Validation of an HIV whole genome sequencing method for HIV drug resistance testing in an Australian clinical microbiology laboratory.

Detection of HIV drug resistance (HIVDR) is vital to successful anti-retroviral therapy (ART). HIVDR testing to determine drug-resistance mutations is routinely performed in Australia to guide ART choice in newly diagnosed people living with HIV or in cases of treatment failure. In 2022, our clinical microbiology laboratory sought to validate a next-generation sequencing (NGS)-based HIVDR assay to replace the previous Sanger-sequencing (SS)-based ViroSeq. NGS solutions for HIVDR offer higher throughput, lower costs and higher sensitivity for variant detection. We sought to validate the previously described low-cost probe-based NGS method (veSEQ-HIV) for whole-genome recovery and HIVDR-testing in a diagnostic setting. veSEQ-HIV displayed 100% and 98% accuracy in major and minor mutation detection, respectively, and 100% accuracy of subtyping (provided > 1000 mapped reads were obtained). Pairwise comparison exhibited low inter-and intrarun variability across the whole-genome (Jaccard index [J] = 0.993; J = 0.972) and the Pol gene (J = 0.999; J = 0.999), respectively. veSEQ-HIV met all our pre-set criteria based on WHO recommendations and successfully replaced ViroSeq in our laboratory. Scaling-down veSEQ-HIV to a limited batch size and sequencing on Illumina iSeq. 100, allowed easy implementation of the assay into the workflow of a small sequencing laboratory with minimal staff and equipment and the ability to meet clinically relevant test turn-around times. As HIVDR-testing moves from SS- to NGS-based methods and new ART drugs come to market (particularly those with targets outside the Pol region), whole-genome recovery using veSEQ-HIV provides a robust, cost-effective and "future-proof" NGS method for HIVDR-testing.

Structural racism and its influence on the severity of atopic dermatitis in African American children.

BACKGROUND/OBJECTIVES: Atopic dermatitis (AD) is the most common skin disease of childhood and is often more severe in African American than white children. The reason for this disparity is unknown, but recent research indicates that it may be due to a combination of environmental and genetic factors. The objective of this article was to explore the relationship between measures of structural racism and residential segregation within pediatric AD. METHODS: An in-office, online survey consisting of 58 questions spanning 5 domains (demographics, in-home crowding, community crowding, air quality, and litter) was administered to a convenience sample of 201 pediatric AD patients (age 0-18 years). Survey data were geocoded and linked to a measure of structural racism (ie, residential segregation). RESULTS: African American children were more likely to live in rented homes, be in lower income families, have caregivers with lower educational attainment, and be exposed to tobacco smoke. The same factors that were associated with worse AD severity in this study were also found in published literature, emphasizing the role of social determinants of health and racial differences in AD severity. Additionally, this study found that living in highly segregated communities was more likely to be associated with severe AD in African American children. CONCLUSIONS: Consistent with reported literature, socioeconomic status, race, and the physical environment appear to affect AD severity. This investigation adds structural racism as an important community characteristic that likely has significant effects on AD severity for African American Children.

Active surveillance of carbapenemase-producing Enterobacterales using genomic sequencing for hospital-based infection control interventions.

BACKGROUND: Whole-genome sequencing (WGS) is increasingly used to characterize hospital outbreaks of carbapenemase-producing Enterobacterales (CPE). However, access to WGS is variable and testing is often centralized, leading to delays in reporting of results. OBJECTIVE: We describe the utility of a local sequencing service to promptly respond to facility needs over an 8-year period. METHODS: The study was conducted at Royal Prince Alfred Hospital in Sydney, Australia. All CPE isolated from patient (screening and clinical) and environmental samples from 2015 onward underwent prospective WGS. Results were notified to the infection control unit in real time. When outbreaks were identified, WGS reports were also provided to senior clinicians and the hospital executive administration. Enhanced infection control interventions were refined based on the genomic data. RESULTS: In total, 141 CPE isolates were detected from 123 patients and 5 environmental samples. We identified 9 outbreaks, 4 of which occurred in high-risk wards (intensive care unit and/or solid-organ transplant ward). The largest outbreak involved Enterobacterales containing an NDM gene. WGS detected unexpected links among patients, which led to further investigation of epidemiological data that uncovered the outpatient setting and contaminated equipment as reservoirs for ongoing transmission. Targeted interventions as part of outbreak management halted further transmission. CONCLUSIONS: WGS has transitioned from an emerging technology to an integral part of local CPE control strategies. Our results show the value of embedding this technology in routine surveillance, with timely reports generated in clinically relevant timeframes to inform and optimize local control measures for greatest impact.

Insights gained from sequencing Australian non-invasive and invasive Streptococcus pyogenes isolates.

Epidemiological data have indicated that invasive infections caused by the Gram-positive cocci Streptococcus pyogenes (group A streptococcus, GAS) have increased in many Australian states over the past two decades. In July 2022, invasive GAS (iGAS) infections became nationally notifiable in Australia via public-health agencies. Surveillance for S. pyogenes infections has been sporadic within the state of New South Wales (NSW). This has led to a lack of genetic data on GAS strains in circulation, particularly for non-invasive infections, which are the leading cause of GAS's burden on the Australian healthcare system. To address this gap, we used whole-genome sequencing to analyse the genomes of 318 S. pyogenes isolates collected within two geographical regions of NSW. Invasive isolates were collected in 2007-2017, whilst non-invasive isolates were collected in 2019-2021. We found that at least 66 different emm-types were associated with clinical disease within NSW. There was no evidence of any Australian-specific clones in circulation. The M1UK variant of the emm1 global pandemic clone (M1global) has been detected in our isolates from 2013 onwards. We detected antimicrobial-resistance genes (mainly tetM, ermA or ermB genes) in less than 10 % of our 318 isolates, which were more commonly associated with non-invasive infections. Superantigen virulence gene carriage was reasonably proportionate between non-invasive and invasive infection isolates. Our study adds rich data on the genetic makeup of historical S. pyogenes infections within Australia. Ongoing surveillance of invasive and non-invasive GAS infections within NSW by whole-genome sequencing is warranted to inform on outbreaks, antimicrobial resistance and vaccine coverage.

The role of real-time, on-site, whole-genome sequencing of severe acute respiratory coronavirus virus 2 (SARS-CoV-2) in guiding the management of hospital outbreaks of coronavirus disease 2019 (COVID-19).

OBJECTIVE: We aimed to demonstrate the role of real-time, on-site, whole-genome sequencing (WGS) of severe acute respiratory coronavirus virus 2 (SARS-CoV-2) in the management of hospital outbreaks of coronavirus disease 2019 (COVID-19). DESIGN: This retrospective study was undertaken at our institutions in Sydney, New South Wales, Australia, between July 2021 and April 2022. We included SARS-CoV-2 outbreaks due to SARS-CoV-2 δ (delta) and ο (omicron) variants. All unexpected SARS-CoV-2-positive cases identified within the hospital were managed by the infection control team. An outbreak was defined as 2 or more cases acquired on a single ward. We included only outbreaks with 2 or more suspected transmission events in which WGS was utilized to assist with outbreak assessment and management. RESULTS: We studied 8 outbreaks involving 266 patients and 486 staff, of whom 73 (27.4%) and 39 (8.0%), respectively, tested positive for SARS-CoV-2 during the outbreak management. WGS was used to evaluate the source of the outbreak, to establish transmission chains, to highlight deficiencies in infection control practices, and to delineate between community and healthcare acquired infection. CONCLUSIONS: Real-time, on-site WGS combined with epidemiologic assessment is a useful tool to guide management of hospital SARS-CoV-2 outbreaks. WGS allowed us (1) to establish likely transmission events due to personal protective equipment (PPE) breaches; (2) to detect inadequacies in infection control infrastructure including ventilation; and (3) to confirm multiple viral introductions during periods of high community SARS-CoV-2 transmission. Insights gained from WGS-guides outbreak management directly influenced policy including modifying PPE requirements, instituting routine inpatient SARS-CoV-2 surveillance, and confirmatory SARS-CoV-2 testing prior to placing patients in a cohort setting.

Centralised or Localised Pathogen Whole Genome Sequencing: Lessons Learnt From Implementation in a Clinical Diagnostic Laboratory.

Whole genome sequencing (WGS) has had widespread use in the management of microbial outbreaks in a public health setting. Current models encompass sending isolates to a central laboratory for WGS who then produce a report for various levels of government. This model, although beneficial, has multiple shortcomings especially for localised infection control interventions and patient care. One reason for the slow rollout of WGS in clinical diagnostic laboratories has been the requirement for professionally trained personal in both wet lab techniques and in the analysis and interpretation of data, otherwise known as bioinformatics. A further bottleneck has been establishment of regulations in order to certify clinical and technical validity and demonstrate WGS as a verified diagnostic test. Nevertheless, this technology is far superior providing information that would normally require several diagnostic tests to achieve. An obvious barrier to informed outbreak tracking is turnaround time and requires isolates to be sequenced in real-time to rapidly identify chains of transmission. One way this can be achieved is through onsite hospital sequencing with a cumulative analysis approach employed. Onsite, as opposed to centralised sequencing, has added benefits including the increased agility to combine with local infection control staff to iterate through the data, finding links that aide in understanding transmission chains and inform infection control strategies. Our laboratory has recently instituted a pathogen WGS service within a diagnostic laboratory, separate to a public health laboratory. We describe our experience, address the challenges faced and demonstrate the advantages of de-centralised sequencing through real-life scenarios.

Gender Bias in Surgical Oncology Fellowship Recommendation Letters: Gaining Progress.

OBJECTIVE: Gender bias has been identified in letters of recommendation (LOR) in many different surgical training fields. Among surgeons, women comprise over 30% of the full-time faculty positions nationally and surgical oncology is one of the most gender diverse surgical subspecialties. We sought to determine if bias existed in LOR submitted to a Complex General Surgical Oncology (CGSO) fellowship. DESIGN: LOR for the CGSO fellowship were retrospectively analyzed from applicants at a single institution over an 8-year period (2013-2020). The linguistic content of the letters was analyzed using Linguistic Inquiry and Word Count (LIWC2015), a validated text analysis program. Using multivariable analysis, LOR were compared by gender of both applicant and letter writer to explore the association between gender and the characteristics of the applicants and letter writers. SETTING: University of North Carolina at Chapel Hill (UNC), Division of Surgical Oncology and Endocrine Surgery. PARTICIPANTS: Applicants interviewed for the CSGO fellowship program at the UNC from 2013 to 2020 as well as all applicants from the 2018 application cycle, regardless of interview status. RESULTS: About 841 letters from 219 interviewed applicants throughout the 2013 to 2020 surgical oncology fellowship application cycles were included. No difference in authenticity, clout, analytic thinking, or emotional tone of the letters was seen when comparing men and women applicants. Of the 41 word categories analyzed, only "references to achievement" in LOR written for women was significantly higher when compared to LOR written for men (p = 0.01). Interestingly, significantly more women applicants had at least 1 LOR written by a woman (p = 0.04). A subset analysis of all applicants regardless of interview status from the 2018 cycle included 294 LOR from 77 applicants. With the inclusion of noninterviewed applicants, LOR for men had more analytic tone than LOR for women (p = 0.02), otherwise there were no significant differences between the groups. CONCLUSIONS: Very few differences in LOR were found for applicants at a CGSO fellowship program based on applicant or letter writer gender. The lack of gender bias demonstrates progress within the field of surgical oncology, likely a result of recent work and educational effort in this area. Efforts to expand this progress into other surgical sub-specialties are necessary.