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.

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.

Impact of COVID-19 Vaccination on Healthcare Worker Infection Rate and Outcome during SARS-CoV-2 Omicron Variant Outbreak in Hong Kong.

Immune escape is observed with SARS-CoV-2 Omicron (Pango lineage B.1.1.529), the predominant circulating strain worldwide. A booster dose was shown to restore immunity against Omicron infection; however, real-world data comparing mRNA (BNT162b2; Comirnaty) and inactivated vaccines' (CoronaVac; Sinovac) homologous and heterologous boosting are lacking. A retrospective study was performed to compare the rate and outcome of COVID-19 in healthcare workers (HCWs) with various vaccination regimes during a territory-wide Omicron BA.2.2 outbreak in Hong Kong. During the study period from 1 February to 31 March 2022, 3167 HCWs were recruited, and 871 HCWs reported 746 and 183 episodes of significant household and non-household close contact. A total of 737 HCWs acquired COVID-19, all cases of which were all clinically mild. Time-dependent Cox regression showed that, compared with two-dose vaccination, three-dose vaccination reduced infection risk by 31.7% and 89.3% in household contact and non-household close contact, respectively. Using two-dose BNT162b2 as reference, two-dose CoronaVac recipient had significantly higher risk of being infected (HR 1.69 p < 0.0001). Three-dose BNT162b2 (HR 0.4778 p< 0.0001) and two-dose CoronaVac + BNT162b2 booster (HR 0.4862 p = 0.0157) were associated with a lower risk of infection. Three-dose CoronaVac and two-dose BNT162b2 + CoronaVac booster were not significantly different from two-dose BNT162b2. The mean time to achieve negative RT-PCR or E gene cycle threshold 31 or above was not affected by age, number of vaccine doses taken, vaccine type, and timing of the last dose. In summary, we have demonstrated a lower risk of breakthrough SARS-CoV-2 infection in HCWs given BNT162b2 as a booster after two doses of BNT162b2 or CoronaVac.

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.

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.

Germline PALB2 Mutation in High-Risk Chinese Breast and/or Ovarian Cancer Patients.

The prevalence of the PALB2 mutation in breast cancer varies across different ethnic groups; hence, it is of intense interest to evaluate the cancer risk and clinical association of the PALB2 mutation in Chinese breast and/or ovarian cancer patients. We performed sequencing with a 6-gene test panel (BRCA1, BRCA2, TP53, PTEN, PALB2, and CDH1) to identify the prevalence of the PALB2 germline mutation among 2631 patients with breast and/or ovarian cancer. In this cohort, 39 mutations were identified with 24 types of mutation variants, where the majority of the mutations were frame-shift mutations and resulted in early termination. We also identified seven novel PALB2 mutations. Most of the PALB2 mutation carriers had breast cancer (36, 92.3%) and were more likely to have family history of breast cancer (19, 48.7%). The majority of the breast tumors were invasive ductal carcinoma (NOS type) (34, 81.0%) and hormonal positive (ER: 32, 84.2%; PR: 23, 60.5%). Pathogenic mutations of PALB2 were found in 39 probands with a mutation frequency of 1.6% and 1% in breast cancer and ovarian cancer patients, respectively. PALB2 mutation carriers were more likely have hormonal positive tumors and were likely to have familial aggregation of breast cancer.

A Case Report of Germline Compound Heterozygous Mutations in the BRCA1 Gene of an Ovarian and Breast Cancer Patient.

The germline carrier of the BRCA1 pathogenic mutation has been well proven to confer an increased risk of breast and ovarian cancer. Despite BRCA1 biallelic pathogenic mutations being extremely rare, they have been reported to be embryonically lethal or to cause Fanconi anemia (FA). Here we describe a patient who was a 48-year-old female identified with biallelic pathogenic mutations of the BRCA1 gene, with no or very subtle FA-features. She was diagnosed with ovarian cancer and breast cancer at the ages of 43 and 44 and had a strong family history of breast and gynecological cancers.

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.

Mutation screening of germline TP53 mutations in high-risk Chinese breast cancer patients.

BACKGROUND: Germline TP53 mutations are associated with Li-Fraumeni syndrome, a severe and rare hereditary cancer syndrome. Despite the rarity of germline TP53 mutations, the clinical implication for mutation carriers and their families is significant. The risk management of TP53 germline mutation carriers is more stringent than BRCA carriers, and radiotherapy should be avoided when possible. METHODS: TP53 gene mutation screening was performed in 2538 Chinese breast cancer patients who tested negative for BRCA mutations. RESULTS: Twenty TP53 mutations were identified with high next-generation sequencing concerning for germline mutations in Chinese breast cancer families. The majorities of the TP53 carriers had early-onset, hormone receptor-positive breast cancer, and had strong family history of cancer. Among all, 11 patients carried a germline mutation and 6 of which were likely de novo germline mutations. In addition, 1 case was suspected to be induced by chemotherapy or radiation, as this patient had no significant family history of cancer and aberrant clonal expansion can commonly include TP53 mutations. Furthermore, we have identified one mosaic LFS case. Two novel mutations (c.524_547dup and c.529_546del) were identified in patients with early-onset. CONCLUSIONS: In view of the high lifetime risk of malignancy, identification of patients with germline TP53 mutations are important for clinicians to aid in accurate risk assessment and offer surveillance for patients and their families.

Somatic mutation profiling in BRCA-negative breast and ovarian cancer patients by multigene panel sequencing.

Targeted therapeutic agents such as poly (ADP-ribose) polymerases (PARP) inhibitors have emerged in treating cancers associated with germline BRCA mutations. Recently studies demonstrated the effectiveness of PARP inhibitors in treating patients with somatic BRCA mutations. Somatic mutations in 122 Chinese breast or ovarian cancer patients without BRCA, PTEN and TP53 mutations were screened using multigene sequencing panel. The five most frequent pathogenic or likely pathogenic mutated genes identified in breast cancer patients were PIK3CA (28.6%), TP53 (16.9%), MAP3K1 (14.3%), GATA3 (14.3%) and PTEN (5.2%). The five most frequently mutated genes identified in ovarian patients were TP53 (52.9%), KRAS (23.5%) and PIK3CA (11.8%), BRCA1 (5.9%) and RB1 (5.9%). Somatic PIK3CA and TP53 mutations were common events in both germline BRCA-negative breast and ovarian cancer patients. In contrast, somatic screening of BRCA mutations in BRCA-negative breast cancer patients has limited value. The results highlight the benefit of somatic testing to guide future research directions on other targeted therapies for breast and ovarian malignancies.

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.

Genome Sequences of SARS-CoV-2 Strains Detected in Hong Kong.

We sequenced severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) genomes from deep throat saliva samples of three imported cases in Hong Kong by Nanopore sequencing. Epidemiological and clinical features of these coronavirus disease 2019 (COVID-19) cases were presented for genomic epidemiology studies.

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.

Sorafenib and omacetaxine mepesuccinate as a safe and effective treatment for acute myeloid leukemia carrying internal tandem duplication of Fms-like tyrosine kinase 3.

BACKGROUND: Omacetaxine mepesuccinate (OME) has antileukemic effects against acute myeloid leukemia (AML) carrying an internal tandem duplication of Fms-like tyrosine kinase 3 (FLT3-ITD). A phase 2 clinical trial was conducted to evaluate a combination treatment of sorafenib and omacetaxine mepesuccinate (SOME). METHODS: Relapsed or refractory (R/R) or newly diagnosed patients were treated with sorafenib (200-400 mg twice daily) and OME (2 mg daily) for 7 (first course) or 5 days (second course onward) every 21 days until disease progression or allogeneic hematopoietic stem cell transplantation (HSCT). The primary endpoint was composite complete remission, which was defined as complete remission (CR) plus complete remission with incomplete hematologic recovery (CRi). Secondary endpoints were leukemia-free survival (LFS) and overall survival (OS). RESULTS: Thirty-nine R/R patients and 5 newly diagnosed patients were recruited. Among the R/R patients, 28 achieved CR or CRi. Two patients showed partial remission, and 9 patients did not respond. Among the 5 newly diagnosed patients, 4 achieved CR, and 1 achieved CRi. The median LFS and OS were 5.6 and 10.9 months, respectively. Prior Fms-like tyrosine kinase 3 (FLT3) inhibitor exposure (P = .007), 2 or more inductions (P = .001), and coexisting IDH2 (P = .008) and RUNX1 mutations (P = .003) were associated with lower CR/CRi rates. HSCT consolidation and deep molecular responses (defined as an FLT3-ITD variant allelic frequency [VAF] ≤ 0.1% or a nucleophosmin 1 [NPM1] mutant VAF ≤ 0.01%) were associated with better OS and LFS. Prior FLT3 inhibitor exposure and 2 or more inductions were associated with inferior LFS. CONCLUSIONS: SOME was safe and effective for R/R and newly diagnosed FLT3-ITD AML.

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.