SARS-CoV-2 Seroprevalence Trends in the Netherlands in the Variant of Concern Era: Input for Future Response.

BACKGROUND: To inform future response planning we aimed to assess SARS-CoV-2 trends in infection- and/or vaccine-induced immunity, including breakthrough infections, among (sub)groups, professions and regions in the Dutch population during the Variant of Concern (VOC)-era. METHODS: In this prospective population-based cohort, randomly selected participants (n = 9985) aged 1-92 years (recruited early-2020) donated home-collected fingerstick-blood samples at six timepoints in 2021/2022, covering waves dominated by Alpha, Delta, and multiple Omicron (sub-)variants. IgG antibody assessment against Spike-S1 and Nucleoprotein was combined with vaccination- and testing data to estimate infection-induced (inf) and total (infection- and vaccination-induced) seroprevalence. RESULTS: Nationwide inf-seroprevalence rose modestly from 12% (95% CI 11-13) since Alpha to 26% (95% CI 24-28) amidst Delta, while total seroprevalence increased rapidly to 87% (95% CI 85-88), particularly in elderly and those with comorbidities (i.e., vulnerable groups). Interestingly, highest infection rates were noticeable among low/middle educated elderly, non-Western, those in contact professions, adolescents and young adults, and in low-vaccination coverage regions. Following Omicron emergence, inf-seroprevalence elevated sharply to 62% (95% CI 59-65) and further to 86% (95% CI 83-90) in late-2022, with frequent breakthrough infections and decreasing seroprevalence dissimilarities between most groups. Whereas > 90% of < 60-year-olds had been infected at least once, 30% of vaccinated vulnerable individuals had still not acquired hybrid immunity. CONCLUSIONS: Groups identified to have been infected disproportionally during the acute phase of the pandemic require specific attention in evaluation of control measures and future response planning worldwide. Furthermore, ongoing tailored vaccination efforts and (sero-)monitoring of vulnerable groups may remain important.

Patient Characteristics Associated with Receiving Gender-Affirming Hormone Therapy in the Veterans Health Administration.

Purpose: This study aimed to examine patient characteristics associated with receipt of gender-affirming hormone therapy in the Veterans Health Administration (VHA). Methods: This cross-sectional study included a national cohort of 9555 transgender and gender diverse (TGD) patients with TGD-related diagnosis codes who received care in the VHA from 2006 to 2018. Logistic regression models were used to determine the association of health conditions and documented social stressors with receipt of gender affirming hormone therapy. Results: Of the 9555 TGD patients, 57.4% received gender-affirming hormone therapy in the VHA. In fully adjusted models, patients who had following characteristics were less likely to obtain gender-affirming hormones in the VHA: Black, non-Hispanic versus white (adjusted odds ratio [aOR]: 0.61; 95% confidence interval [CI]: 0.52-0.72), living in the Northeast versus the West (aOR: 0.72; 95% CI: 0.62-0.84), a documented drug use disorder (aOR: 0.56; 95% CI: 0.47-0.68), ≥3 versus no comorbidities (aOR: 0.44; 95% CI: 0.34-0.57), and ≥3 versus no social stressors (aOR: 0.42; 95% CI: 0.30-0.58; all p<0.001). Younger patients aged 21-29 years were almost 3 times more likely to receive gender affirming hormone therapy in the VHA than those aged ≥60 (aOR: 2.98; 95% CI: 2.55-3.47; p<0.001). Conclusion: TGD individuals who were older, Black, non-Hispanic, and had more comorbidities and documented social stressors were less likely to receive gender-affirming hormone therapy in the VHA. Further understanding of patient preferences in addition to clinician- and site-level determinants that may impact access to gender-affirming hormone therapy for TGD individuals in the VHA is needed.

Protective mucosal SARS-CoV-2 antibodies in the majority of the general population in the Netherlands.

Antibodies to SARS-CoV-2 on the mucosal surfaces of the respiratory tract are understood to contribute to protection against SARS-CoV-2 infection. We aimed to describe the prevalence, levels, and functionality of mucosal antibodies in the general Dutch population. Nasal samples were collected from 778 randomly selected participants, 1-90 years of age, nested within the nationwide prospective SARS-CoV-2 PIENTER corona serosurvey in the Netherlands. Spike-specific immunoglobulin (Ig)G was detected in the nasal samples of 94.6% (in case of the wild-type S1 variant) and 94.9% (Omicron BA.1) of the individuals, whereas 44.2% and 62.7% of the individuals were positive for wild-type and Omicron BA.1 S1 IgA, respectively. The lowest prevalence of mucosal antibodies was observed in children under 12 years of age. The prevalence and levels of IgA and IgG were higher in individuals with a history of SARS-CoV-2 infection. Mucosal antibodies inhibited the binding of Wuhan, Delta, and Omicron BA.1 receptor binding domain to human angiotensin-converting enzyme 2 in 94.4%, 95.4%, and 92.6% of the participants, respectively. Higher levels of mucosal antibodies were associated with a lower risk of future infection.

The Use of Phentermine for Obesity in Psychiatric Patients With Antipsychotics.

OBJECTIVE: Phentermine is a commonly used weight-loss agent in the United States, but there is a little information about the use of phentermine for patients with obesity taking antipsychotic medications. METHODS: We gathered 57 patients with obesity taking antipsychotic medications whose phentermine treatment was simultaneous with or after any type of antipsychotic exposure and collected data of clinical information, initial/follow-up anthropometric variables, and adverse events (AEs) for the 6-month study period. RESULTS: In total, the mean body weight reduction (BWR) was 4.45 (7.04) kg, and the mean BWR percent (BWR%) was 3.92% (6.96%) at 6 months. Based on the response to phentermine, the patients were classified into two groups: the responder (n=25; BWR% ≥5%) and nonresponder (n=32; BWR% <5%) groups. The responder group's mean BWR and BWR% were 10.13 (4.43) kg and 9.35% (4.09%), respectively, at 6 months. The responders had higher rates of anticonvulsant combination therapy (ACT; responder, 72.0% vs. non-responder, 43.8%; p=0.033) and a shorter total antipsychotic exposure duration (responder, 23.9 [16.9] months vs. non-responder, 37.2 [27.6] months; p= 0.039). After adjusting age, sex, and initial body weight, ACT maintained a significant association with phentermine response (odds ratio=3.840; 95% confidence interval: 1.082-13.630; p=0.037). In the final cohort, there was no report of adverse or new-onset psychotic symptoms, and the common AEs were sleep disturbances, dry mouth, and dizziness. CONCLUSION: Overall, phentermine was effective and tolerable for patients with obesity taking antipsychotic medications, and ACT (predominantly topiramate) augmented the weight-loss effect of phentermine.

Outcomes and predictors of survival for tracheal cancer.

INTRODUCTION: Primary tracheal cancer is uncommon, with poor survival. While surgical resection is the mainstay of therapy, the role of chemotherapy and radiation is poorly defined. We aimed to study the impact of treatment modalities on survival. METHODS: Patients with primary tracheal cancer were identified from the National Cancer Database over 12 years, 2004-2015. Patient characteristics, tumor characteristics, treatment modalities, and survival were recorded. Factors associated with survival were assessed using Cox Regression. RESULTS: Of the 1726 patients identified, 59% were male, 83% White race, 62% had a comorbidity index of zero, median age 64 years, median tumor size 2.7 cm, and median survival was 28.5 months (89 months for patients undergoing surgical resection). Cox Regression for all patients found adenoid cystic carcinoma (ACC) (p < 0.001), radiation (p < 0.001), and surgical resection (p < 0.001) to be associated with improved survival, while increasing age (p < 0.001) decreased survival. For patients receiving resection, ACC (p < 0.001) was associated with improved survival, while increasing age (p < 0.001) and positive margins (p = 0.002) were associated with worse survival. For R0 resections, ACC (p < 0.001) was associated with improved survival, while increasing age (p < 0.001) decreased survival, with chemotherapy and radiation having no impact. For R1/2 resections, ACC (p < 0.001) and radiation (p < 0.001) were associated with improved survival, while increasing age (p < 0.001) decreased survival, with chemotherapy having no impact on survival. CONCLUSIONS: Primary tracheal cancer is highly lethal, with surgical resection leading to the best chance of survival. For patients undergoing resection, radiation provided survival benefits for R1/2 but not R0, while chemotherapy did not impact survival regardless of margin status.

Evaluating the Compliance, Efficacy, and Cost of Formulated Vitamins Versus Separate Supplements in Post-bariatric Surgery Patients.

OBJECTIVE: Micronutrient deficiencies are common complications after bariatric surgery as alterations to the gastrointestinal tract change absorption. Patients are recommended to take supplements including multivitamins, B complex, calcium, vitamin D, and iron after bariatric surgery, and can take these as specifically formulated vitamins for post-bariatric patients or separate vitamin supplements. We investigated the compliance, efficacy, and cost of specifically formulated vitamins for post-bariatric patients in comparison to separate vitamin supplements. METHODS: We surveyed 126 post-bariatric adult patients between February 1, 2022, and August 31, 2022, who had undergone bariatric surgery between 2014 and 2021 to assess the type of supplements taken, compliance, and cost. Demographics, type of bariatric surgery, and serum micronutrient levels were evaluated for all patients. RESULTS: There were 51 patients taking formulated vitamins and 75 patients taking separate vitamins. The formulated vitamin group demonstrated greater adherence to optimal vitamin dosage (formulated vitamin group, 76.5% vs. separate supplement group, 30.7%; p < 0.001) and higher compliance (formulated vitamin group, 90.2% vs. separate supplement group, 66.7%; p = 0.002). The costs associated with both groups were comparable. No significant difference was observed in the incidence of micronutrient deficiency between the groups. CONCLUSIONS: Our study shows that formulated vitamins do not offer significant difference in micronutrient levels or cost compared to separate standard vitamin supplements. However, formulated bariatric vitamins have improved compliance compared to separate vitamin supplements.

Use of Structured Templates to Improve Completeness of Endocrinology Referrals.

OBJECTIVE: Endocrinology referrals frequently lack important clinical information, which may increase the risk of inefficiency and adverse outcomes. This quality improvement project aimed to improve the completeness of new referrals by utilizing structured referral templates for common endocrine conditions at a large Veterans Health Administration medical center. Our target was of at least a 30% improvement in referral completeness for each condition after the intervention. METHODS: Electronic structured referral templates were designed utilizing existing resources and input from primary care providers and endocrinologists. Essential elements were identified and included in the templates. We conducted a retrospective chart review to compare referrals for 125 patients referred between January 1, 2021 and September 1, 2021 (preintervention) and 125 patients referred between October 1, 2021 and September 30, 2022 (postintervention). Each referral was rated using a scoring system derived from the criteria in the data abstraction tool formulated by the investigators. RESULTS: On average, preintervention referrals included 52% of the essential elements and postintervention referrals included 93%. Improvements in referral scores for each condition all met the prespecified 30% improvement target. The greatest improvement was for the element "type of visit preference." A separate analysis excluding that element showed an average improvement from 64% of essential elements preintervention to 92% postintervention. CONCLUSION: Structured referral templates, designed with the input of primary care providers and endocrinologists and embedded into an electronic referral system, can improve the availability of essential information and increase the quality of referrals. Future work should examine the effect of structured referral templates on efficiency, specialist experience, patient experience of care, and clinical outcomes.

Dynamics of non-household contacts during the COVID-19 pandemic in 2020 and 2021 in the Netherlands.

The COVID-19 pandemic was in 2020 and 2021 for a large part mitigated by reducing contacts in the general population. To monitor how these contacts changed over the course of the pandemic in the Netherlands, a longitudinal survey was conducted where participants reported on their at-risk contacts every two weeks, as part of the European CoMix survey. The survey included 1659 participants from April to August 2020 and 2514 participants from December 2020 to September 2021. We categorized the number of unique contacted persons excluding household members, reported per participant per day into six activity levels, defined as 0, 1, 2, 3-4, 5-9 and 10 or more reported contacts. After correcting for age, vaccination status, risk status for severe outcome of infection, and frequency of participation, activity levels increased over time, coinciding with relaxation of COVID-19 control measures.

Laparoscopic Heller Myotomy With EGD and Balloon Dilation: A Durable Solution for Achalasia.

BACKGROUND: Laparoscopic Heller myotomy (LHM) and esophageal balloon dilation (BD) are the two mainstays of achalasia treatment-this study examines the outcomes when they are performed simultaneously without fundoplication. METHODS: All patients undergoing LHM&BD were reviewed for demographic and procedural data, and to see if additional procedures for achalasia had been performed. Patients were surveyed using the Eckardt score and the GERD quality-of-life score (GERD-HRQL) to assess the durability of repair. RESULTS: From 2013-2020, 66 patients underwent LHM&BD. There were no esophageal perforations and a median LOS of 1 day. Seven patients have required additional operations or procedures at median 4-years follow up. 31 patients (47%) responded to the survey. The average Eckardt score was 2.9 (goal<4) with mean GERD-HRQL of 14.4 (goal<25). CONCLUSIONS: LHM&BD allows for a safe, durable repair of achalasia. Reflux symptoms are manageable with PPI without fundoplication and the re-intervention rate similar to published values.

The Impact of an Enhanced Recovery Protocol in a High-Risk Population Undergoing Colon Cancer Surgery.

OBJECTIVE: Enhanced Recovery ERP protocols (ERP) have improved surgical outcomes in patients undergoing elective colon cancer (CC) surgery; however, efficacy in different populations may vary. We examined the impact of an ERP in a population with high rates of obesity and multiple comorbidities. METHODS: We performed a retrospective analysis of factors associated with postoperative complications (PoC) and length of stay (LOS) following CC surgery from 2011 to 2019 in a 5-hospital healthcare system which serves a population with higher rates of obesity (body mass index ≥30kg/m2) and multi-comorbidities, as compared to published studies. Univariable and multivariable analyses were performed. RESULTS: A total of 408 elective CC surgery patients with complete oncologic surgical data were identified. Of these, 191 (46.81%) were under ERP. Factors independently associated with PoC included obesity (OR=1.66, P=.029), laparoscopic (OR=.52, P=.020), and hybrid (OR=.38, P=.012) versus open surgery and ASA (American Society of Anesthesiologists) class ≥3 (OR=1.98, P=.006). ERP did not impact PoC but was associated with a reduction in LOS (ß=-1.02 days, 95%CI: -1.75 - -.30, P=.006). ERP had an impact on LOS in both the non-obese and obese groups (P<.001 and P=.034, respectively). PoC significantly increased LOS (ß=6.67 days, 95%CI: 5.41-7.03, P<.001). CONCLUSIONS: Following elective CC surgery, obesity and medical comorbidities were associated with increased PoC and in turn, as expected, increased LOS. ERP was associated with a reduction in LOS in both obese and non-obese patients. In high-risk populations, application of ERP may be particularly important to optimize surgical outcomes following CC surgery.

Endocrinologists' Experiences With Telehealth: A Qualitative Study With Implications for Promoting Sustained Use.

OBJECTIVE: Endocrinology is well-suited to telehealth, with high rates of use and known benefits. Clinician attitudes toward telehealth will be critical to ensuring sustained use after the pandemic. We examined endocrinologists' experiences with synchronous telehealth to identify factors affecting experiences with and acceptance of the technology. METHODS: We conducted qualitative interviews and directed-content analysis with a purposive sample of 26 U.S. endocrinologists. Factors affecting clinicians' experiences were mapped to the human-organization-technology fit (HOT-fit) framework. RESULTS: We found that clinicians' experiences with synchronous telehealth were influenced by: (1) Clinician factors: Clinicians welcomed telehealth but expressed concerns about patient interest, rapport building, and clinical appropriateness, desiring more data to support its use. Many clinicians feared missing clinical findings on virtual examination, despite no such personal experiences. Effects on professional and personal life contributed to satisfaction, through increased flexibility but also increased workload. (2) Organizational factors: Departmental meetings and trainings supported clinicians' technical, logistical, and clinical needs, reducing resistance to telehealth use. Shifting staff responsibilities in clinical workflows improved clinicians' experiences and supported telehealth use, while mixed telehealth and in-person schedules impeded workflow. (3) Technology factors: Most clinicians preferred video visits to telephone. Usability and reliability of telehealth platforms, integration of patient self-monitoring data, and availability of IT support were crucial to a positive experience. CONCLUSION: Clinician acceptance of telehealth is influenced by clinician, organizational, and technology factors that can be leveraged to improve buy-in. Organizational leaders' attention to addressing these factors will be critical to support endocrinologists' continued provision of telehealth for their patients.

Clinical Appropriateness of Telehealth: A Qualitative Study of Endocrinologists' Perspectives.

Background: Outpatient endocrinology care delivered by telehealth is likely to remain common after the pandemic. There are few data to guide endocrinologists' judgments of clinical appropriateness (safety and effectiveness) for telehealth by synchronous video. We examined how, in the absence of guidelines, endocrinologists determine clinical appropriateness for telehealth, and we identified their strategies to navigate barriers to safe and effective use. Methods: We conducted qualitative, semi-structuredinterviews with 26 purposively selected US endocrinologists. We used a directed content analysis to characterize participant perceptions of which patients and situations were clinically appropriate for telehealth and to identify adaptations they made to accommodate telehealth visits. Results: Endocrinologists' perspectives about appropriateness for telehealth were influenced by clinical considerations, nonclinical patient factors, and the type and timing of the visit. These factors were weighed differently across individual participants according to their risk tolerance, values related to the physical examination and patient relationships, and impressions of patient capabilities and preferences. Some participants made practice adaptations that increased their comfort offering telehealth to a wider swath of patients. Conclusions: Endocrinologists' judgments about clinical appropriateness of telehealth for different patient situations varied widely across participants. The risk of such divergent approaches to determining appropriateness is unintended and clinically unwarranted variation in use of telehealth, compromising quality of care. Expert consensus is needed to guide endocrinologists now, along with studies to anchor future evidence-based guidelines for determining clinical appropriateness of telehealth in endocrinology.

SARS-CoV-2 Spike S1-specific IgG kinetic profiles following mRNA or vector-based vaccination in the general Dutch population show distinct kinetics.

mRNA- and vector-based vaccines are used at a large scale to prevent COVID-19. We compared Spike S1-specific (S1) IgG antibodies after vaccination with mRNA-based (Comirnaty, Spikevax) or vector-based (Janssen, Vaxzevria) vaccines, using samples from a Dutch nationwide cohort. In adults 18-64 years old (n = 2412), the median vaccination interval between the two doses was 77 days for Vaxzevria (interquartile range, IQR: 69-77), 35 days (28-35) for Comirnaty and 33 days (28-35) for Spikevax. mRNA vaccines induced faster inclines and higher S1 antibodies compared to vector-based vaccines. For all vaccines, one dose resulted in boosting of S1 antibodies in adults with a history of SARS-CoV-2 infection. For Comirnaty, two to four months following the second dose (n = 196), S1 antibodies in adults aged 18-64 years old (436 BAU/mL, IQR: 328-891) were less variable and median concentrations higher compared to those in persons ≥ 80 years old (366, 177-743), but differences were not statistically significant (p > 0.100). Nearly all participants seroconverted following COVID-19 vaccination, including the aging population. These data confirm results from controlled vaccine trials in a general population, including vulnerable groups.

Visceral angiosarcoma: A nationwide analysis of treatment factors and outcomes.

BACKGROUND AND OBJECTIVES: Visceral angiosarcoma is rare and aggressive, accounting for 2% of soft tissue sarcomas. Using a national data set, we examine determinants of outcomes for patients presenting with this rare disease. METHODS: The 2004-2015 National Cancer Database was queried for patients with visceral angiosarcoma. Trends in treatment and outcomes were examined. Factors affecting overall survival (OS) were assessed with log-rank and Cox regression. RESULTS: Eight hundred and ninety-three patients with visceral angiosarcoma were identified (median age 65 years, male [63%], Charlson comorbidity index <1 [86%]). Tumor size was <5 cm in 20.7%, and 34.2% were moderate/high grade. Median OS was 3.8 months (95% CI: 3.4-4.4). By multivariate analysis, increased tumor grade and size, and liver/biliary origin demonstrated worse OS while surgery, radiation, and systemic chemotherapy demonstrated improved OS (all p < 0.001). Survival was similar between patients achieving R0 resection and those with R1/2 resection receiving chemotherapy by Kaplan-Meier analysis. CONCLUSIONS: Visceral angiosarcomas are rare tumors with poor outcomes. Liver/biliary origin, higher tumor grade, and larger tumor size demonstrate worse outcomes. While R0 resection remains the mainstay of treatment, patients with R1/R2 resection have improved survival with addition of chemotherapy. Consideration should be made for multimodal therapy in these patients.

Seropositivity to Nucleoprotein to detect mild and asymptomatic SARS-CoV-2 infections: A complementary tool to detect breakthrough infections after COVID-19 vaccination?

BACKGROUND: With COVID-19 vaccine roll-out ongoing in many countries globally, monitoring of breakthrough infections is of great importance. Antibodies persist in the blood after a severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. Since COVID-19 vaccines induce immune response to the Spike protein of the virus, which is the main serosurveillance target to date, alternative targets should be explored to distinguish infection from vaccination. METHODS: Multiplex immunoassay data from 1,513 SARS-CoV-2 RT-qPCR-tested individuals (352 positive and 1,161 negative) without COVID-19 vaccination history were used to determine the accuracy of Nucleoprotein-specific immunoglobulin G (IgG) in detecting past SARS-CoV-2 infection. We also described Spike S1 and Nucleoprotein-specific IgG responses in 230 COVID-19 vaccinated individuals (Pfizer/BioNTech). RESULTS: The sensitivity of Nucleoprotein seropositivity was 85% (95% confidence interval: 80-90%) for mild COVID-19 in the first two months following symptom onset. Sensitivity was lower in asymptomatic individuals (67%, 50-81%). Participants who had experienced a SARS-CoV-2 infection up to 11 months preceding vaccination, as assessed by Spike S1 seropositivity or RT-qPCR, produced 2.7-fold higher median levels of IgG to Spike S1 ≥ 14 days after the first dose as compared to those unexposed to SARS-CoV-2 at ≥ 7 days after the second dose (p = 0.011). Nucleoprotein-specific IgG concentrations were not affected by vaccination in infection-naïve participants. CONCLUSIONS: Serological responses to Nucleoprotein may prove helpful in identifying SARS-CoV-2 infections after vaccination. Furthermore, it can help interpret IgG to Spike S1 after COVID-19 vaccination as particularly high responses shortly after vaccination could be explained by prior exposure history.

Characterization of Epigenomic Alterations in HPV16+ Head and Neck Squamous Cell Carcinomas.

BACKGROUND: Epigenetic changes associated with human papillomavirus (HPV)-driven tumors have been described; however, HPV type-specific alterations are less well understood. We sought to compare HPV16-specific methylation changes with those in virus-unassociated head and neck squamous cell carcinomas (HNSCC). METHODS: Within The Cancer Genome Atlas, 59 HPV16+ HNSCC, 238 nonviral HNSCC (no detectable HPV or other viruses), and 50 normal head and neck tissues were evaluated. Significant differentially methylated regions (DMR) were selected, and key associated genes were identified. Partial least squares models were generated to predict HPV16 status in additional independent samples. RESULTS: HPV infection in HNSCC is associated with type-specific methylomic profiles. Multiple significant DMRs were identified between HPV16+, nonviral, and normal samples. The most significant differentially methylated genes, SYCP2, MSX2, HLTF, PITX2, and GRAMD4, demonstrated HPV16-associated methylation patterns with corresponding alterations in gene expression. Phylogenetically related HPV types (alpha-9 species; HPV31, HPV33, and HPV35) demonstrated a similar methylation profile to that of HPV16 but differed from those seen in other types, such as HPV18 and 45 (alpha-7). CONCLUSIONS: HNSCC linked to HPV16 and types from the same alpha species are associated with a distinct methylation profile. This HPV16-associated methylation pattern is also detected in cervical cancer and testicular germ cell tumors. We present insights into both shared and unique methylation alterations associated with HPV16+ tumors and may have implications for understanding the clinical behavior of HPV-associated HNSCC. IMPACT: HPV type-specific methylomic changes may contribute to understanding biologic mechanisms underlying differences in clinical behavior among different HPV+ and HPV- HNSCC.

A low-cost TaqMan minor groove binder probe-based one-step RT-qPCR assay for rapid identification of N501Y variants of SARS-CoV-2.

The increasing prevalence of N501Y variants of SARS-CoV-2 has kindled global concern due to their enhanced transmissibility. Genome sequencing is the gold standard method to identify the emerging variants of concern. But it is time-consuming and expensive, limiting the widespread deployment of genome surveillance in some countries. Health authorities surge the development of alternative assay to expand screening capacity with reduced time and cost. In this study, we developed an in-house TaqMan minor groove binder (MGB) probe-based one-step RT-qPCR assay to detect the presence of N501Y mutation in SARS-CoV-2. A total of 168 SARS-CoV-2 positive respiratory specimens were collected to determine diagnostic accuracy of the RT-qPCR assay. As a reference standard, PANGO lineages and the mutation patterns of all samples were characterised by whole-genome sequencing. The analytical sensitivity and the ability of the assay to detect low frequency of N501Y variants were also evaluated. A total of 31 PANGO lineages were identified from 168 SARS-CoV-2 positive cases, in which 34 samples belonged to N501Y variants, including B.1.1.7 (n = 20), B.1.351 (n = 12) and P.3 (n = 2). The N501Y RT-qPCR correctly identified all 34 samples as N501Y-positive and the other 134 samples as wildtype. The limit-of-detection of the assay consistently achieved 1.5 copies/µL on four different qPCR platforms. N501Y mutation was successfully detected at an allele frequency as low as 10 % in a sample with mixed SARS-CoV-2 lineage. The N501Y RT-qPCR is simple and inexpensive (US$1.6 per sample). It enables robust high-throughput screening for surveillance of SARS-CoV-2 variants of concern harbouring N501Y mutation.

The Clinical Utility of Two High-Throughput 16S rRNA Gene Sequencing Workflows for Taxonomic Assignment of Unidentifiable Bacterial Pathogens in Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry.

Bacterial pathogens that cannot be identified using matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) are occasionally encountered in clinical laboratories. The 16S rRNA gene is often used for sequence-based analysis to identify these bacterial species. Nevertheless, traditional Sanger sequencing is laborious, time-consuming, and low throughput. Here, we compared two commercially available 16S rRNA gene sequencing tests that are based on Illumina and Nanopore sequencing technologies, respectively, in their ability to identify the species of 172 clinical isolates that failed to be identified by MALDI-TOF MS. Sequencing data were analyzed by the respective built-in programs (MiSeq Reporter software of Illumina and Epi2me of Nanopore) and BLAST+ (v2.11.0). Their agreement with Sanger sequencing on species-level identification was determined. Discrepancies were resolved by whole-genome sequencing. The diagnostic accuracy of each workflow was determined using the composite sequencing result as the reference standard. Despite the high base-calling accuracy of Illumina sequencing, we demonstrated that the Nanopore workflow had a higher taxonomic resolution at the species level. Using built-in analysis algorithms, the concordance of Sanger 16S with the Illumina and Nanopore workflows was 33.14% and 87.79%, respectively. The agreement was 65.70% and 83.14%, respectively, when BLAST+ was used for analysis. Compared with the reference standard, the diagnostic accuracy of Nanopore 16S was 96.36%, which was identical to that of Sanger 16S and better than that of Illumina 16S (69.07%). The turnaround time of the Illumina workflow and the Nanopore workflow was 78 h and 8.25 h, respectively. The per-sample cost of the Illumina and Nanopore workflows was US$28.5 and US$17.7, respectively.

Rapid cell counting and viability detection method of Escherichia coli Nissle using image cytometry.

The improvement of cell enumeration methods for the counting of Escherichia coli (E. coli) is important as E. coli gains in popularity as a basis for biopharmaceutical applications. In the biopharmaceutical industry, enumerating, characterizing, and dosing the accurate number of cells is imperative. In this work, we demonstrate the utilization of a chip-based image cytometer using a thin-gap, low volume counting chamber consumable to directly enumerate E. coli in bright field and fluorescence, and measure their viability using SYTOX™ Green. The total E. coli particles can be counted accurately label-free by adjusting the focus and targeting the linear range of the instrument. The E. coli are stained with SYTOX™ Green to count the membrane-compromised dead bacterial cells in the green fluorescence channel, while the total cells are counted using the bright field channel. Optimization of the system settings, image focus, cell counting range, and staining conditions have yielded a precise, rapid, and accurate E. coli cell enumeration and viability measurement.