Objective: We designed and tested a Nanopore sequencing panel for direct tuberculosis drug resistance profiling. The panel targeted 10 resistance-associated loci. We assessed the feasibility of amplifying and sequencing these loci from 23 clinical specimens with low bacillary burden. Results: At least 8 loci were successfully amplified from the majority for predicting first- and second-line drug resistance (14/23, 60.87%), and the 12 specimens yielding all 10 targets were sequenced with Nanopore MinION and Illumina MiSeq. MinION sequencing data was corrected by Nanopolish and recurrent variants were filtered. A total of 67,082 bases across all consensus sequences were analyzed, with 67,019 bases called by both MinION and MiSeq as wildtype. For the 41 single nucleotide variants (SNVs) called by MiSeq with 100% variant allelic frequency (VAF), 39 (95.1%) were called by MinION. For the 22 mixed bases called by MiSeq, a SNV with the highest VAF (70%) was called by MinION. With short assay time, reasonable reagent cost as well as continuously improving sequencing chemistry and signal correction pipelines, this Nanopore method can be a viable option for direct tuberculosis drug resistance profiling in the near future.

Background: Diversified etiology of lower respiratory tract infection renders diagnosis challenging. The mainstay microbial culture is time-consuming and constrained by variable growth requirements. In this study, we explored the use of Nanopore sequencing as a supplementary tool to alleviate this diagnostic bottleneck. Methods: We developed a targeted Nanopore method based on amplification of bacterial 16S rRNA gene and fungal internal transcribed spacer region. The performance was compared with routine infectious disease workups on 43 respiratory specimens. Results: Nanopore successfully identified majority of microbes (47/54, 87.04%) and 7 possible pathogens not detected by routine workups, which were attributable to the content of microbiological investigations (n = 5) and negative culture (n = 2). The average sequencing time for first target reads was 7 min (1–43 min) plus 5 h of pre-sequencing preparation. Conclusions: The Nanopore method described here was rapid, economical and hypothesis-free, which might provide valuable hints to further microbiological follow-up for opportunistic pathogens missed or not detectable by conventional tests.

Background: Human papillomavirus (HPV) testing has been employed by several European countries to augment cytology-based cervical screening programs. A number of research groups have demonstrated potential utility of next-generation sequencing (NGS) for HPV genotyping, with comparable performance and broader detection spectrum than current gold standards. Nevertheless, most of these NGS platforms may not be the best choice for medium sample throughput and laboratories with less resources and space. In light of this, we developed a Nanopore sequencing assay for HPV genotyping and compared its performance with cobas HPV Test and Roche Linear Array HPV Genotyping Test (LA).Methods: Two hundred and one cervicovaginal swabs were routinely tested for Papanicolaou smear, cobas HPV Test and LA. Residual DNA was used for Nanopore protocol after routine testing. Briefly, HPV L1 region was amplified using PGMY and MGP primers, and PCR-positive specimens were sequenced on MinION flow cells (R9.4.1). Data generated in first 2 h were aligned with reference sequences from Papillomavirus Episteme database for genotyping.Results: Nanopore detected 96 HPV-positive (47.76%) and 95 HPV-negative (47.26%) specimens, with 10 lacking β-globin band and not further analyzed (4.98%). Substantial agreement was achieved with cobas HPV Test and LA (κ: 0.83–0.93). In particular, Nanopore appeared to be more sensitive than cobas HPV Test for HPV 52 (n = 7). For LA, Nanopore revealed higher concordance for high-risk (κ: 0.93) than non-high risk types (κ: 0.83), and with similar high-risk positivity in each cytology grading. Nanopore also provided better resolution for HPV 52 in 3 specimens co-infected with HPV 33 or 58, and for HPV 87 which was identified as HPV 84 by LA. Interestingly, Nanopore identified 5 additional HPV types, with an unexpected high incidence of HPV 90 (n = 12) which was reported in North America and Belgium but not in Hong Kong.Conclusions: We developed a Nanopore workflow for HPV genotyping which was economical (about USD 50.77 per patient specimen for 24-plex runs), and with comparable or better performance than 2 reference methods in the market. Future prospective study with larger sample size is warranted to further evaluate test performance and streamline the protocol.

Coronavirus disease 2019 (COVID-19) pandemic has been a catastrophic burden to global healthcare systems. The fast spread of the etiologic agent, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), highlights the need to identify unknown coronaviruses rapidly for prompt clinical and public health decision making. Moreover, owing to the high mutation rate of RNA viruses, periodic surveillance on emerging variants of key virus components is essential for evaluating the efficacy of antiviral drugs, diagnostic assays and vaccines. These 2 knowledge gaps formed the basis of this study. In the first place, we evaluated the feasibility of characterizing coronaviruses directly from respiratory specimens. We amplified partial RdRP gene, a stable genetic marker of coronaviruses, from a collection of 57 clinical specimens positive for SARS-CoV-2 or other human coronaviruses, and sequenced the amplicons with Nanopore Flongle and MinION, the fastest and the most scalable massively-parallel sequencing platforms to-date. Partial RdRP sequences were successfully amplified and sequenced from 82.46% (47/57) of specimens, ranging from 75 to 100% by virus type, with consensus accuracy of 100% compared with Sanger sequences available (n = 40). In the second part, we further compared 19 SARS-CoV-2 RdRP sequences collected from the first to third waves of COVID-19 outbreak in Hong Kong with 22,173 genomes from GISAID EpiCoV™ database. No single nucleotide variants (SNVs) were found in our sequences, and 125 SNVs were observed from global data, with 56.8% being low-frequency (n = 1–47) missense mutations affecting the rear part of RNA polymerase. Among the 9 SNVs found on 4 conserved domains, the frequency of 15438G > T was highest (n = 34) and was predominantly found in Europe. Our data provided a glimpse into the sequence diversity of a primary antiviral drug and diagnostic target. Further studies are warranted to investigate the significance of these mutations.

Coronavirus disease 2019 (COVID-19) pandemic has progressed into second year. The etiologic agent severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has evolved into numerous lineages, some of which are gifted with transmissibility advantages. This underscores the importance of variant surveillance, to keep public health countermeasures and our understanding on the virus up-to-date. In the light of this, we extracted one-year genomic data (August 2020 to July 2021) from Global Initiative on Sharing All Influenza Data (GISAID) EpiCoV™ database and estimated monthly proportions of 11 SARS-CoV-2 variants in various geographical regions. From continental perspective, delta variant of concern (VOC) predominated in Africa, Asia, Europe, North America and Oceania, with proportions ranging from 67.58 to 98.31% in July 2021. In South America, proportion of the expanding delta VOC (23.24%) has been getting closer to the predominant yet diminishing gamma VOC (56.86%). We further analyzed monthly data on new COVID-19 cases, new deaths, vaccination status and variant proportions of 6 countries. Similar to continental data, delta VOC predominated in all countries except Brazil in July 2021. In most occasions, rise and predominance of alpha, beta, gamma, delta and zeta variants were accompanied with surges of new cases, especially after the time points of major lineage interchange. The ascending phases of new cases lasted for 1 to 5 months with 1.69- to 40.63-fold peak growth, whereas new death toll varied with regional vaccination status. Our data suggested surges of COVID-19 cases might be predicted from variant surveillance data. Despite vaccine breakthroughs by delta VOC, death tolls were more stable in countries with better immunization coverage. Another takeaway is the urgent need to improve vaccine efficacy against delta and emerging variants.