Continued improvement in the development of the SARS-CoV-2 whole genome sequencing proficiency testing program.

Application of whole genome sequencing (WGS) has allowed monitoring of the emergence of variants of concern (VOC) of severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) globally. Genomic investigation of emerging variants and surveillance of clinical progress has reduced the public health impact of infection during the COVID-19 pandemic. These steps required developing and implementing a proficiency testing program (PTP), as WGS has been incorporated into routine reference laboratory practice. In this study, we describe how the PTP evaluated the capacity and capability of one New Zealand and 14 Australian public health laboratories to perform WGS of SARS-CoV-2 in 2022. The participants' performances in characterising a specimen panel of known SARS-CoV-2 isolates in the PTP were assessed based on: (1) genome coverage, (2) Pango lineage, and (3) sequence quality, with the choice of assessment metrics refined based on a previously reported assessment conducted in 2021. The participants' performances in 2021 and 2022 were also compared after reassessing the 2021 results using the more stringent metrics adopted in 2022. We found that more participants would have failed the 2021 assessment for all survey samples and a significantly higher fail rate per sample in 2021 compared to 2022. This study highlights the importance of choosing appropriate performance metrics to reflect better the laboratories' capacity to perform SARS-CoV-2 WGS, as was done in the 2022 PTP. It also displays the need for a PTP for WGS of SARS-CoV-2 to be available to public health laboratories ongoing, with continuous refinements in the design and provision of the PTP to account for the dynamic nature of the COVID-19 pandemic as SARS-CoV-2 continues to evolve.

Shrinking in the dark: Parallel endosymbiont genome erosions are associated with repeated host transitions to an underground life.

Microbial symbioses have had profound impacts on the evolution of animals. Conversely, changes in host biology may impact the evolutionary trajectory of symbionts themselves. Blattabacterium cuenoti is present in almost all cockroach species and enables hosts to subsist on a nutrient-poor diet. To investigate if host biology has impacted Blattabacterium at the genomic level, we sequenced and analyzed 25 genomes from Australian soil-burrowing cockroaches (Blaberidae: Panesthiinae), which have undergone at least seven separate subterranean, subsocial transitions from above-ground, wood-feeding ancestors. We find at least three independent instances of genome erosion have occurred in Blattabacterium strains exclusive to Australian soil-burrowing cockroaches. These shrinkages have involved the repeated inactivation of genes involved in amino acid biosynthesis and nitrogen recycling, the core role of Blattabacterium in the host-symbiont relationship. The most drastic of these erosions have occurred in hosts thought to have transitioned underground the earliest relative to other lineages, further suggestive of a link between gene loss in Blattabacterium and the burrowing behavior of hosts. As Blattabacterium is unable to fulfill its core function in certain host lineages, these findings suggest soil-burrowing cockroaches must acquire these nutrients from novel sources. Our study represents one of the first cases, to our knowledge, of parallel host adaptations leading to concomitant parallelism in their mutualistic symbionts, further underscoring the intimate relationship between these two partners.

Human cytomegalovirus (CMV) dysregulates neurodevelopmental pathways in cerebral organoids.

Human cytomegalovirus (CMV) infection is the leading non-genetic aetiology of congenital malformation in developed countries, causing significant fetal neurological injury. This study investigated potential CMV pathogenetic mechanisms of fetal neural malformation using in vitro human cerebral organoids. Cerebral organoids were permissive to CMV replication, and infection dysregulated cellular pluripotency and differentiation pathways. Aberrant expression of dual-specificity tyrosine phosphorylation-regulated kinases (DYRK), sonic hedgehog (SHH), pluripotency, neurodegeneration, axon guidance, hippo signalling and dopaminergic synapse pathways were observed in CMV-infected organoids using immunofluorescence and RNA-sequencing. Infection with CMV resulted in dysregulation of 236 Autism Spectrum Disorder (ASD)-related genes (p = 1.57E-05) and pathways. This notable observation suggests potential links between congenital CMV infection and ASD. Using DisGeNET databases, 103 diseases related to neural malformation or mental disorders were enriched in CMV-infected organoids. Cytomegalovirus infection-related dysregulation of key cerebral cellular pathways potentially provides important, modifiable pathogenetic mechanisms for congenital CMV-induced neural malformation and ASD.

Clinical, Genomic, and Immunological Characterization of RSV Surge in Sydney, Australia, 2022.

OBJECTIVES: The 2022 seasonal respiratory syncytial virus (RSV) epidemic in Sydney, Australia saw an unprecedented number of RSV detections. We aimed to characterize genomic and immunologic factors associated with the surge in RSV cases. METHODS: Whole genome sequences of RSV were generated from 264 RSV-infected infants and linked to case-matched clinical data from the 2022 southern hemisphere RSV season. We then performed an immunologic analysis of baseline RSV-specific humoral immunity in women of childbearing age before and throughout the coronavirus disease 2019 pandemic. RESULTS: Clinical analysis revealed a high burden of disease across patients of all health backgrounds. More than one-half of RSV-related health care visits by infants resulted in hospitalization, and one-quarter required high-flow respiratory support or a higher level of care. Viral phylogenetic analyses revealed that 2022 Sydney RSV sequences were closely related to viruses that had been circulating globally since 2017, including those detected in recent US outbreaks. Nonsynonymous mutations within the palivizumab and nirsevimab binding sites were detected at low frequencies. There was no difference in baseline RSV-neutralizing antibody titers between 2020 and 2022. CONCLUSIONS: Collectively, these findings suggest that neither the emergence of a novel RSV genotype nor hypothesized immune debt was associated with the surge of RSV cases and hospitalizations in 2022. Continued genomic and immunologic surveillance is required to further understand the factors driving outbreaks of RSV globally, and to inform guidelines for the rollout and ongoing use of recently developed immunotherapeutics and vaccines.

Bioinformatic investigation of discordant sequence data for SARS-CoV-2: insights for robust genomic analysis during pandemic surveillance.

The COVID-19 pandemic has necessitated the rapid development and implementation of whole-genome sequencing (WGS) and bioinformatic methods for managing the pandemic. However, variability in methods and capabilities between laboratories has posed challenges in ensuring data accuracy. A national working group comprising 18 laboratory scientists and bioinformaticians from Australia and New Zealand was formed to improve data concordance across public health laboratories (PHLs). One effort, presented in this study, sought to understand the impact of the methodology on consensus genome concordance and interpretation. SARS-CoV-2 WGS proficiency testing programme (PTP) data were retrospectively obtained from the 2021 Royal College of Pathologists of Australasia Quality Assurance Programmes (RCPAQAP), which included 11 participating Australian laboratories. The submitted consensus genomes and reads from eight contrived specimens were investigated, focusing on discordant sequence data and findings were presented to the working group to inform best practices. Despite using a variety of laboratory and bioinformatic methods for SARS-CoV-2 WGS, participants largely produced concordant genomes. Two participants returned five discordant sites in a high-Cτ replicate, which could be resolved with reasonable bioinformatic quality thresholds. We noted ten discrepancies in genome assessment that arose from nucleotide heterogeneity at three different sites in three cell-culture-derived control specimens. While these sites were ultimately accurate after considering the participants' bioinformatic parameters, it presented an interesting challenge for developing standards to account for intrahost single nucleotide variation (iSNV). Observed differences had little to no impact on key surveillance metrics, lineage assignment and phylogenetic clustering, while genome coverage <90 % affected both. We recommend PHLs bioinformatically generate two consensus genomes with and without ambiguity thresholds for quality control and downstream analysis, respectively, and adhere to a minimum 90 % genome coverage threshold for inclusion in surveillance interpretations. We also suggest additional PTP assessment criteria, including primer efficiency, detection of iSNVs and minimum genome coverage of 90 %. This study underscores the importance of multidisciplinary national working groups in informing guidelines in real time for bioinformatic quality acceptance criteria. It demonstrates the potential for enhancing public health responses through improved data concordance and quality control in SARS-CoV-2 genomic analysis during pandemic surveillance.

Emergence and antibody evasion of BQ, BA.2.75 and SARS-CoV-2 recombinant sub-lineages in the face of maturing antibody breadth at the population level.

BACKGROUND: The Omicron era of the COVID-19 pandemic commenced at the beginning of 2022 and whilst it started with primarily BA.1, it was latter dominated by BA.2 and the related sub-lineage BA.5. Following resolution of the global BA.5 wave, a diverse grouping of Omicron sub-lineages emerged derived from BA.2, BA.5 and recombinants thereof. Whilst emerging from distinct lineages, all shared similar changes in the Spike glycoprotein affording them an outgrowth advantage through evasion of neutralising antibodies. METHODS: Over the course of 2022, we monitored the potency and breadth of antibody neutralization responses to many emerging variants in the Australian community at three levels: (i) we tracked over 420,000 U.S. plasma donors over time through various vaccine booster roll outs and Omicron waves using sequentially collected IgG pools; (ii) we mapped the antibody response in individuals using blood from stringently curated vaccine and convalescent cohorts. (iii) finally we determine the in vitro efficacy of clinically approved therapies Evusheld and Sotrovimab. FINDINGS: In pooled IgG samples, we observed the maturation of neutralization breadth to Omicron variants over time through continuing vaccine and infection waves. Importantly, in many cases, we observed increased antibody breadth to variants that were yet to be in circulation. Determination of viral neutralization at the cohort level supported equivalent coverage across prior and emerging variants with isolates BQ.1.1, XBB.1, BR.2.1 and XBF the most evasive. Further, these emerging variants were resistant to Evusheld, whilst increasing neutralization resistance to Sotrovimab was restricted to BQ.1.1 and XBF. We conclude at this current point in time that dominant variants can evade antibodies at levels equivalent to their most evasive lineage counterparts but sustain an entry phenotype that continues to promote an additional outgrowth advantage. In Australia, BR.2.1 and XBF share this phenotype and, in contrast to global variants, are uniquely dominant in this region in the later months of 2022. INTERPRETATION: Whilst the appearance of a diverse range of omicron lineages has led to primary or partial resistance to clinically approved monoclonal antibodies, the maturation of the antibody response across both cohorts and a large donor pools importantly observes increasing breadth in the antibody neutralisation responses over time with a trajectory that covers both current and known emerging variants. FUNDING: This work was primarily supported by Australian Medical Foundation research grants MRF2005760 (SGT, GM & WDR), Medical Research Future Fund Antiviral Development Call grant (WDR), the New South Wales Health COVID-19 Research Grants Round 2 (SGT & FB) and the NSW Vaccine Infection and Immunology Collaborative (VIIM) (ALC). Variant modeling was supported by funding from SciLifeLab's Pandemic Laboratory Preparedness program to B.M. (VC-2022-0028) and by the European Union's Horizon 2020 research and innovation programme under grant agreement no. 101003653 (CoroNAb) to B.M.

Persistence of a Frameshifting Deletion in SARS-CoV-2 ORF7a for the Duration of a Major Outbreak.

Australia experienced widespread COVID-19 outbreaks from infection with the SARS-CoV-2 Delta variant between June 2021 and February 2022. A 17-nucleotide frameshift-inducing deletion in ORF7a rapidly became represented at the consensus level (Delta-ORF7aΔ17del) in most Australian outbreak cases. Studies from early in the COVID-19 pandemic suggest that frameshift-inducing deletions in ORF7a do not persist for long in the population; therefore, Delta-ORF7aΔ17del genomes should have disappeared early in the Australian outbreak. In this study, we conducted a retrospective analysis of global Delta genomes to characterise the dynamics of Delta-ORF7aΔ17del over time, determined the frequency of all ORF7a deletions worldwide, and compared global trends with those of the Australian Delta outbreak. We downloaded all GISAID clade GK Delta genomes and scanned them for deletions in ORF7a. For each deletion we identified, we characterised its frequency, the number of countries it was found in, and how long it persisted. Of the 4,018,216 Delta genomes identified globally, 134,751 (~3.35%) possessed an ORF7a deletion, and ORF7aΔ17del was the most common. ORF7aΔ17del was the sole deletion in 28,014 genomes, of which 27,912 (~99.6%) originated from the Australian outbreak. During the outbreak, ~87% of genomes were Delta-ORF7aΔ17del, and genomes with this deletion were sampled until the outbreak's end. These data demonstrate that, contrary to suggestions early in the COVID-19 pandemic, genomes with frameshifting deletions in ORF7a can persist over long time periods. We suggest that the proliferation of Delta-ORF7aΔ17del genomes was likely a chance founder effect. Nonetheless, the frequency of ORF7a deletions in SARS-CoV-2 genomes worldwide suggests they might have some benefit for virus transmission.

Comparative study of two Saccharomyces cerevisiae strains with kinetic models at genome-scale.

The parameterization of kinetic models requires measurement of fluxes and/or metabolite levels for a base strain and a few genetic perturbations thereof. Unlike stoichiometric models that are mostly invariant to the specific strain, it remains unclear whether kinetic models constructed for different strains of the same species have similar or significantly different kinetic parameters. This important question underpins the applicability range and prediction limits of kinetic reconstructions. To this end, herein we parameterize two separate large-scale kinetic models using K-FIT with genome-wide coverage corresponding to two distinct strains of Saccharomyces cerevisiae: CEN.PK 113-7D strain (model k-sacce306-CENPK), and growth-deficient BY4741 (isogenic to S288c; model k-sacce306-BY4741). The metabolic network for each model contains 306 reactions, 230 metabolites, and 119 substrate-level regulatory interactions. The two models (for CEN.PK and BY4741) recapitulate, within one standard deviation, 77% and 75% of the fitted dataset fluxes, respectively, determined by 13C metabolic flux analysis for wild-type and eight single-gene knockout mutants of each strain. Strain-specific kinetic parameterization results indicate that key enzymes in the TCA cycle, glycolysis, and arginine and proline metabolism drive the metabolic differences between these two strains of S. cerevisiae. Our results suggest that although kinetic models cannot be readily used across strains as stoichiometric models, they can capture species-specific information through the kinetic parameterization process.

Appraisal of vitreous syphilis antibody as a novel biomarker for the diagnosis of syphilitic uveitis: a prospective case-control study.

PURPOSE: To determine the sensitivity and specificity of syphilis antibody tests in vitreous samples and to propose an algorithm using vitreous syphilis antibody as a supplementary test to confirm syphilitic uveitis (SU). METHODS: A prospective case-control study was conducted at the Retina and Uveitis Clinic from May 2017 to January 2020. Initially, patients were classified based on syphilis serology into group 1 (positive testing) and group 2 (negative testing). Group 1 was further divided into 2 subgroups (group 1A and 1B) depending on their relevant clinical manifestations and clinical improvement. Group 2 served as a control group. RESULTS: Thirty-eight patients were enrolled in the study: 14 in group 1A, 5 in group 1B, and 19 in group 2B. No patient was assigned to group 2A. All patients in group 1A, representing definite SU, completed syphilis test (rapid plasma reagin [RPR], enzyme immunoassay [EIA], and fluorescent treponemal antibody-absorption [FTA-ABS]) for vitreous, and all vitreous samples yielded positive results. Of the 5 subjects in group 1B, 3 cases were considered to be not SU with different conditions, and 2 were indeterminate for SU. They presented with different features not typical of SU, and they had variable and fewer positive syphilis antibody responses. The most sensitive test for detecting syphilis antibodies in vitreous was EIA (90.9%), followed by RPR (80.0%) and FTA-ABS IgG (78.9%). EIA and FTA-ABS had the highest specificity, detecting 100% of the syphilis antibody. CONCLUSIONS: Vitreous analysis of syphilis antibody can serve as a supplementary test to confirm SU in selected cases as the proposed algorithm.

NONPARANEOPLASTIC AUTOIMMUNE RETINOPATHY VERSUS PERICENTRAL RETINAL DEGENERATION PHENOTYPE: WHICH CAME FIRST? A CASE REPORT.

PURPOSE: To report a case of nonparaneoplastic autoimmune retinopathy with phenotypical features of pericentral retinal degeneration (PRD) who responded to IV immunoglobulin therapy. METHODS: A case report. A 27-year-old man presented with recent subacute progressive nyctalopia and photopsia. RESULTS: Dilated fundoscopy demonstrated confluent yellow-white patches along the main temporal vascular arcades with sparing of the central island in the posterior pole. Color vision, fundus autofluorescence, fluorescein angiography, static visual field, and electroretinographic studies were inconclusive for retinal degeneration. Subsequent genetic testing for known mutations was negative. Workup for paraneoplastic autoimmune retinopathy was negative. Antiretinal antibodies were positive. The patient was diagnosed with nonparaneoplastic autoimmune retinopathy and was treated with IV immunoglobulin, which resulted in objective and subjective improvement on electroretinography, visual field, and optical coherence tomography of the retina. CONCLUSION: Nonparaneoplastic autoimmune retinopathy may present in a patient with the clinical phenotype of PRD. It is essential to rule out nonparaneoplastic autoimmune retinopathy in patients with subacute changes in the natural course of pericentral retinal degeneration because treatment with IV immunoglobulin may be helpful.

Central-line-associated bloodstream infections and central-line-associated non-CLABSI complications among pediatric oncology patients.

OBJECTIVE: To assess central venous catheter (CVC) harm in pediatric oncology patients, we explored risks for central-line-associated bloodstream infections (CLABSIs) and central-line-associated non-CLABSI complications (CLANCs). DESIGN: Retrospective cohort study. SETTING: Midwestern US pediatric oncology program. PATIENTS: The study cohort comprised 592 pediatric oncology patients seen between 2006 and 2016. METHODS: CLABSIs were defined according to Centers for Disease Control and Prevention (CDC)/National Health Safety Network (NHSN) definitions. CLANCs were classified using a novel definition requiring CVC removal. Patient-level and central-line-level risks were calculated using a negative binomial model to adjust for correlations between total events and line numbers. RESULTS: CVCs were inserted in 62% of patients, with 175,937 total catheter days. The inpatient CLABSI and CLANC rates were 5.8 and 8.5 times higher than outpatient rates. At the patient level, shared risks included acute myeloid leukemia (AML) and age <1 year at diagnosis. At the line level, shared risks included age <1 year at diagnosis, non-mediports, and >1 lumen. AML was a CLABSI-specific risk. CLANC-specific risks included non-brain-tumor diagnosis, younger age at diagnosis or central-line placement, and age <1 year at diagnosis or line placement. Multivariable risks were for CLABSI >1 lumen and for CLANC age <1 year at placement. CONCLUSIONS: Among patients with CVCs, CLABSI and CLANC rates were similar, higher among inpatients than outpatients. For both CLABSIs and CLANCs, infants and patients with AML were at higher risk. In both univariate and multivariate models, lines with >1 lumen were associated with CLABSIs and placement during infancy with CLANCs.

Differential transcriptomic responses to heat stress in surface and subterranean diving beetles.

Subterranean habitats are generally very stable environments, and as such evolutionary transitions of organisms from surface to subterranean lifestyles may cause considerable shifts in physiology, particularly with respect to thermal tolerance. In this study we compared responses to heat shock at the molecular level in a geographically widespread, surface-dwelling water beetle to a congeneric subterranean species restricted to a single aquifer (Dytiscidae: Hydroporinae). The obligate subterranean beetle Paroster macrosturtensis is known to have a lower thermal tolerance compared to surface lineages (CTmax 38 °C cf. 42-46 °C), but the genetic basis of this physiological difference has not been characterized. We experimentally manipulated the thermal environment of 24 individuals to demonstrate that both species can mount a heat shock response at high temperatures (35 °C), as determined by comparative transcriptomics. However, genes involved in these responses differ between species and a far greater number were differentially expressed in the surface taxon, suggesting it can mount a more robust heat shock response; these data may underpin its higher thermal tolerance compared to subterranean relatives. In contrast, the subterranean species examined not only differentially expressed fewer genes in response to increasing temperatures, but also in the presence of the experimental setup employed here alone. Our results suggest P. macrosturtensis may be comparatively poorly equipped to respond to both thermally induced stress and environmental disturbances more broadly. The molecular findings presented here have conservation implications for P. macrosturtensis and contribute to a growing narrative concerning weakened thermal tolerances in obligate subterranean organisms at the molecular level.

SARS-CoV-2 Omicron BA.5: Evolving tropism and evasion of potent humoral responses and resistance to clinical immunotherapeutics relative to viral variants of concern.

BACKGROUND: Genetically distinct viral variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have been recorded since January 2020. The introduction of global vaccine programs has contributed to lower COVID-19 hospitalisation and mortality rates, particularly in developed countries. In late 2021, Omicron BA.1 emerged, with substantially altered genetic differences and clinical effects from other variants of concern. Shortly after dominating global spread in early 2022, BA.1 was supplanted by the genetically distinct Omicron lineage BA.2. A sub-lineage of BA.2, designated BA.5, presently has an outgrowth advantage over BA.2 and other BA.2 sub-lineages. Here we study the neutralisation of Omicron BA.1, BA.2 and BA.5 and pre-Omicron variants using a range of vaccine and convalescent sera and therapeutic monoclonal antibodies using a live virus neutralisation assay. Using primary nasopharyngeal swabs, we also tested the relative fitness of BA.5 compared to pre-Omicron and Omicron viral lineages in their ability to use the ACE2-TMPRSS2 pathway. METHODS: Using low passage clinical isolates of Clade A.2.2, Beta, Delta, BA.1, BA.2 and BA.5, we determined humoral neutralisation in vitro in vaccinated and convalescent cohorts, using concentrated human IgG pooled from thousands of plasma donors, and licensed monoclonal antibody therapies. We then determined infectivity to particle ratios in primary nasopharyngeal samples and expanded low passage isolates in a genetically engineered ACE2/TMPRSS2 cell line in the presence and absence of the TMPRSS2 inhibitor Nafamostat. FINDINGS: Peak responses to 3 doses of BNT162b2 vaccine were associated with a 9-fold reduction in neutralisation for Omicron lineages BA.1, BA.2 and BA.5. Concentrated pooled human IgG from convalescent and vaccinated donors and BNT162b2 vaccination with BA.1 breakthrough infections were associated with greater breadth of neutralisation, although the potency was still reduced 7-fold across all Omicron lineages. Testing of clinical grade antibodies revealed a 14.3-fold reduction using Evusheld and 16.8-fold reduction using Sotrovimab for the BA.5. Whilst the infectivity of BA.1 and BA.2 was attenuated in ACE2/TMPRSS2 entry, BA.5 was observed to be equivalent to that of an early 2020 circulating clade and had greater sensitivity to the TMPRSS2 inhibitor Nafamostat. INTERPRETATION: Observations support all Omicron variants to significantly escape neutralising antibodies across a range of vaccination and/or convalescent responses. Potency of therapeutic monoclonal antibodies is also reduced and differs across Omicron lineages. The key difference of BA.5 from other Omicron sub-variants is the reversion in tropism back to using the well-known ACE2-TMPRSS2 pathway, utilised efficiently by pre-Omicron lineages. Monitoring if these changes influence transmission and/or disease severity will be key for ongoing tracking and management of Omicron waves globally. FUNDING: This work was primarily supported by Australian Medical Foundation research grants MRF2005760 (ST, GM & WDR), MRF2001684 (ADK and ST) and Medical Research Future Fund Antiviral Development Call grant (WDR), Medical Research Future Fund COVID-19 grant (MRFF2001684, ADK & SGT) and the New South Wales Health COVID-19 Research Grants Round 2 (SGT).

Outcomes After Positive Syphilis Screening.

BACKGROUND: Syphilis screening during pregnancy helps prevent congenital syphilis. The harms associated with false positive (FP) screens and whether screening leads to correct treatments has not been well determined. METHODS: The population included mothers and infants from 75 056 pregnancies. Using laboratory-based criteria we classified initial positive syphilis screens as FP or true positive (TP) and calculated false discovery rates. For mothers and infants we determined treatments, clinical characteristics, and syphilis classifications. RESULTS: There were 221 positive screens: 183 FP and 38 TP. The false discovery rate was 0.83 (95% confidence interval [CI], 0.78-0.88). False discovery rates were similar for traditional 0.83 [95% CI, 0.72-0.94] and reverse algorithms 0.83 (95% CI, 0.77-0.88), and for syphilis Immunoglobin (Ig) G 0.79 (95% CI, 0.71-0.86) and total 0.90 (95% CI, 0.82-0.97) assays. FP screens led to treatment in 2 women and 1 infant. Two high-risk women were not rescreened at delivery and were diagnosed after hospital discharge; 1 infant developed congenital syphilis. Among 15 TP women with new syphilis, the diagnosis was before the late third trimester in 14 (93%). In one-half of these women, there was concern for reinfection, treatment failure, inadequate treatment or follow-up care, or late treatment, and their infants did not achieve an optimal syphilis classification. CONCLUSIONS: Syphilis screening identifies maternal syphilis, but limitations include FP screens, which occasionally lead to unnecessary treatment, inconsistent risk-based rescreening, and among TP mothers failure to optimize care to prevent birth of infants at higher risk for congenital syphilis.

Evolution of the SARS-CoV-2 omicron variants BA.1 to BA.5: Implications for immune escape and transmission.

The first dominant SARS-CoV-2 Omicron variant BA.1 harbours 35 mutations in its Spike protein from the original SARS-CoV-2 variant that emerged late 2019. Soon after its discovery, BA.1 rapidly emerged to become the dominant variant worldwide and has since evolved into several variants. Omicron is of major public health concern owing to its high infectivity and antibody evasion. This review article examines the theories that have been proposed on the evolution of Omicron including zoonotic spillage, infection in immunocompromised individuals and cryptic spread in the community without being diagnosed. Added to the complexity of Omicron's evolution are the multiple reports of recombination events occurring between co-circulating variants of Omicron with Delta and other variants such as XE. Current literature suggests that the combination of the novel mutations in Omicron has resulted in the variant having higher infectivity than the original Wuhan-Hu-1 and Delta variant. However, severity is believed to be less owing to the reduced syncytia formation and lower multiplication in the human lung tissue. Perhaps most challenging is that several studies indicate that the efficacy of the available vaccines have been reduced against Omicron variant (8-127 times reduction) as compared to the Wuhan-Hu-1 variant. The administration of booster vaccine, however, compensates with the reduction and improves the efficacy by 12-35 fold. Concerningly though, the broadly neutralising monoclonal antibodies, including those approved by FDA for therapeutic use against previous SARS-CoV-2 variants, are mostly ineffective against Omicron with the exception of Sotrovimab and recent reports suggest that the Omicron BA.2 is also resistant to Sotrovimab. Currently two new Omicron variants BA.4 and BA.5 are emerging and are reported to be more transmissible and resistant to immunity generated by previous variants including Omicron BA.1 and most monoclonal antibodies. As new variants of SARS-CoV-2 will likely continue to emerge it is important that the evolution, and biological consequences of new mutations, in existing variants be well understood.

Detection of Ganciclovir-Resistant Cytomegalovirus in a Prospective Cohort of Kidney Transplant Recipients Receiving Subtherapeutic Valganciclovir Prophylaxis.

Cytomegalovirus infection during antiviral prophylaxis occurs in transplant recipients despite individualized regimens based on renal function. Fifty kidney transplant recipients were assessed between 2016 and 2019 for valganciclovir dosing, ganciclovir exposure, cytomegalovirus infection, and genotypic resistance markers during the first year posttransplant. Ganciclovir plasma concentrations were measured using mass spectrometry. Population pharmacokinetics was used to determine individual ganciclovir exposure and to evaluate the ability of manufacturer dosing guidelines to meet therapeutic target daily area under the curve (AUC24) of 40 to 50 µg·h/mL. Full-length UL54 and UL97 were assessed using high-throughput sequencing in cytomegalovirus DNA-positive patient specimens. Valganciclovir doses administered to recipients with creatinine clearance of <40 mL/min were higher than specified by guidelines, and they were lower for recipients with creatinine clearance of ≥40 mL/min. The mean ganciclovir AUC24 was 33 ± 13 µg·h/mL, and 82% of subjects did not attain the therapeutic target. Pharmacokinetic simulations showed that the guidelines similarly could not attain the therapeutic target in 79% of individuals. Cytomegalovirus breakthrough occurred in 6% (3/50) of recipients, while 12% (6/50) developed late-onset infection. The mean AUC24s of recipients with (n = 3) and without (n = 47) infection were not significantly different (P = 0.528). However, one recipient with an AUC24 of 20 µg·h/mL acquired two UL97 ganciclovir resistance mutations. Current prophylaxis guidelines resulted in subtherapeutic ganciclovir exposure in several study recipients, including the emergence of resistance genotypes. IMPORTANCE This study examined the pharmacokinetics and viral genomic data from a prospective cohort of kidney transplant recipients undergoing valganciclovir prophylaxis for cytomegalovirus (CMV) prevention. We showed for the first time using high-throughput sequencing the detection of ganciclovir resistance mutations in breakthrough CMV infection during subtherapeutic plasma ganciclovir as indicated by the pharmacokinetic parameter daily area under the curve (AUC24). In addition, we found that current valganciclovir dosing guidelines for CMV prophylaxis are predicted to attain therapeutic targets in only 21% of recipients, which is consistent with previous pharmacokinetic studies. The novel findings of resistance mutations during subtherapeutic ganciclovir exposure presented here can inform future studies investigating the dynamics of drug selection pressure and the emergence of resistance mutations in vivo.