19Laboratories are currently witnessing extraordinary demand globally for sampling devices, 20 reagents, consumables, and diagnostic instruments needed for timely diagnosis of SARS-CoV-2 21 infection. To meet diagnostic needs as the pandemic grows, the US Food and Drug 22 Administration (FDA) recently granted several commercial SARS-CoV-2 tests Emergency Use 23 Authorization (EUA), but manufacturer-independent evaluation data are scarce. We performed 24 the first manufacturer-independent evaluation of the fully automated sample-to-result two-25 target test cobas 6800 SARS-CoV-2 (cobas) (Roche Molecular Systems, Branchburg, NJ), which 26 received US FDA EUA on March 12, 2020. The comparator was a standardized 3-hour SARS-CoV-27 2 protocol, consisting of RNA extraction using an automated portable instrument, followed by a 28 two-target RT-PCR, which our laboratory has routinely used since January 2020 (Corman VM et 29 al. EuroSurveill 25(3):2000045). Cobas and the comparator showed overall agreement of 98.1% 30 and a kappa value of 0.95 on an in-house validation panel consisting of 217 well-characterized 31 retrospective samples. Immediate prospective head-to-head comparative evaluation followed 32 on 502 samples, and the diagnostic approaches showed overall percent agreement of 99.6% 33 and a kappa value of 0.98. A good correlation (r² = 0.96) between cycle threshold values for 34 SARS-CoV-2 specific targets obtained by cobas and the comparator was observed. Our results 35 showed that cobas is a reliable assay for qualitative detection of SARS-CoV-2 in nasopharyngeal 36 swab samples collected in the UTM-RT system. Under the extraordinary circumstances that 37 laboratories are facing worldwide, a safe diagnostic platform switch is feasible in only 48 hours 38 and in the midst of the COVID-19 pandemic if carefully planned and executed. 39 on June 9, 2020 by guest http://jcm.asm.org/ Downloaded from 3 40 Keywords: SARS-CoV-2, COVID-19, cobas, cobas 6800 41 on June 9, 2020 by guest http://jcm.asm.org/ Downloaded from 58 12), followed by the launch of a range of commercial SARS-CoV-2 PCR-based assays in the last 3 59 months. Despite the fact that the US Food and Drug Administration (FDA) granted several 60 commercial SARS-CoV-2 amplification assays Emergency Use Authorization (EUA), as of March 61 29, 2020 no manufacturer-independent evaluation data for any commercial SARS-CoV-2 assay 62 with US FDA EUA is available in peer-reviewed literature. 63 on June 9, 2020 by guest http://jcm.asm.org/ Downloaded from 5 Here we present the results of the first manufacturer-independent evaluation of the fully 64 automated sample-to-result two-target test cobas 6800 SARS-CoV-2 (cobas) (Roche Molecular 65 Systems, Branchburg, NJ, USA), which received US FDA EUA on March 12, 2020. The 66 performance of cobas was first evaluated on a well-characterized in-house validation panel 67 consisting of 217 samples. The comparator was a standardized 3-hour SARS-CoV-2 detection 68 protocol, consisting of RNA extraction using an automated por...
e Mammalian orthoreoviruses (MRVs) are known to cause mild enteric and respiratory infections in humans. They are widespread and infect a broad spectrum of mammals. We report here the first case of an MRV detected in a child with acute gastroenteritis, which showed the highest similarity to an MRV reported recently in European bats. An examination of a stool sample from the child was negative for most common viral and bacterial pathogens. Reovirus particles were identified by electron microscopic examination of both the stool suspension and cell culture supernatant. The whole-genome sequence was obtained with the Ion Torrent next-generation sequencing platform. Prior to sequencing, the stool sample suspension and cell culture supernatant were pretreated with nucleases and/or the convective interaction medium (CIM) monolithic chromatographic method to purify and concentrate the target viral nucleic acid. Whole-genome sequence analysis revealed that the Slovenian SI-MRV01 isolate was most similar to an MRV found in a bat in Germany. High similarity was shared in all genome segments, with nucleotide and amino acid identities between 93.8 to 99.0% and 98.4 to 99.7%, respectively. It was shown that CIM monolithic chromatography alone is an efficient method for enriching the sample in viral particles before nucleic acid isolation and next-generation sequencing application.
Human papillomavirus type 6 (HPV6) is the major etiological agent of anogenital warts and laryngeal papillomas and has been included in both the quadrivalent and nonavalent prophylactic HPV vaccines. This study investigated the global genomic diversity of HPV6, using 724 isolates and 190 complete genomes from six continents, and the association of HPV6 genomic variants with geographical location, anatomical site of infection/disease, and gender. Initially, a 2,800-bp E5a-E5b-L1-LCR fragment was sequenced from 492/530 (92.8%) HPV6-positive samples collected for this study. Among them, 130 exhibited at least one single nucleotide polymorphism (SNP), indel, or amino acid change in the E5a-E5b-L1-LCR fragment and were sequenced in full. A global alignment and maximum likelihood tree of 190 complete HPV6 genomes (130 fully sequenced in this study and 60 obtained from sequence repositories) revealed two variant lineages, A and B, and five B sublineages: B1, B2, B3, B4, and B5. HPV6 (sub)lineage-specific SNPs and a 960-bp representative region for whole-genome-based phylogenetic clustering within the L2 open reading frame were identified. Multivariate logistic regression analysis revealed that lineage B predominated globally. Sublineage B3 was more common in Africa and North and South America, and lineage A was more common in Asia. Sublineages B1 and B3 were associated with anogenital infections, indicating a potential lesion-specific predilection of some HPV6 sublineages. Females had higher odds for infection with sublineage B3 than males. In conclusion, a global HPV6 phylogenetic analysis revealed the existence of two variant lineages and five sublineages, showing some degree of ethnogeographic, gender, and/or disease predilection in their distribution. IMPORTANCEThis study established the largest database of globally circulating HPV6 genomic variants and contributed a total of 130 new, complete HPV6 genome sequences to available sequence repositories. Two HPV6 variant lineages and five sublineages were identified and showed some degree of association with geographical location, anatomical site of infection/disease, and/or gender. We additionally identified several HPV6 lineage-and sublineage-specific SNPs to facilitate the identification of HPV6 variants and determined a representative region within the L2 gene that is suitable for HPV6 whole-genome-based phylogenetic analysis. This study complements and significantly expands the current knowledge of HPV6 genetic diversity and forms a comprehensive basis for future epidemiological, evolutionary, functional, pathogenicity, vaccination, and molecular assay development studies.
Human papillomavirus 11 (HPV11) is an etiological agent of anogenital warts and laryngeal papillomas and is included in the 4-valent and 9-valent prophylactic HPV vaccines. We established the largest collection of globally circulating HPV11 isolates to date and examined the genomic diversity of 433 isolates and 78 complete genomes (CGs) from six continents. The genomic variation within the 2,800-bp E5a-E5b-L1-upstream regulatory region was initially studied in 181/207 (87.4%) HPV11 isolates collected for this study. Of these, the CGs of 30 HPV11 variants containing unique single nucleotide polymorphisms (SNPs), indels (insertions or deletions), or amino acid changes were fully sequenced. A maximum likelihood tree based on the global alignment of 78 HPV11 CGs (30 CGs from our study and 48 CGs from GenBank) revealed two HPV11 lineages (lineages A and B) and four sublineages (sublineages A1, A2, A3, and A4). HPV11 (sub)lineage-specific SNPs within the CG were identified, as well as the 208-bp representative region for CG-based phylogenetic clustering within the partial E2 open reading frame and noncoding region 2. Globally, sublineage A2 was the most prevalent, followed by sublineages A1, A3, and A4 and lineage B. IMPORTANCEThis collaborative international study defined the global heterogeneity of HPV11 and established the largest collection of globally circulating HPV11 genomic variants to date. Thirty novel complete HPV11 genomes were determined and submitted to the available sequence repositories. Global phylogenetic analysis revealed two HPV11 variant lineages and four sublineages. The HPV11 (sub)lineage-specific SNPs and the representative region identified within the partial genomic region E2/noncoding region 2 (NCR2) will enable the simpler identification and comparison of HPV11 variants worldwide. This study provides an important knowledge base for HPV11 for future studies in HPV epidemiology, evolution, pathogenicity, prevention, and molecular assay development. Human papillomavirus 11 (HPV11), which belongs to species 10 of the Alphapapillomavirus genus (Alpha-PV), is etiologically associated with approximately 20% of anogenital warts and 30 to 40% of laryngeal papillomas (1-11). HPV11 is generally considered a low-risk HPV type due to its rare presence in HPVrelated cancers in humans, especially cervical cancer (9). HPV11 is present in 0.5% of samples from HPV-positive women with a normal cytology worldwide and causes 2.3% of cervical low-grade squamous cell intraepithelial lesions (12, 13). However, rare case reports of HPV11-positive cases of cervical and anal squamous cell carcinomas, malignantly transformed laryngeal papillomas, and sinonasal inverted papillomas associated with squamous cell carcinoma can be found in the literature (14-21). Due to its clinical significance, HPV11 has been included in the current quadrivalent and nonavalent prophylactic HPV vaccines (22,23).The genetic diversity of HPV11 was studied for the first time in 1995, when Heinzel et al. sequenced the noncoding upstre...
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