Clinical Evaluation of the BIOFIRE SPOTFIRE Respiratory Panel.

The BIOFIRE SPOTFIRE Respiratory (R) Panel is a novel, in vitro diagnostic PCR assay with 15 pathogen targets. The runtime is about 15 min which is the shortest among similar panels in the market. We evaluated the performance of the SPOTFIRE R Panel with 151 specimens, including 133 collected from the upper respiratory tract (URT), 13 from the lower respiratory tract (LRT) and 5 external quality assessment program (EQAP) samples. The respiratory specimens were enrolled throughout the first two post-COVID-19 influenza seasons in Hong Kong (March to December 2023). For URT specimens, full concordance was observed between the SPOTFIRE R Panel and the standard-of-care FilmArray Respiratory 2.1 plus Panel (RP2.1plus) for 109 specimens (109/133, 81.95%). After discrepant analysis, the SPOTFIRE R Panel identified more pathogens than the RP2.1plus in 15 specimens and vice versa in 3 specimens. The per-target negative and positive percentage agreement (NPA and PPA) were 92.86-100% except the PPA of adenovirus (88.24%). For LRT and EQAP samples, all results were fully concordant. To conclude, the performance of the SPOTFIRE R Panel was comparable to the RP2.1plus.

Clinical Evaluation of Xpert Xpress CoV-2/Flu/RSV plus and Alinity m Resp-4-Plex Assay.

The performance of the Xpert Xpress CoV-2/Flu/RSV plus and Alinity m Resp-4-Plex Assays were evaluated using 167 specimens, including 158 human respiratory specimens and 9 external quality assessment program (EQAP) samples. For respiratory specimens, CoV-2/Flu/RSV plus exhibited perfect agreement with the standard-of-care (SOC) methods (Cohen's κ: 1, 100% agreement). The overall positive and negative percent agreement (PPA and NPA) were 100%, with 95% confidence intervals of 96.50 to 100% and 85.70 to 100%, respectively. On the other hand, Resp-4-Plex revealed an almost perfect agreement with the SOC methods (Cohen's κ: 0.92, 97.71% agreement). The overall PPA and NPA were 100% (95.76 to 100%) and 88.46% (70.20 to 96.82%), respectively. For EQAP samples, the results of CoV-2/Flu/RSV plus (9/9) and Resp-4-Plex (4/4) were concordant with the expected results. The experimental limit of detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was the lowest (25 copies/mL for both methods), and that of the respiratory syncytial virus was the highest (400 copies/mL for CoV-2/Flu/RSV plus and 100 copies/mL for Resp-4-Plex). Threshold cycle (Ct) value correlation showed a large positive linear association between CoV-2/Flu/RSV plus and Resp-4-Plex, with R-squared values of 0.92-0.97, and on average, the Ct values of CoV-2/Flu/RSV plus were higher than that of Resp-4-Plex by 1.86-2.78, except for Flu A1 target (-0.66). To conclude, the performance of both assay was comparable to the SOC methods for both upper and lower respiratory specimens. Implementation of these rapid assay may reinforce the diagnostic capacity for the post-pandemic co-circulation of SARS-CoV-2 and other respiratory viruses.

Evaluation of a Commercial Point-of-Care RT-LAMP Assay for Rapid Detection of SARS-CoV-2.

The goal of this study was to evaluate the performance of a commercial reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay (Detect COVID-19 Test) in the detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). A total of 202 human respiratory and viral culture specimens were tested retrospectively. The performance of the Detect COVID-19 Test was comparable to that of commercial real-time polymerase chain reaction assays (sensitivity: 93.42%; specificity: 100%), and better than that of the rapid antigen test (sensitivity: 48.00%; specificity: 100%) for specimens with threshold cycle (Ct) values of less than 30. The Beta, Delta, and Omicron variants of concern were successfully detected. With their simplicity of use and good assay sensitivity, point-of-care RT-LAMP assays may be a viable option for SARS-CoV-2 testing at home, or in regions without sophisticated laboratory facilities.

The Seasonality of Respiratory Viruses in a Hong Kong Hospital, 2014-2023.

We reviewed the multiplex PCR results of 20,127 respiratory specimens tested in a hospital setting from January 2014 to April 2023. The seasonal oscillation patterns of 17 respiratory viruses were studied. Compared with 2014-2019, a prominent drop in PCR positivity (from 64.46-69.21% to 17.29-29.89%, p < 0.001) and virus diversity was observed during the COVID-19 pandemic, with predominance of rhinovirus/enterovirus, sporadic spikes of parainfluenza viruses 3 and 4, respiratory syncytial virus and SARS-CoV-2, and rare detection of influenza viruses, metapneumovirus, adenovirus and coronaviruses. The suppressed viruses appeared to regain activity from the fourth quarter of 2022 when pandemic interventions had been gradually relaxed in Hong Kong. With the co-circulation of SARS-CoV-2 and seasonal respiratory viruses, surveillance of their activity and an in-depth understanding of the clinical outcomes will provide valuable insights for improved public health measures and reducing disease burden.

Consensus Statements on Precision Oncology in the China Greater Bay Area.

BACKGROUND: Next-generation sequencing comprehensive genomic panels (NGS CGPs) have enabled the delivery of tailor-made therapeutic approaches to improve survival outcomes in patients with cancer. Within the China Greater Bay Area (GBA), territorial differences in clinical practices and health care systems and strengthening collaboration warrant a regional consensus to consolidate the development and integration of precision oncology (PO). Therefore, the Precision Oncology Working Group (POWG) formulated standardized principles for the clinical application of molecular profiling, interpretation of genomic alterations, and alignment of actionable mutations with sequence-directed therapy to deliver clinical services of excellence and evidence-based care to patients with cancer in the China GBA. METHODS: Thirty experts used a modified Delphi method. The evidence extracted to support the statements was graded according to the GRADE system and reported according to the Revised Standards for Quality Improvement Reporting Excellence guidelines, version 2.0. RESULTS: The POWG reached consensus in six key statements: harmonization of reporting and quality assurance of NGS; molecular tumor board and clinical decision support systems for PO; education and training; research and real-world data collection, patient engagement, regulations, and financial reimbursement of PO treatment strategies; and clinical recommendations and implementation of PO in clinical practice. CONCLUSION: POWG consensus statements standardize the clinical application of NGS CGPs, streamline the interpretation of clinically significant genomic alterations, and align actionable mutations with sequence-directed therapies. The POWG consensus statements may harmonize the utility and delivery of PO in China's GBA.

Molecular characteristics of Asian male BRCA-related cancers.

PURPOSE: Germline mutations of BRCA1 or BRCA2 predispose men to develop various cancers, including breast cancers and prostate cancers. Male breast cancer (MBC) is a rare disease while prostate cancer (PRC) is uncommon in young men at the age of less than 40. The prevalence of BRCA genes in Asian male patients has to be elevated. METHODS: Germline mutations screening was performed in 98 high-risk Chinese MBC and PRC patients. RESULT: We have identified 16 pathogenic BRCA2 mutation carriers, 12 were MBC patients, 2 were PRC patients and 2 were patients with both MBC and PRC. The mutation percentages were 18.8%, 6.7% and 50% for MBC, PRC and both MBC and PRC patients, respectively. BRCA2 gene mutations confer a significantly higher risk of breast/prostate cancers in men than those with BRCA1 mutations. BRCA mutated MBC patients had a younger age of diagnosis and strong family histories of breast cancers while BRCA mutated PRC patients had strong family histories of ovarian cancers. CONCLUSION: Male BRCA carriers with breast cancers or prostate cancers showed distinct clinical and molecular characteristics, a male-specific genetic screening model would be useful to identify male cancer patients who have a high risk of BRCA mutation.

Genomic characteristics and viral load dynamics of a SARS-CoV-2 Omicron BA.2.2 variant from a hospitalized patient treated with molnupiravir.

We sequenced severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) genomes from nasal and throat swabs of a hospitalized patient during the fifth wave of coronavirus disease 2019 (COVID-19) pandemic in Hong Kong. Genomic characteristics and viral load dynamics of an Omicron BA.2.2 variant before and after molnupiravir treatment were presented.

NGS4THAL, a One-Stop Molecular Diagnosis and Carrier Screening Tool for Thalassemia and Other Hemoglobinopathies by Next-Generation Sequencing.

Thalassemia is one of the most common genetic diseases and a major health threat worldwide. Accurate, efficient, and scalable analysis of next-generation sequencing (NGS) data is much needed for its molecular diagnosis and carrier screening. We developed NGS4THAL, a bioinformatics analysis pipeline analyzing NGS data to detect pathogenic variants for thalassemia and other hemoglobinopathies. NGS4THAL realigns ambiguously mapped NGS reads derived from the homologous Hb gene clusters for accurate detection of point mutations and small insertions/deletions. It uses a combination of complementary structural variant (SV) detection tools and an in-house database of control data containing specific SVs to achieve accurate detection of the complex SV types. Detected variants are matched with those in HbVar (A Database of Human Hemoglobin Variants and Thalassemia Mutations), allowing recognition of known pathogenic variants, including disease modifiers. Tested on simulation data, NGS4THAL achieved high sensitivity and specificity. For targeted NGS sequencing data from samples with laboratory-confirmed pathogenic Hb variants, it achieved 100% detection accuracy. Application of NGS4THAL on whole genome sequencing data from unrelated studies revealed thalassemia mutation carrier rates for Hong Kong Chinese and Northern Vietnamese that were consistent with previous reports. NGS4THAL is a highly accurate and efficient molecular diagnosis tool for thalassemia and other hemoglobinopathies based on tailored analysis of NGS data and may be scaled for population carrier screening.

How does re-classification of variants of unknown significance (VUS) impact the management of patients at risk for hereditary breast cancer?

BACKGROUND: The popularity of multigene testing increases the probability of identifying variants of uncertain significance (VUS). While accurate variant interpretation enables clinicians to be better informed of the genetic risk of their patients, currently, there is a lack of consensus management guidelines for clinicians on VUS. METHODS: Among the BRCA1 and BRCA2 mutations screening in 3,544 subjects, 236 unique variants (BRCA1: 86; BRCA2: 150) identified in 459 patients were being reviewed. These variants consist of 231 VUS and 5 likely benign variants at the initial classification. RESULTS: The variants in 31.8% (146/459) patients were reclassified during the review, which involved 26 unique variants (11.0%). Also, 31 probands (6.8%) and their family members were offered high-risk surveillance and related management after these variants were reclassified to pathogenic or likely pathogenic. At the same time, 69 probands (15%) had their VUS downgraded to cancer risk equivalent to the general population level. CONCLUSION: A review of archival variants from BRCA1 and BRCA2 genetic testing changed the management for 31.8% of the families due to increased or reduced risk. We encourage regular updates of variant databases, reference to normal population and collaboration between research laboratories on functional studies to define the clinical significances of VUS better.

Germline mutations in Chinese ovarian cancer with or without breast cancer.

BACKGROUND: Ovarian and breast cancers are known to have significant genetic components. Considering the differences in the mutation spectrum across ethnicity, it is important to identify hereditary breast and ovarian cancer (HBOC) genes mutation in Chinese for clinical management. METHODS: Two cohorts of 451 patients with ovarian cancer only (OV) and 93 patients with both breast and ovarian (BROV) cancers were initially screened for BRCA1, BRCA2, TP53, and PTEN. 109 OV and 43 BROV patients with extensive clinical risk and were being tested negative, were then further characterized by 30-gene panel analysis. RESULTS: Pathogenic BRCA1/2 variants were identified in 45 OV patients and 33 BROV patients, giving a prevalence of 10% and 35.5%, respectively. After the extended screening, mutations in other HBOC genes were identified in an additional 12.8% (14/109) of the OV cohort and 14% (6/43) in the BROV cohort. The most commonly mutated genes in the OV cohort were MSH2 (4.6%) while in the BROV cohort were MSH2 (4.7%) and PALB2 (4.7%). With this extended multigene testing strategy, pathogenic mutations were detected in 12.8% of OV patients (BRCAs: 10%; additional genes: 12.8%) and 40.9% (BRCAs: 35.5%; additional genes: 14%) of BROV patients. CONCLUSION: Extended characterization of the contributions of HBOC genes to OV and BROV patients has significant impacts on further management in patients and their families, expanding the screening net for more asymptomatic individuals.

Safety and Immunogenicity of a Booster Vaccination by CoronaVac or BNT162b2 in Previously Two-Dose Inactivated Virus Vaccinated Individuals with Negative Neutralizing Antibody.

COVID-19 has swept across the globe since 2019 and repeated waves of infection have been caused by different variants of the original SARS-CoV-2 (wild type), with the Omicron and Delta variants having dominated recently. Vaccination is among the most important measures in the absence of widespread use of antivirals for prevention of morbidity and mortality. Inactivated virus vaccine has been abundantly used in many countries as the primary two-dose regimen. We aim to study the safety and immunogenicity of CoronaVac (three-dose inactivated virus vaccine) and the BNT162b2 (two-dose inactivated virus vaccine followed by an mRNA vaccine) booster. Both CoronaVac and BNT162b2 boosters are generally safe and have good immunogenicity against the wild type SARS-CoV-2 and the Delta variant with the majority having neutralizing antibodies (NAb) on day 30 and day 90. However, the BNT162b2 booster is associated with a much higher proportion of positive NAb against the Omicron variant. Only 8% of day 30 and day 90 samples post CoronaVac booster have NAb against the Omicron variant. In addition, more BNT162b2 booster recipients are having positive T-cell responses using interferon gamma release assay. In places using inactivated virus vaccine as the primary two-dose scheme, the heterologous mRNA vaccine booster is safe and more immunogenic against the Omicron variant and should be considered as a preferred option during the current outbreak.

Geographical prevalence of SARS-CoV-2 variants, August 2020 to July 2021.

We extracted one-year genomic data (August 2020-July 2021) from GISAID EpiCoV™ database and estimated monthly proportions of 11 SARS-CoV-2 variants in various geographical regions. From continental perspective, Delta VOC predominated in Africa, Asia, Europe, North America and Oceania, with proportions of 67.58-98.31% in July 2021. In South America, proportion of Delta VOC (23.24%) has been approaching 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. Delta VOC predominated in all countries except Brazil (Gamma VOC) 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 point of major lineage interchange. The ascending phases of new cases lasted for 1-5 months with 1.69- to 40.63-fold peak growth, whereas new death tolls 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.

Evaluation of the INDICAID COVID-19 Rapid Antigen Test in Symptomatic Populations and Asymptomatic Community Testing.

As the COVID-19 pandemic progresses, there is an increasing need for rapid, accessible assays for SARS-CoV-2 detection. We present a clinical evaluation and real-world implementation of the INDICAID COVID-19 rapid antigen test (INDICAID rapid test). A multisite clinical evaluation of the INDICAID rapid test using prospectively collected nasal (bilateral anterior) swab samples from symptomatic subjects was performed. The INDICAID rapid test demonstrated a positive percent agreement (PPA) and negative percent agreement (NPA) of 85.3% (95% confidence interval [95% CI], 75.6% to 91.6%) and 94.9% (95% CI, 91.6% to 96.9%), respectively, compared to laboratory-based reverse transcriptase PCR (RT-PCR) using nasal specimens. The INDICAID rapid test was then implemented at COVID-19 outbreak screening centers in Hong Kong as part of a testing algorithm (termed "dual-track") to screen asymptomatic individuals for prioritization for confirmatory RT-PCR testing. In one approach, preliminary positive INDICAID rapid test results triggered expedited processing for laboratory-based RT-PCR, reducing the average time to confirmatory result from 10.85 h to 7.0 h. In a second approach, preliminary positive results triggered subsequent testing with an onsite rapid RT-PCR, reducing the average time to confirmatory result to 0.84 h. In 22,994 asymptomatic patients, the INDICAID rapid test demonstrated a PPA of 84.2% (95% CI, 69.6% to 92.6%) and an NPA of 99.9% (95% CI, 99.9% to 100%) compared to laboratory-based RT-PCR using combined nasal/oropharyngeal specimens. The INDICAID rapid test has excellent performance compared to laboratory-based RT-PCR testing and, when used in tandem with RT-PCR, reduces the time to confirmatory positive result. IMPORTANCE Laboratory-based RT-PCR, the current gold standard for COVID-19 testing, can require a turnaround time of 24 to 48 h from sample collection to result. The delayed time to result limits the effectiveness of centralized RT-PCR testing to reduce transmission and stem potential outbreaks. To address this, we conducted a thorough evaluation of the INDICAID COVID-19 rapid antigen test, a 20-minute rapid antigen test, in both symptomatic and asymptomatic populations. The INDICAID rapid test demonstrated high sensitivity and specificity with RT-PCR as the comparator method. A dual-track testing algorithm was also evaluated utilizing the INDICAID rapid test to screen for preliminary positive patients, whose samples were then prioritized for RT-PCR testing. The dual-track method demonstrated significant improvements in expediting the reporting of positive RT-PCR test results compared to standard RT-PCR testing without prioritization, offering an improved strategy for community testing and controlling SARS-CoV-2 outbreaks.

Evaluation on the use of Nanopore sequencing for direct characterization of coronaviruses from respiratory specimens, and a study on emerging missense mutations in partial RdRP gene of SARS-CoV-2.

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.

Rapid and economical drug resistance profiling with Nanopore MinION for clinical specimens with low bacillary burden of Mycobacterium tuberculosis.

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.

An economical Nanopore sequencing assay for human papillomavirus (HPV) genotyping.

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 ß-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.

Potential utility of targeted Nanopore sequencing for improving etiologic diagnosis of bacterial and fungal respiratory infection.

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.

Potential utility of metagenomic sequencing for improving etiologic diagnosis of infective endocarditis.

Aim: To explore potential utility of metagenomic sequencing for improving etiologic diagnosis of infective endocarditis (IE) caused by fastidious bacteria. Materials & methods: Plasma and heart valves of two patients, who were diagnosed with IE caused by Bartonella quintana and Propionibacterium species, were sequenced by using Illumina MiSeq and Nanopore MinION. Results: For patient 1, B. quintana was detected in the plasma pool collected 4 days before valvular replacement surgery. For patient 2, Propionibacterium sp. oral taxon 193 was detected in the plasma sample collected on hospital day 1. Nearly complete bacterial genomes (>98%) were retrieved from resected heart valves of both patients, enabling detection of antibiotic resistance-associated features. Real-time sequencing of heart valves identified both pathogens within the first 16 min of sequencing runs. Conclusion: Metagenomic sequencing may be a helpful supplement to IE diagnostic workflow, especially when conventional tests fail to yield a diagnosis.

Draft Genome Sequence of Helicobacter cinaedi, Compiled by Direct Whole-Genome Sequencing of a Blood Culture-Positive Isolate in Hong Kong.

Isolation of Helicobacter cinaedi from a positive blood culture requires prolonged and stringent subculture conditions. Direct whole-genome sequencing (WGS) of a positive blood culture may provide timely treatment-associated genetic information. Here, we report a draft genome sequence of H. cinaedi compiled by direct WGS, which was 1,995,911 bp in length with 39.1% GC content.

Frequent functional activation of RAS signalling not explained by RAS/RAF mutations in relapsed/refractory multiple myeloma.

RAS mutations are frequent in relapsed/refractory multiple myeloma (RRMM) but functional study in primary samples is scanty. Herein, in primary myeloma plasma cells of 17 suspected RRMM, functional activation of RAS signalling was studied by Western blot of phosphorylated ERK1/2 (phospho-ERK1/2). Moreover, activating mutations in KRAS, NRAS, BRAF, and ALK were studied by PCR and bidirectional direct sequencing. Furthermore, methylation of negative RAS signalling regulator genes, RASSF1A and RASD1, were analyzed by methylation-specific PCR. As evidenced by phospho-ERK1/2 over-expression, functional RAS activation was detected in 12 (75.0%) RRMM. Of patients with functional RAS activation, sequencing data showed only seven (58.3%) patients with one each had NRAS Q61H, NRAS Q61K, KRAS G12D, KRAS G12V, KRAS G13D, KRAS Q61P, or BRAF V600E mutation, whereas five (41.7%) patients had no RAS/RAF mutation. Conversely, patients without functional RAS activation had no RAS/RAF mutation. Moreover, none of the patients with functional RAS activation had ALK mutations, or methylation of RASSF1A and RASD1. Collectively, functional activation of RAS signalling was present in majority of RRMM but only about half (58.3%) accountable by RAS/RAF mutations. If verified in larger studies, clinical investigations of MEK inhibitors are warranted regardless of RAS/RAF mutations.