There were 11 case reports of C. auris clinical cases that included clade determination. Two studies focused on the detection and clade determination of C. auris from non-healthcare environments. Clade-specific characteristics were described or analyzed in 14 studies. One study applied machine learning to C. auris drug resistance analysis, but not for clade determination. Four studies focused on the description of potentially new C. auris lineages, subclades, or clades. All publications described isolates that belong to one of the five known C. auris clades (I-V). All publications found that strains from different clades differed by more than 35,000 SNPs, and that there are clade-specific differences in geographical distribution, phenotypic characteristics, antifungal susceptibility profile, outbreak potential, and clinical manifestations. The NCBI Pathogen Detection system contained 4,506 C. auris genomes on July 1, 2023. There were ten (0·22%) submissions from Southeast Asian countries and 92 (2·04%) submissions from South Asia and the Indian subcontinent, which are parts of the Indomalayan biogeographic realm. Added value of this study To the best of our knowledge, we are the first group to perform hybrid assemblies on three representative isolates in a new C. auris clade, which is separated from all other existing clades (I-V) by >36,000 SNPs. Whole-genome SNP analysis and phenotypic characterization of these epidemiologically unlinked isolates detected in Singapore suggest that they represent a previously unreported sixth major clade. High-resolution SNP analysis of 3,651 international whole-genome sequences from the NCBI database, which generated the final dataset consisting of more than 6.6 million genome pairs, revealed six distinct genetic clusters representing the five known clades and the new sixth clade (Indomalayan). In addition, we demonstrate that a machine learning approach can be used to flag these outlier genomes for further investigations as soon as they become available, thus providing the earliest possible alert for potential new public health threats. Implications of all the available evidence Despite the high antimicrobial resistance burden in Southeast Asia and South Asia, these regions are disproportionately underrepresented in terms of genomic surveillance of C. auris , a multidrug-resistant fungal pathogen. The detection of three epidemiologically unlinked C. auris isolates in Singapore belonging to a new C. auris clade suggests that yet-to-be-reported strains may be circulating in the region. Given the propensity for multidrug resistance, healthcare-associated infection outbreaks, and the associated high mortality, active surveillance and continued vigilance is necessary. ### Competing Interest Statement The authors have declared no competing interest. ### Funding Statement This work was supported by the Singapore National Medical Research Council (NMRC) research training fellowship (MOH-FLWSHP19may-0005), the NCRS Duke-NUS Academic Medical Center Academic Clinical Program grant (09/FY2022/P1/17-A32, GRDUKP003401), and the Genedant-GIS Innovation Program grant. ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: This study used pre-existing retrospective collections of isolates and our analyses led to no clinical intervention. Epidemiological data collection was previously performed as part of routine surveillance and infection prevention measures and hence constituted a non-research infection control surveillance activity. Institutional review board exemption was granted by the SingHealth Centralised Institutional Review Board (Reference number 2017-2576). I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable. Yes Reads and genome assemblies from this study have been deposited in the National Centre for Biotechnology Information (NCBI) Sequence Read Archive (SRA) database () under BioProject accession number PRJNA1000034. All data produced in the present study are available upon reasonable request to the authors.

Introduction Kidney transplant recipients (KTRs) suffer from immunosuppression-related adverse events (iRAEs), such as infections and malignancy from chronic immunosuppression, but are also at risk of graft loss from rejection with underimmunosuppression. Biomarkers that predict both iRAEs and rejection while allowing individualisation of immunosuppression exposure are lacking. Although plasma viral DNA levels of torque teno virus (TTV), a widely prevalent, non-pathogenic virus, have been shown to predict both iRAE and rejection in newly transplanted KTRs within the first year after transplant, its role for prevalent KTRs on stable immunosuppression is less clear.This study aims to determine the prognostic value of TTV levels for severe infections (defined as infections requiring hospitalisation) in prevalent KTRs on stable immunosuppression for at least 3 months and compare it against that of other commonly available biomarkers. The study also aims to explore the relationship between TTV levels and factors affecting the ‘net state of immunosuppression’ as well as other clinical outcomes.Methods and analysis This is a single-centre, prospective, observational cohort study of 172 KTRs on stable immunosuppression for more than 3 months. TTV levels will be measured using the TTV R-GENE kit upon recruitment when study subjects are admitted and when kidney allograft biopsies are performed. Subjects will be monitored for iRAEs and rejection for at least 12 months. The relationship between TTV load and clinical outcomes such as severe infections will be analysed and compared against that from other common biomarkers and previously published predictive scores.Ethics and dissemination The study was approved by the SingHealth Centralised Institutional Review Board (2023/2170). The results will be presented at conferences and submitted for publication in peer-reviewed journals.Trial registration number NCT05836636.

Although laboratory issues were considered part of pandemic preparedness in many countries, it was difficult to anticipate the unexpected challenges and unprecedented demands in delivering SARS-CoV-2 testing during the COVID-19 pandemic. As the pandemic evolved, particularly during the first 2 years, significant pressures were placed on laboratories worldwide to provide timely SARS-CoV-2 testing, in addition to the routine tests required for other illnesses. Rapidly changing government and central laboratory mandates, obscure tendering processes [[1]British Medical Association Outsourced and undermined: the COVID-19 windfall for private providers.2020https://www.bma.org.uk/news-and-opinion/outsourced-and-undermined-the-covid-19-windfall-for-private-providersGoogle Scholar,[2]Dyer C. Covid-19: "Impossible" to be confident that government awarded Randox contracts properly, say MPs.BMJ. 2022; 378o1893https://doi.org/10.1136/bmj.o1893Crossref Scopus (2) Google Scholar], requirements for rapid reporting of results to guide individual patient management (in both hospital and community settings), infection control mandates placed additional stress on diagnostic laboratories. The demands for testing targets were not based on strong scientific evidence as the pandemic evolved but more on the numbers of daily cases reported by national surveillance systems and hospital admissions. Early during the pandemic, many questions were asked about the diagnostic performance of SARS-CoV-2 tests. Evolving, real-time data on asymptomatic and pre-symptomatic SARS-CoV-2 infection and re-infection as well as viral kinetics and shedding of replication-competent viruses made regular communication with clinicians, public health physicians, policymakers, the general public and the media necessary. However, these were often limited by time pressures on laboratory scientists and microbiologists. Shortages due to supply chain issues of reagents, consumables, personal protective equipment, and staff placed additional significant strain on laboratories. Here, we summarize our shared experiences and suggest solutions to some of the problems which we encountered during the COVID-19 pandemic, with the aim of informing and improving future pandemic preparedness of diagnostic laboratories. This is not intended to be an exhaustive review; however, in presenting our individual laboratory experiences, we hope to demonstrate to stakeholders the key role of diagnostic laboratories in the face of any new pandemic. This was a common theme to varying degrees across our laboratories. Policymakers and government officials, who were unfamiliar with how diagnostic testing worked in public and private sectors, were demanding accurate results of the nucleic acid amplification test (NAAT), with rapid but often unrealistic turnaround times to screen people with suspected SARS-CoV-2 infection. Initially, diagnostic testing was centralized in many countries, which was understandable as new NAAT assays were developed and optimized. Following this, the timely roll-out to other hospitals (where patients with suspected or confirmed COVID-19 were managed) or non-hospital-based laboratories (serving community-based patients, prisons, elderly care homes, or the general public) was delayed, until central laboratories were overwhelmed. Although urgent and rapid reporting of results is important during the early stages of any evolving pandemic, expert virology oversight is required to assist with the interpretation of test results and enable appropriate patient management by clinical, infection control and public health teams [[3]Boseley S. Coronavirus: UK virologists criticise handling of Covid testing contracts. The Guardian, 2020https://www.theguardian.com/world/2020/aug/03/uk-virologists-criticise-handling-coronavirus-testing-contractsGoogle Scholar,[4]The UK Clinical Virology Network. 2022https://clinicalvirology.org/Google Scholar]. Governments should appoint laboratory-trained advisors with relevant diagnostic expertise to engage local diagnostic networks early in the process because they can help guide assay design, interpret results, co-ordinate supplies of testing reagents, work with regulators, and reduce the burden on central diagnostic laboratories. After the roll-out to non-central laboratories, the impact and burden of SARS-CoV-2 testing during the pandemic on local diagnostic laboratories was immediate and severe. Many staff members themselves were absent because of COVID-19 or had to self-quarantine when they were identified as close contacts of cases. This occurred particularly during the early stages of the pandemic, when the incidence of COVID-19 was high in the community, mask wearing was not mandatory, and COVID-19 vaccines were not yet available. Staffing issues took several of the following forms:i)Absenteeism because of COVID-19, which led to staff fatigue (including increased repetitive strain injuries from manual sample processing and data entry) and longer working hours to cover for those absent.ii)Extended work hours (including night shifts in some laboratories) because of slow recruitment of additional staff, who were often recruited from other laboratory specialties, resulting in knock-on effects on those services.iii)Training of new staff by staff involved in SARS-CoV-2 testing. Those who had worked in other laboratories (such as research or non-human) often took longer to be trained because they were unfamiliar with workflows, information systems and quality assurance procedures in clinical diagnostic laboratories.iv)In some countries, the inability to use non-clinically trained staff (e.g. academic or research) who could perform SARS-CoV-2 testing but were not registered with or accredited by relevant authorities (or equivalent organizations) for human health diagnostic testing.v)The importance of maintaining a minimum number of staff members with expertise in developing, verifying and validating diagnostic assays, separate from those performing routine testing. In order to alleviate some of these pressures, other diagnostic tests, including respiratory virus multiplex NAAT, were rationalized in some laboratories, which led to reduced information on the presence of other important pathogens. Testing of pooled samples during the early stages of the pandemic was helpful in improving the efficiency of testing and conserving testing reagents; however, this was not sustainable once the incidence of infection increased. Although less skilled staff could be used to register samples and prepare them for testing on more fully automated platforms [[5]Yau F. Ferreira R. Kamali R. Bird P.W. Halliwell R. Patel H. et al.Clinical utility of a rapid 'on-demand' laboratory-based SARS-CoV-2 diagnostic testing service in an acute hospital setting admitting COVID-19 patients.Clin Infect Pract. 2021; 12100086https://doi.org/10.1016/j.clinpr.2021.100086Crossref PubMed Scopus (6) Google Scholar], they needed to be recruited and trained, adding to the burden on administrative and existing staff. In some laboratories, incentives or rewards, such as one-off staff bonuses, were given. Learning from and co-ordinating existing diagnostic laboratory networks will help inform decisions on how to manage staff burnout and shortages. Establishment of a register of 'locum' staff (including those working in academic institutions) across the country could provide support to local or distant (for those willing to travel) laboratories at short notice. The use of automated processes and platforms (e.g. liquid-handling robots) should be increased once such assays become available. Staff morale should be maintained with team-building activities (in which social distancing is allowed), confidence in leadership should be improved, and, where appropriate, salary bonuses should be provided. Many laboratories ran out of nucleic acid extraction and amplification reagents, plastic ware (e.g. pipette tips and test tubes), swabs and sample transport media. With a high demand globally, countries became protectionist, with manufacturers refusing or legally unable to supply goods outside of their native countries to prioritize support for local diagnostic testing. It was not always possible to assess reagents sourced from alternative suppliers, and of those which were, some were not 'fit for purpose'. Although this might be understandable, on a larger, international scale, this becomes detrimental because undiagnosed or poor surveillance in potential hotspots of infection can lead to delayed identification and control of the spread of novel SARS-CoV-2 variants of concern, including Alpha (B.1.1.7), Beta (B.1.351), Delta (B.1.617.2) and Omicron (B.1.1.529) [[6]Vandenberg O. Martiny D. Rochas O. van Belkum A. Kozlakidis Z. Considerations for diagnostic COVID-19 tests.Nat Rev Microbiol. 2021; 19: 171-183https://doi.org/10.1038/s41579-020-00461-zCrossref PubMed Scopus (458) Google Scholar]. Unfortunately, there is no simple solution to supply issues which will benefit all laboratories. If each country utilized their local laboratory network to form a larger customer base to lobby suppliers, other laboratories elsewhere will lose out. Ideally, these supplies should be linked to need; however, during pandemics, the need is global. Suppliers will tend to prioritize laboratories with which they have long, existing and close commercial relationships. Individual laboratories may have to rely on personal relationships with existing suppliers to support them when the demand is high. Richer countries could, theoretically, outbid other countries for reagents; so, there may have to be some pre-established or rapidly established global principles which suppliers would need to follow to control this somewhat unethical practice. Jurisdictional laboratories with greater purchasing power because of economies of scale were more successful at leveraging manufacturers for supplies. Some countries urgently considered on-shore manufacturing of consumables, including the use of novel technologies such as three-dimensional-printed swabs [[7]Williams E. Bond K. Isles N. Chong B. Johnson D. Druce J. et al.Pandemic printing: evaluation of a novel 3D printed swab for detecting SARS-CoV-2.Med J Aust. 2020; 213: 276-279https://doi.org/10.5694/mja2.50726Crossref PubMed Scopus (25) Google Scholar]. The use of multiple testing platforms in each laboratory will help to mitigate reagent shortages. Stockpile options need to be considered. Diagnostic testing for SARS-CoV-2 during the early phases of the COVID-19 pandemic reflected a chaotic and disorganized scramble for the development and roll-out of assays to meet urgent government and public health demands. This led to many errors, even by experienced laboratories [[8]Cohen J. The United States badly bungled coronavirus testing—but things may soon improve. Science Insider, 2020https://www.science.org/content/article/united-states-badly-bungled-coronavirus-testing-things-may-soon-improveGoogle Scholar], and bottlenecks in testing hampered effective community surveillance and public health control of the virus [[9]Iacobucci G. Covid-19: lack of capacity led to halting of community testing in March, admits deputy chief medical officer.BMJ. 2020; 369: m1845https://doi.org/10.1136/bmj.m1845Crossref PubMed Scopus (20) Google Scholar]. With this experience and hindsight, we highlight potential mitigation strategies for future pandemics and suggest more co-ordinated responses from relevant stakeholders and governments. Governments should consult their national public health laboratory networks (where such networks exist) and work with them to deliver optimal testing strategies for novel pathogens with pandemic potential [[3]Boseley S. Coronavirus: UK virologists criticise handling of Covid testing contracts. The Guardian, 2020https://www.theguardian.com/world/2020/aug/03/uk-virologists-criticise-handling-coronavirus-testing-contractsGoogle Scholar]. Such laboratory networks can also utilize their collective existing industry contacts and 'buy power' to secure supplies of reagents, plastics and test kits. Finally, these networks can establish a register of laboratory staff who can be rapidly trained and deployed where needed to assist with any required laboratory workforce surge. JWT initially conceived the idea for the article and proposed this to all authors. All authors (MD, PWB, JK, CKL, HN, LO, GA, DED, CWH, LCJ) were supportive of the idea and sent accounts of their own experiences of virology diagnostic testing during the COVID-19 pandemic. JWT summarized each account in an initial draft and returned it to all authors for checking or verification. JWT then drafted the final version, and all authors critically reviewed the manuscript before submission. The authors declare that they have no conflicts of interest.

Background: Post-mRNA vaccination-associated cardiac complication is a rare but life-threatening adverse event. Its risk has been well balanced by the benefit of vaccination-induced protection against se-vere COVID-19. As the rate of severe COVID-19 has consequently declined, future booster vaccination to sustain immunity, especially against infection with new severe acute respiratory syndrome corona -virus 2 (SARS-CoV-2) variants, may encounter benefit-risk ratios that are less favorable than at the start of the COVID-19 vaccination campaign. Understanding the pathogenesis of rare but severe vaccine-associated adverse events to minimize its risk is thus urgent. Methods: Here, we report a serendipitous finding of a case of cardiac complication following a third shot of COVID-19 mRNA vaccine. As this case was enrolled in a cohort study, pre-vaccination and pre -symptomatic blood samples were available for genomic and multiplex cytokine analyses. Findings: These analyses revealed the presence of subclinical chronic inflammation, with an elevated expression of RNASE2 at pre-booster baseline as a possible trigger of an acute-on-chronic inflammation that resulted in the cardiac complication. RNASE2 encodes for the ribonu-clease RNase2, which cleaves RNA at the 30 side of uridine, which may thus remove the only Toll-like receptor (TLR)-avoidance safety feature of current mRNA vaccines. Conclusions: These pre-booster and pre-symptomatic gene and cytokine expression data provide unique insights into the possible pathogenesis of vaccine-associated cardiac complication and suggest the incorporation of additional nucleoside modification for an added safety margin.

Rapid onsite whole-genome sequencing of two suspected severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) N gene diagnostic escape samples revealed a previously unreported N gene point mutation at genome position 29195. Because the G29195T mutation occurs within a region probed by a commonly referenced U.S. CDC N gene reverse transcription (RT)-PCR assay, we hypothesize that the G29195T mutation rendered the N gene target of a proprietary commercial assay undetectable. The putative diagnostic escape G29195T mutation demonstrates the need for nearly real-time surveillance, as emergence of a novel SARS-CoV-2 variant with the potential to escape diagnostic tests continues to be a threat. IMPORTANCE Accurate diagnostic detection of SARS-CoV-2 currently depends on the large-scale deployment of RT-PCR assays. SARS-CoV-2 RT-PCR assays target predetermined regions in the viral genomes by complementary binding of primers and probes to nucleic acid sequences in the clinical samples. Potential diagnostic escapes, such as those of clinical samples harboring the G29195T mutation, may result in false-negative SARS-CoV-2 RT-PCR results. The rapid detection and sharing of potential diagnostic escapes are essential for diagnostic laboratories and manufacturers around the world, to optimize their assays as SARS-CoV-2 continues to evolve.

BackgroundProlonged shedding/relapse of COVID-19 infection has been reported, particularly in patients who received anti-CD20 agents (eg. rituximab). However, cases of occult COVID-19, in which SARS-CoV-2 persistence in lung parenchyma is diagnosed despite clearance from nasopharyngeal (NP) specimens, are uncommon.Case summaryWe describe two cases of occult COVID-19 in immunocompromised patients. Both patients had received rituximab previously. Both cases initially presented as ground-glass infiltrates on lung imaging; the diagnosis was originally not suspected due to repeated demonstration of negative SARS-CoV-2 from NP specimens, and alternative etiologies were originally considered. Persistence of SARS-CoV-2 in lung parenchyma, however, was demonstrated on bronchoalveolar lavage (BAL) specimens; additionally, isolation of viable SARS-CoV-2 virus and detection of SARS-CoV-2 nucleocapsid and spike-protein antigen in lung tissue on immunohistochemistry close to 3-months from primary infection strongly suggested ongoing viral persistence and replication as a driver of the lung parenchymal changes, which resolved after antiviral treatment.DiscussionOccult COVID-19 can be a cause of unexplained ground-glass infiltrates on lung imaging; negative NP samples do not rule out SARS-CoV-2 persistence and invasive sampling must be considered. The unsuspected presence of viable virus on BAL, however, highlights that procedurists perfoming aerosol-generating-procedures during an ongoing pandemic wave must also practise appropriate infection-prevention precautions to limit potential exposure.

The emergence of clinically important mutations described in this report highlights the need for sustained vigilance and containment measures when managing immunocompromised patients with persistent COVID-19. Even as jurisdictions across the globe start lifting pandemic control measures, immunocompromised patients with persistent COVID-19 constitute a unique group that requires close genomic monitoring and enhanced infection control measures, to ensure early detection and containment of mutations and variants of therapeutic and public health importance.

Importance Three-dimensionally printed nasopharyngeal swabs (3DP swabs) have been used to mitigate swab shortages during the coronavirus disease 2019 (COVID-19) pandemic. Clinical validation for diagnostic accuracy and consistency, as well as patient acceptability, is crucial to evaluate the swab's performance. Objective To determine the accuracy and acceptability of the 3DP swab for identifying severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Design, Setting, and Participants A diagnostic study was conducted from May to July 2020 at 2 tertiary care centers in Singapore with different reference swabs (FLOQSwab [COPAN Diagnostics] or Dacron swab [Deltalab]) and swab processing techniques (wet or dry) to evaluate the performance of the 3DP swab compared with traditional, standard-of-care nasopharyngeal swabs used in health care institutions. The participants were patients with COVID-19 in the first 2 weeks of illness and controls with acute respiratory illness with negative test results for SARS-CoV-2. Paired nasopharyngeal swabs were obtained from the same nostril and tested for SARS-CoV-2 by reverse-transcriptase polymerase chain reaction. The sequence of swabs was randomized based on odd and even participant numbers. Main Outcomes and Measures Primary outcome measures were overall agreement (OA), positive percentage agreement (PPA), and negative percentage agreement of the 3DP swab compared with reference swabs. Secondary outcome measures were the correlation of cycle threshold (Ct) values of both swabs. Results The mean (SD) age of participants was 45.4 (13.1) years, and most participants were men (87 of 89 [97.8%]), in keeping with the epidemiology of the COVID-19 pandemic in Singapore. A total of 79 patients with COVID-19 and 10 controls were recruited. Among the patients with COVID-19, the overall agreement and PPA of the 3DP swab was 91.1% and 93.5%, respectively, compared with reference swabs. The PPA was 100% for patients with COVID-19 who were tested within the first week of illness. All controls tested negative. The reverse-transcriptase polymerase chain reaction Ct values for theORF1abandE-genetargets showed a strong correlation (intraclass correlations coefficient, 0.869-0.920) between the 3DP and reference swab on independent testing at each institution despite differences in sample processing. Discordant results for both gene targets were observed only at high Ct values. Conclusions and Relevance In this diagnostic study of 79 patients with COVID-19 and 10 controls, the 3DP swab performed accurately and consistently across health care institutions and could help mitigate strained resources in the escalating COVID-19 pandemic.

Background: Effective management of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) requires large-scale testing to identify and isolate infectious carriers. Self-administered buccal swab and saliva collection are convenient, painless, and safe alternatives to the current healthcare worker (HCW)-collected nasopharyngeal swab (NPS). Methods: A cross-sectional single-centre study was conducted on 42 participants who had tested positive for SARS-CoV-2 via an NPS within the past 7 days. Real-time polymerase chain reaction (RT-PCR) was performed and cycle threshold (Ct) values were obtained for each test. The positive percent agreement (PPA), negative percent agreement (NPA), and overall agreement (OA) were calculated for the saliva samples and buccal swabs, and compared with NPS. Results: Among the 42 participants, 73.8% (31/42) tested positive by any one of the three tests. With reference to NPS, the saliva test had PPA 66.7%, NPA 91.7%, and OA 69.0%; the buccal swab had PPA 56.7%, NPA 100%, and OA 73.8%. Conclusion: Self-collected saliva tests and buccal swabs showed only moderate agreement with HCW-collected NPS. Primary screening for SARS-CoV-2 may be performed with a saliva test or buccal swab, with a negative test warranting a confirmatory NPS to avoid false-negatives, minimize discomfort, and reduce the risk of spread to the community and HCWs.

This cross-sectional study assessed the changes in respiratory virus prevalence in 2020 vs 2019 associated with the COVID-19 pandemic.

AbstractImportanceCOVID-19 pandemic control measures affect the prevalence of other respiratory viruses. Effects on some viruses have been described; however, the broader impact and temporal relationship of control measures on virus decline and subsequent re-emergence have not been thoroughly documented. Understanding these phenomena may influence health policies.ObjectiveTo examine the prevalence of unrelated respiratory viruses in relation to population-wide pandemic response measures and phases in 2020 in Singapore.Design, Setting, and ParticipantsData from respiratory multiplex PCRs from 3 major hospitals (total 3700 beds) in Singapore were collated. The full dataset consisted of 42,558 test results, 19,898 from 2019 and 22,660 from 2020.Main Outcomes and MeasuresWeekly virus prevalence data were mapped onto prevailing pandemic response measures, in order to elucidate temporal relationships and differential virus responses. Pre-pandemic data from 2019 were compared with data from 2020.ResultsEarly response measures, even before national lockdown, were followed by a dramatic reduction of influenza viruses and a more gradual decline of other respiratory viruses, including respiratory syncytial virus, parainfluenza viruses, endemic coronaviruses and metapneumovirus. Marked decline of enterovirus/rhinovirus and adenovirus, however, was only observed during lockdown. About 13 weeks into phased reopening, enterovirus/rhinovirus re-emerged, followed by adenovirus, the latter mainly in the pediatric population. All other viruses remained at low levels until the end of 2020.Conclusions and RelevanceCOVID-19 control measures in Singapore had a significant impact on a broad range of respiratory viruses. Effects of various control measures varied between phases and different viruses. Influenza viruses declined earliest and most dramatically; relaxation of measures was followed by re-emergence of enterovirus/rhinovirus and adenovirus. These patterns are presumably a result of different propensities for contact versus droplet and overall ease of transmission, and different virus reservoirs. Further studies into these phenomena are a matter of public health importance.Key PointsQuestionWhat were the effects of COVID-19 pandemic control measures in Singapore on the prevalence of other respiratory viruses?FindingsViruses responded differently to control measures. Influenza viruses declined rapidly after early control measures and remained near-absent during reopening after lockdown. Enterovirus/rhinovirus and adenovirus declined later and re-emerged earlier than other viruses during phased reopening.MeaningPopulation-wide interventions resulted in a broad decline and subsequent differential re-emergence of non-targeted respiratory viruses, corresponding to different patterns of virus response to control measures.

We aimed to test the sensitivity of naso-oropharyngeal saliva and self-administered nasal (SN) swab compared to nasopharyngeal (NP) swab for COVID-19 testing in a large cohort of migrant workers in Singapore. We also tested the utility of next-generation sequencing (NGS) for diagnosis of COVID-19. Saliva, NP and SN swabs were collected from subjects who presented with acute respiratory infection, their asymptomatic roommates, and prior confirmed cases who were undergoing isolation at a community care facility in June 2020. All samples were tested using RT-PCR. SARS-CoV-2 amplicon-based NGS with phylogenetic analysis was done for 30 samples. We recruited 200 subjects, of which 91 and 46 were tested twice and thrice respectively. In total, 62.0%, 44.5%, and 37.7% of saliva, NP and SN samples were positive. Cycle threshold (Ct) values were lower during the earlier period of infection across all sample types. The percentage of test-positive saliva was higher than NP and SN swabs. We found a strong correlation between viral genome coverage by NGS and Ct values for SARS-CoV-2. Phylogenetic analyses revealed Clade O and lineage B.6 known to be circulating in Singapore. We found saliva to be a sensitive and viable sample for COVID-19 diagnosis.