Katherine Bond

Background: The gold standard for identifying non-tuberculous mycobacteria (NTM) is partial sequencing of one or more distinct gene loci and determining the closest match through reference database matching. The advent of whole-genome sequencing (WGS) allows for higher accuracy in resolving Mycobacterium species, by matching a larger genome, although at increased cost. Determining when to employ WGS for NTM identification in diagnostic laboratories is a challenge, particularly when only a minority of isolated NTM are clinically significant. Case Summary: A 5-year-old patient with inflammatory arthritis, previously responsive to intra-articular glucocorticoid injections, presented with a chronic, painful, hot, swollen left knee. An NTM isolated from synovial fluid was initially reported as Mycobacterium intracellulare subsp. chimaera , based on gene-loci 16S rRNA and internal transcribed spacer (rRNA 16S-23S ITS) PCR and sequencing. WGS subsequently determined the NTM to best match a single, novel Mycobacterium species collected from a patient with lung disease from South Korea, 2018 (NCBI upload 2022). Phylogenetic analysis determined the isolate from our case to share the most recent ancestry with Mycobacterium nebraskense , and clustered in a sister clade to that of Mycobacterium avium intracellulare complex. The patient’s symptoms improved after a prolonged course of rifabutin, azithromycin, and moxifloxacin, with samples culture-negative 7 months following treatment. Conclusion: We describe a rare NTM as the cause of a significant joint infection in a 5-year-old child. WGS was instrumental in defining the diagnosis and should be considered early for accurate NTM identification in significant infections, those non-responsive to treatment, or where iatrogenic or outbreak-related cases are a consideration.

The WHO mutation catalog is the reference for genotypic antimicrobial susceptibility predictions (ASPs) for Mycobacterium tuberculosis . To increase sensitivity, WHO endorsed additional grading rules to classify mutations with insufficient experimental evidence. For rifampin, this includes any non-silent rpoB mutation in the rifampin resistance-determining region (RRDR). For delamanid and pretomanid, the rules include loss-of-function (LoF) variants in six resistance genes, including fbiC . Here, we describe three edge cases in which these additional grading rules overcalled resistance. The first case was a low-frequency rpoB variant in RRDR caused by a sequencing artifact. The second case involved a tandem repeat region starting at the end of fbiC . Because the H37Rv reference genome carries 2.7 repeats, deletion of one or two full repeats caused mapping errors, making these deletions appear as minority variants that trigger the fbiC LoF rule. However, assembly showed that these deletions did not affect the fbiC coding region because the remaining repeat substituted for the deleted repeats. The third case was a fbiC frameshift in lineage 4.6.1 Uganda genotype strains, caused by a 19 bp duplication upstream of the same tandem repeat region. Although this frameshift affects the fbiC coding region and meets the fbiC LoF rule, sequence assembly revealed only a modest impact on the end of fbiC . Although catalog performance remains high, WHO and others have acknowledged that, in specific situations, exceptions to its additional grading rules may result in unintended overcalling of resistance. Thus, systematic external quality control and communication of edge cases are essential. IMPORTANCE: Antimicrobial susceptibility predictions (ASPs) for Mycobacterium tuberculosis using next-generation sequencing and the WHO mutation catalog are increasingly used for clinical decision-making. This avoids systematic false-resistant results of real-time PCR- or hybridization-based ASP assays caused by mutations that do not confer resistance (e.g., synonymous rifampin resistance-determining region [RRDR] mutations) and, potentially, subsequent inappropriate treatment adjustments. However, we demonstrate that false-resistant results occur even using the WHO mutation catalog for rifampin, delamanid, and pretomanid. Similar exceptions linked to the additional grading rules of the catalog are possible for other drugs, underlining the importance of monitoring the performance of the catalog continuously.

Streptococcus dysgalactiae subspecies equisimilis (SDSE) is increasingly recognized as a cause of human disease. Despite decades of penicillin use, SDSE is near universally penicillin-susceptible. Here we report and characterize by whole genome sequencing (WGS) three penicillin-resistant SDSE isolates (minimum inhibitory concentration [MIC] ≥0.256 mg/L) isolated from blood and skin in an individual with recurrent cellulitis on penicillin prophylaxis. The isolates carried 11 penicillin-binding protein (PBP) PBP2x, five PBP1a, and one PBP2b mutations that were distinct from the only previously reported WGS-characterized penicillin-resistant SDSE isolates ( n = 4). We then took a genotype-led approach to test the MICs of penicillin and amoxicillin for 63 isolates with PBP2x, PBP1a, and PBP2b variants selected from a well-characterized database of invasive SDSE isolates from Australia (2011–2023, n = 436). PBPs were highly conserved in Australian invasive SDSE genomes. Only a single isolate carrying a PBP2x variant with five mutations around the penicillin-binding cleft found in 1/436 invasive isolates demonstrated a four-fold rise in penicillin MIC, although remaining below clinical breakpoints. Strains with PBP2b and PBP1a variants alone were not associated with raised MICs. To provide broader context for PBP variants, we then examined a global database of SDSE genomes ( n = 1,160). PBP2x mutations in isolates with raised and/or resistant penicillin MICs were found in only nine global SDSE database genomes. SDSE penicillin resistance is rare and associated with multiple PBP mutations, particularly PBP2x. WGS with phenotypic testing of novel PBP variants could be used to monitor emerging mutations associated with raised penicillin MICs.

SUMMARY: Pathogen genomics, including whole-genome sequencing (WGS) and clinical metagenomics, is a transformative technology increasingly being implemented in clinical microbiology, including in hospital laboratories. Pathogen genomics can improve the control of healthcare-associated infections, provide rapid infection diagnosis, and could enable replacement of laborious microbiology tests. To date, real-world implementation of pathogen genome sequencing has primarily been limited to public health laboratories, but sequencing in the clinical microbiology setting has the potential to provide advantages, including turnaround time and ability to focus on local priorities. In this review, we consider the factors that represent barriers to, and potential enablers of, the implementation of pathogen genomics in clinical microbiology, including the availability of funding and genomics-trained staff. We outline key use cases and implementation models of pathogen genomics in clinical microbiology and suggest a broad framework for labs commencing sequencing. Finally, we consider future opportunities, including direct-from-specimen sequencing, the role of machine learning in genomics analysis, and the application of pathogen genomics to clinical decision support.

Background: In the absence of commercial assays, laboratories have relied on laboratory-developed tests (LDTs) for BK polyomavirus (BKPyV) monitoring; however, variation in assay designs and calibration hinder consistent interpretation. Objective: To evaluate analytical performance (linearity, precision, specificity) of Alinity m BKV and compare qualitative, quantitative agreement and workflow efficiency with two validated LDTs. Methods: Linearity (1 - 6 log10 IU/mL), precision (%CV), and analytical specificity were assessed. Plasma (n=390) and urine (n=57) specimens were tested. Agreement was evaluated using overall/positive/negative percent agreement (OPA/PPA/NPA), Cohen’s kappa (κ), Deming regression and Bland-Altman analysis. Workflow metrics were derived from Alinity m system logs and LDT’s standard operating times. Results: Alinity m BKV showed excellent linearity (r=1.000) and robust precision across matrices, with within-/between-run variability ≤7.5% CV, and no cross-reactivity. In plasma, OPA was 92% (κ = 0.77) versus RealStar and 97.1% (κ=0.90) versus ELITe BKV LDT. Quantitative correlation was strong (r =0.931 and r =0.873, respectively) with negative mean biases (Alinity m BKV – LDT) of -1.31 and -0.84 log10 IU/mL. In urine, qualitative agreement was 100%, quantitative correlation r = 0.900, and minimal mean bias (+0.11 log10 IU/mL). Total analytic processing time was reduced by ~111 minutes (~46%) versus RealStar and ~81 minutes (~39%) compared to ELITe LDT. Conclusion: Alinity m BKV assay provides robust analytical performance, high qualitative agreement and shorter processing time than LDTs. Systematic quantitative differences were observed in plasma, likely reflecting differences in calibration strategy and assay processing as well as unit conversion (copies/mL to IU/mL for LDTs).

The availability of early serological assays for COVID-19 infection allows investigation of aspects of SARS-CoV-2 transmission which may not be evident on PCR testing. Here we describe two serological surveys in different settings in 2020 which assess the effectiveness of local infection prevention practices: i) a retrospective cohort of household contacts previously quarantined; and ii) health care workers in a tertiary hospital. Serological testing of household contacts did not diagnose any additional cases to that which had been notified by testing symptomatic contacts with PCR. The secondary household attack rate for these 53 contacts from 42 households was 36.2% (95% CI: 37.4, 74.5%). Sero-positivity in health care workers increased from 0.5% in May 2020 to 3.8-5.5% in August to October 2020. Multivariate analysis demonstrated that male HCW were less likely to be sero-positive (OR 0.19 95% CI 0.03-0.7, p=0.04), while there was an increased risk of sero-positivity associated with nursing staff (OR 8.1 95% CI 1.7-145.6, p=0.04), staff who experience anosmia (OR 22.7, 95% CI 5.4-112.4, p < 0.001); and those caring for COVID-19 patients (OR 4.1 95% CI 1.8-10.5, p=0.01). In this low prevalence setting with rapid access to PCR testing, serological testing provided a small additional benefit to PCR testing alone. Measures to detect COVID-19 cases in household contacts were found to be sufficient, while infection control measures during a period with a high burden of cases in a tertiary hospital were found to be inadequate to prevent COVID-19 infections in health care workers.

Solid tumor malignancy (STM) patients experience increased risk of breakthrough SARS-CoV-2 infection owing to reduced COVID-19 vaccine immunogenicity. However, the underlying immunological causes of impaired neutralization remain poorly characterized. Furthermore, non-neutralizing antibody functions can contribute to reduced disease severity but remain understudied within high-risk populations. We dissected polyfunctional antibody responses in STM patients and age-matched controls who received adenoviral vector- or mRNA-based COVID-19 vaccine regimens. Elevated inflammatory biomarkers, including agalactosylated IgG, interleukin (IL)-6, IL-18, and an expanded population of CD11c ⁻ CD21 ⁻ double negative 3 (DN3) B cells were observed in STM patients. These inflammatory biomarkers were associated with impaired neutralization and reduced IgG targeting the antigenically novel receptor binding domain. In contrast, mRNA vaccination induced Fc effector functions that were comparable in patients and controls and were cross-reactive against SARS-CoV-2 variants, supported by robust generation of IgG against the antigenically conserved spike 2 domain in STM patients. These data highlight the resilience of Fc functional antibodies and identify systemic inflammatory biomarkers that may underpin impaired neutralizing antibody responses, suggesting potential avenues for immunomodulation via rational vaccine design.

Repeated mRNA COVID-19 vaccination increases spike-specific immunoglobulin G4 (IgG4) titers. Here, we characterized the influence of increased IgG4 titers on a range of Fc-mediated responses. Elevated spike-specific IgG4 reduced binding to FcγRIIIa and decreased antibody-dependent cellular cytotoxicity. However, in individuals with lower total spike-specific IgG, IgG4 acted in synergy with other IgG subclasses to improve FcγRI and FcγRIIa binding and consequently antibody-dependent cellular phagocytosis. Furthermore, this trend was more pronounced with more recent SARS-CoV-2 variants where vaccination induced comparably lower total spike-specific titers. These observations were further confirmed by in silico modeling where antibody subclass concentrations and FcγR polymorphisms were modulated. Collectively, we illustrate that the impact of elevated IgG4 titers upon Fc functions is dependent on multiple interconnected antibody and antigen factors, which should be taken into consideration when dissecting the mechanisms driving an effective Fc-mediated response following vaccination.

Introduction: Substantial population-level variation in vaccine-specific antibody responses has been observed following global coronavirus disease 2019 (COVID-19) vaccination efforts. Beyond the influence of clinical and demographic features, immunogenetic variation is suggested to underlie divergent serological responses following COVID-19 vaccination of distinct populations. Methods: Immunoglobulin G1 (IgG1) allotypic markers (G1m) for 121 COVID-19 vaccinated healthy adults were genotyped via Sanger sequencing. Vaccine-specific IgG and Fc gamma receptor (FcγR) engagement were characterised via bead-based multiplex array. Results: Following two COVID-19 vaccine doses, G1m1,17 ^+/+ compared to G1m-1,3 ^+/+ vaccinees had increased IgG and FcγR engagement specific for the antigenically conserved SARS-CoV-2 Spike 2 (S2) domain. IgG targeting antigenically novel SARS-CoV-2 receptor binding domain (RBD) trended higher in G1m1,17 ^+/+ vaccinees, facilitating increased RBD-specific FcγR2a-R131 and FcγR2b binding. Conclusion: Primary COVID-19 vaccination induced increased S2-specific IgG in G1m1,17 ^+/+ vaccinees, potentially facilitating enhanced anti-viral FcγR activation. Validation in larger cohorts may inform optimisation of next-generation vaccination strategies.

Outcomes are presented for a multisite retrospective case series, describing a contemporary cohort of 22 immunocompromised patients with persistent coronavirus disease 2019 (COVID-19) polymerase chain reaction positivity who were retreated with antiviral therapy. For those with data available 14 and 30 days after commencement of antiviral therapy, 41% (9 of 22) and 68% (15 of 22), respectively, cleared COVID-19.