SARS-CoV-2 Genome Sequencing Methods Differ in Their Abilities To Detect Variants from Low-Viral-Load Samples.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) genomic surveillance has been vital in understanding the spread of coronavirus disease 2019 (COVID-19), the emergence of viral escape mutants, and variants of concern. However, low viral loads in clinical specimens affect variant calling for phylogenetic analyses and detection of low-frequency variants, important in uncovering infection transmission chains. We systematically evaluated three widely adopted SARS-CoV-2 whole-genome sequencing methods for their sensitivity, specificity, and ability to reliably detect low-frequency variants. Our analyses reveal that the ARTIC v3 protocol consistently displays high sensitivity for generating complete genomes at low viral loads compared with the probe-based Illumina Respiratory Viral Oligo panel and a pooled long-amplicon method. We show substantial variability in the number and location of low-frequency variants detected using the three methods, highlighting the importance of selecting appropriate methods to obtain high-quality sequence data from low-viral-load samples for public health and genomic surveillance purposes.

Interpret with caution: An evaluation of the commercial AusDiagnostics versus in-house developed assays for the detection of SARS-CoV-2 virus.

INTRODUCTION: There is limited data on the analytical performance of commercial nucleic acid tests (NATs) for laboratory confirmation of COVID-19 infection. METHODS: Nasopharyngeal, combined nose and throat swabs, nasopharyngeal aspirates and sputum was collected from persons with suspected SARS-CoV-2 infection, serial dilutions of SARS-CoV-2 viral cultures and synthetic positive controls (gBlocks, Integrated DNA Technologies) were tested using i) AusDiagnostics assay (AusDiagnostics Pty Ltd); ii) in-house developed assays targeting the E and RdRp genes; iii) multiplex PCR assay targeting endemic respiratory viruses. Discrepant SARS-CoV-2 results were resolved by testing the N, ORF1b, ORF1ab and M genes. RESULTS: Of 52 clinical samples collected from 50 persons tested, respiratory viruses were detected in 22 samples (42 %), including SARS CoV-2 (n = 5), rhinovirus (n = 7), enterovirus (n = 5), influenza B (n = 4), hMPV (n = 5), influenza A (n = 2), PIV-2 (n = 1), RSV (n = 2), CoV-NL63 (n = 1) and CoV-229E (n = 1). SARS-CoV-2 was detected in four additional samples by the AusDiagnostics assay. Using the in-house assays as the "gold standard", the sensitivity, specificity, positive and negative predictive values of the AusDiagnostics assay was 100 %, 92.16 %, 55.56 % and 100 % respectively. The Ct values of the real-time in-house-developed PCR assay targeting the E gene was significantly lower than the corresponding RdRp gene assay when applied to clinical samples, viral culture and positive controls (mean 21.75 vs 28.1, p = 0.0031). CONCLUSIONS: The AusDiagnostics assay is not specific for the detection SARS-CoV-2. Any positive results should be confirmed using another NAT or sequencing. The case definition used to investigate persons with suspected COVID-19 infection is not specific.

Detection of Recently Discovered Human Polyomaviruses in a Longitudinal Kidney Transplant Cohort.

A large number of human polyomaviruses have been discovered in the last 7 years. However, little is known about the clinical impact on vulnerable immunosuppressed patient populations. Blood, urine, and respiratory swabs collected from a prospective, longitudinal adult kidney transplant cohort (n = 167) generally pre-operatively, at day 4, months 1, 3, and 6 posttransplant, and at BK viremic episodes within the first year were screened for 12 human polyomaviruses using real-time polymerase chain reaction. Newly discovered polyomaviruses were most commonly detected in the respiratory tract, with persistent shedding seen for up to 6 months posttransplant. Merkel cell polyomavirus was the most common detection, but was not associated with clinical symptoms or subsequent development of skin cancer or other skin abnormalities. In contrast, KI polyomavirus was associated with respiratory disease in a subset of patients. Human polyomavirus 9, Malawi polyomavirus, and human polyomavirus 12 were not detected in any patient samples.

Association of micropapillary urothelial carcinoma of the bladder and BK viruria in kidney transplant recipients.

INTRODUCTION: BK virus (BKV) is an ubiquitous human polyomavirus that establishes latency in urothelium. BKV is known to re-activate in immunosuppressed individuals, and is an increasingly important cause of nephropathy and graft loss in kidney transplant recipients. Animal studies have demonstrated BKV has a potential role as a tumor virus. However, its role in precipitating or facilitating oncogenesis in humans is still debated. REPORT: We report 2 cases of aggressive micropapillary urothelial carcinoma of the bladder in kidney transplant recipients with persistent BK viruria and preserved graft function. RESULTS: In both cases, polyomavirus immunohistochemistry performed on the tumor specimens was strongly positive, and limited to the malignant tissue. BKV DNA, viral protein 1, and large T antigen mRNA were detected in the tumor; however, no viral particles were seen on electron microscopy. CONCLUSION: In one of the cases, BKV integration into the host genome was identified, leading to the truncation of the major viral capsid gene. This finding raises the concern that persisting BK viruria may be a risk factor for this aggressive form of bladder cancer. Further studies to determine screening and management strategies are required.