Molecular and clinical epidemiology of carbapenem resistant Acinetobacter baumannii, Pseudomonas aeruginosa and Enterobacterales in Fiji: a multicentre prospective observational study.

Background: Carbapenem resistant organisms (CROs) such as Acinetobacter baumannii (CRAb), Pseudomonas aeruginosa (CRPa), Escherichia coli (CREc), and Klebsiella pneumoniae (CRKp) have been identified by the World Health Organization (WHO) as global priority pathogens. The dissemination of these pathogens and clonal outbreaks within healthcare facilities are of serious concern, particularly in regions with limited resources. In Fiji, where healthcare services are primarily provided by public hospitals, understanding the extent and nature of this problem is essential for the development of effective patient management, prevention interventions and control strategies. Methods: CROs isolated from 211 (77.3%) non-sterile (urinary catheters, urine, sputum, wound swab, and endotracheal tube) and 62 (22.7%) normally sterile (blood, cerebrospinal fluid, intravascular catheter, and aspirates) body sites of 272 patients treated at the three major hospitals in Fiji, the Colonial War Memorial Hospital (CWMH), Lautoka Hospital (LTKH), and Labasa Hospital (LBSH), and outer peripheral health centres around Fiji, were analysed. Clinical and demographic patient data such as age, sex, admission diagnosis, admission and discharge dates, patient outcomes, date of death, start and end date of meropenem and colistin treatment were reviewed. These CRO isolates comprised A. baumannii, P. aeruginosa, E. coli, and K. pneumoniae, that were prospectively collected at the microbiology laboratory of CWMH and LBSH from January 2020 through August 2021 and at the LTKH from January 2020 to December 2021. In addition, 10 retrospectively stored CRPa isolates collected from patients at the CWMH from January through December 2019, were also included in the study. All isolates were characterised using mass spectrometry, antimicrobial susceptibility testing, and whole genome sequencing. Phylogenetic relationships among the CROs were assessed through core genome single nucleotide polymorphism (SNP) analysis. The CRAb isolates were also compared to the CRAb isolates from CWMH isolated in 2016/2017 and 2019, along with CRAb isolates obtained from Fijian patients admitted to New Zealand hospitals in 2020 and 2021 from our retrospective study. Findings: Of 272 patients, 140 (51.5%) were male, the median (range) age of patients was 45 (<1-89) years, 161 (59.2%) were I-Taukei, 104 (38.2%) Fijians of Indian descent, and 7 (2.6%) were from other ethnic backgrounds. 234 (86.0%) of these 272 patients, had their first positive CRO sample collected ≥72 h following admission and the remaining 38 (14.0%) were isolated within 72 h following admission. Of the 273 CROs, 146 (53.5%) were collected at the CWMH, 66 (24.2%) LTKH, and 61 (22.3%) LBSH, while 62 (22.7%) were isolated from normally sterile sites and 211 (77.3%) from sites that are not sterile. Of 273 isolates, 131 (48.0%) were CRAb, 90 (33.0%) CRPa, 46 (16.8%) CREc, and 6 (2.2%) CRKp. Of 131 CRAb, 108 (82.4%) were ST2, with three distinct clones, all encoding bla OXA-23 and bla OXA - 66, while clone 3 also encoded bla NDM-1; bla OXA-23 was associated with two copies of ISAba1 insertion element, forming the composite transposon Tn2006. The first two CRAb ST2 clones were genetically linked to those isolated at CMWH 2016 through 2019, while the third was genetically linked to isolates from Fijian patients admitted to New Zealand hospitals in 2020 and 2021. Of CRPa, 65 (72.2%) were ST773 and carried ß-lactamase genes bla NDM-1, bla OXA-50, and bla OXA-395. Of 10 retrospective CRPa isolates, all belonged to CRPa ST773 and carried bla NDM-1, bla OXA-50, and bla OXA-395. Of 46 CREc, 44 (95.7%) were ST410 and encoded bla NDM-7 on an IncX3 plasmid. Of 6 CRKp, 4 (66.7%) were ST16 and carried bla NDM-5 on an IncX3 plasmid. Other sequence types of CRPa (ST9, ST357, ST654, ST664), CRAb (ST25, ST374, ST499), CREc (ST167), and CRKp (ST45, ST336) were also detected. Of those receiving meropenem treatment in the prospective study, 30 (57.7%) received it inappropriately. Of 272 patients, 65 (23.9%) died within the 30 days after first positive CRO isolation. Interpretation: We identified nosocomial transmission of distinct clones of CRAb ST2, CRPa ST773, CREc ST410, and CRKp ST16 within and between the three major hospitals in Fiji. Moreover, community onset infections associated with CRPa, CREc, and CRAb were also detected. Of note, cross-border transmission of CRAb ST2 clone 3 strain between Fiji and New Zealand was also detected. These clones encoded an array of carbapenem resistance genes associated with mobile genetic elements, including plasmids, transposons, and integrative and conjugative elements, signifying their potential for increased mobility, further acquisition of resistance genes, and spread. Inappropriate use of meropenem was common. Of note, the majority of patients who died had acquired CRO during their hospital stay. These findings highlight the need for stringent IPC strategies focusing on catheter and ventilator management, meticulous wound care, rigorous sepsis control, consistent hand hygiene, effective use of disinfectants, and thorough sanitisation of both hospital environments and medical equipment in the three major hospitals in Fiji. Additionally, diligent surveillance of AMR and robust antimicrobial stewardship are crucial for effectively managing nosocomial infections. Funding: This project was funded by the Otago Medical School Foundations Trust (Dean's Bequest Fund) and a Fiji National University seed grant. The funders of the study had no role in the study design, data collection, data analysis, data interpretation, or writing of the report.

Molecular and clinical epidemiology of carbapenem resistant Acinetobacter baumannii ST2 in Oceania: a multicountry cohort study.

Background: Carbapenem resistant Acinetobacter baumannii (CRAb) is categorised by the World Health Organization (WHO) as a pathogen of critical concern. However, little is known about CRAb transmission within the Oceania region. This study addresses this knowledge gap by using molecular epidemiology to characterise the phylogenetic relationships of CRAb isolated in hospitals in Fiji, Samoa, and other countries within the Oceania region including Australia and New Zealand, and India from South Asia. Methods: In this multicountry cohort study, we analysed clinical isolates of CRAb collected from the Colonial War Memorial Hospital (CWMH) in Fiji from January through December 2019 (n = 64) and Tupua Tamasese Mea'ole Hospital (TTMH) in Samoa from November 2017 through June 2021 (n = 32). All isolates were characterised using mass spectrometry, antimicrobial susceptibility testing, and whole-genome sequencing. For CWMH, data were collected on clinical and demographic characteristics of patients with CRAb, duration of hospital stay, mortality and assessing the appropriateness of meropenem use from the treated patients who had CRAb infections. To provide a broader geographical context, CRAb strains from Fiji and Samoa were compared with CRAb sequences from Australia collected in 2016-2018 (n = 22), New Zealand in 2018-2021 (n = 13), and India in 2019 (n = 58), a country which has close medical links with Fiji. Phylogenetic relationships of all these CRAb isolates were determined using differences in core genome SNPs. Findings: Of CRAb isolates, 49 (77%) of 64 from Fiji and all 32 (100%) from Samoa belonged to CRAb sequence type 2 (ST2). All ST2 isolates from both countries harboured blaOXA-23, blaOXA-66 and ampC-2 genes, mediating resistance to ß-lactam antimicrobials, including cephalosporins and carbapenems. The blaOXA-23 gene was associated with two copies of ISAba1 insertion element, forming the composite transposon Tn2006, on the chromosome. Two distinct clusters (group 1 and group 2) of CRAb ST2 were detected in Fiji. The first group shared common ancestral linkage to all CRAb ST2 collected from Fiji's historic outbreak in 2016/2017, Samoa, Australia and 54% of total New Zealand isolates; they formed a single cluster with a median (range) SNP difference of 13 (0-102). The second group shared common ancestral linkage to 3% of the total CRAb ST2 isolated from India. Fifty eight of the 64 patients with CRAb infections at the CWMH had their first positive CRAb sample collected 72 h or more following admission. Meropenem use was deemed inappropriate in 15 (48%) of the 31 patients that received treatment with meropenem in Fiji. Other strains of CRAb ST1, ST25, ST107, and ST1112 were also detected in Fiji. Interpretation: We identified unrecognised outbreaks of CRAb ST2 in Fiji and Samoa that linked to strains in other parts of Oceania and South Asia. The existence of Tn2006, containing the blaOXA-23 and ISAba1 insertion element, within CRAb ST2 from Fiji and Samoa indicates the potential for high mobility and dissemination. This raises concerns about unmitigated prolonged outbreaks of CRAb ST2 in the two major hospitals in Fiji and Samoa. Given the magnitude of this problem, there is a need to re-evaluate the current strategies used for infection prevention and control, antimicrobial stewardship, and public health measures locally and internationally. Moreover, a collaborative approach to AMR surveillance within the Oceania region with technical, management and budgetary support systems is required to prevent introduction and control transmission of these highly problematic strains within the island nation health systems. Funding: This project was funded by an Otago Global Health Institute seed grant and Maurice Wilkins Centre of Research Excellence (CoREs) grant (SC0000169653, RO0000002300).

Robust immunogenicity of a third BNT162b2 vaccination against SARS-CoV-2 Omicron variant in a naïve New Zealand cohort.

The ability of a third dose of the Pfizer-BioNTech BNT162b2 SARS-CoV-2 vaccine to stimulate immune responses against subvariants, including Omicron BA.1, has not been assessed in New Zealand populations. Unlike many overseas populations, New Zealanders were largely infection naïve at the time they were boosted. This adult cohort of 298 participants, oversampled for at-risk populations, was composed of 29% Maori and 28% Pacific peoples, with 40% of the population aged 55+. A significant proportion of the cohort was obese and presented with at least one comorbidity. Sera were collected 28 days and 6 months post second vaccination and 28 days post third vaccination. SARS-CoV-2 anti-S IgG titres and neutralising capacity using surrogate viral neutralisation assays against variants of concern, including Omicron BA.1, were investigated. The incidence of SARS-CoV-2 infection, within our cohort, prior to third vaccination was very low (<6%). This study found a third vaccine significantly increased the mean SARS-CoV-2 anti-S IgG titres, for every demographic subgroup, by a minimum of 1.5-fold compared to titres after two doses. Diabetic participants experienced a greater increase (∼4-fold) in antibody titres after their third vaccination, compared to non-diabetics (increase of âˆ¼ 2-fold). This corrected for the deficiency in antibody titres within diabetic participants which was observed following two doses. A third dose also induced a neutralising response against Omicron variant BA.1, which was absent after two doses. This neutralising response improved regardless of age, BMI, ethnicity, or diabetes status. Participants aged ≥75 years consistently had the lowest SARS-CoV-2 anti-S IgG titres at each timepoint, however experienced the greatest improvement after three doses compared to younger participants. This study shows that in the absence of prior SARS-CoV-2 infection, a third Pfizer-BioNTech BNT162b2 vaccine enhances immunogenicity, including against Omicron BA.1, in a cohort representative of at-risk groups in the adult New Zealand population.

Incorporation of SARS-CoV-2 spike NTD to RBD protein vaccine improves immunity against viral variants.

Emerging SARS-CoV-2 variants pose a threat to human health worldwide. SARS-CoV-2 receptor binding domain (RBD)-based vaccines are suitable candidates for booster vaccines, eliciting a focused antibody response enriched for virus neutralizing activity. Although RBD proteins are manufactured easily, and have excellent stability and safety properties, they are poorly immunogenic compared to the full-length spike protein. We have overcome this limitation by engineering a subunit vaccine composed of an RBD tandem dimer fused to the N-terminal domain (NTD) of the spike protein. We found that inclusion of the NTD (1) improved the magnitude and breadth of the T cell and anti-RBD response, and (2) enhanced T follicular helper cell and memory B cell generation, antibody potency, and cross-reactive neutralization activity against multiple SARS-CoV-2 variants, including B.1.1.529 (Omicron BA.1). In summary, our uniquely engineered RBD-NTD-subunit protein vaccine provides a promising booster vaccination strategy capable of protecting against known SARS-CoV-2 variants of concern.

Development of real-time RT-PCR assays to detect measles virus on the Hologic Panther Fusion® System.

BACKGROUND: In 2019, Aotearoa New Zealand (NZ) experienced its worst measles outbreak since 1997. Due to declining childhood vaccination rates since the beginning of the SARS-CoV-2 pandemic, NZ is at serious risk of another major measles outbreak. Our laboratory provides diagnostic services to NZ's Southern region. In 2019 the Southern region experienced the greatest number of cases outside of Auckland and Northland, however we did not have a validated measles PCR assay in our laboratory. OBJECTIVES: We sought to develop reverse transcription real-time polymerase chain reaction (RT-PCR) assays for measles on the Hologic Panther Fusion® System by utilising its open access function. STUDY DESIGN: Previously published real-time RT-PCR assays were modified and optimised to detect wild-type measles virus (LDT-Mea), and the vaccine strain of measles virus (LDT-MeaVacA), on the Hologic Panther Fusion® System. The assays were clinically validated. RESULTS: The LDT-Mea assay has a limit of detection (LoD) of 0.1 CCID50, while the LDT-MeaVacA assay is less sensitive with a LoD of 1 CCID50. Using 27 samples, the clinical sensitivity and specificity was 100% for both assays. Other common respiratory viruses were found not to cross-react with either the LDT-Mea or LDT-MeaVacA assays. CONCLUSION: We have successfully adapted and validated for diagnostic use on the Hologic Panther Fusion® System previously published assays to detect wild-type and vaccine strains of the measles virus. The implementation of measles testing on this system will greatly improve the turn-around time for measles testing, and better support the measles public health response, for our region.

Antimicrobial Resistance in New Zealand-A One Health Perspective.

Antimicrobial resistance (AMR) is an increasing global threat that affects human, animal and, often less acknowledged, environmental health. This complex issue requires a multisectoral One Health approach to address the interconnectedness of humans, animals and the natural environment. The prevalence of AMR in these reservoirs varies widely among countries and thus often requires a country-specific approach. In New Zealand (NZ), AMR and antimicrobial usage in humans are relatively well-monitored and -understood, with high human use of antimicrobials and the frequency of resistant pathogens increasing in hospitals and the community. In contrast, on average, NZ is a low user of antimicrobials in animal husbandry systems with low rates of AMR in food-producing animals. AMR in New Zealand's environment is little understood, and the role of the natural environment in AMR transmission is unclear. Here, we aimed to provide a summary of the current knowledge on AMR in NZ, addressing all three components of the One Health triad with a particular focus on environmental AMR. We aimed to identify knowledge gaps to help develop research strategies, especially towards mitigating AMR in the environment, the often-neglected part of the One Health triad.

Characterization of the First SARS-CoV-2 Isolates from Aotearoa New Zealand as Part of a Rapid Response to the COVID-19 Pandemic.

SARS-CoV-2, the virus responsible for the COVID-19 pandemic, has wreaked havoc across the globe for the last two years. More than 300 million cases and over 5 million deaths later, we continue battling the first real pandemic of the 21st century. SARS-CoV-2 spread quickly, reaching most countries within the first half of 2020, and New Zealand was not an exception. Here, we describe the first isolation and characterization of SARS-CoV-2 variants during the initial virus outbreak in New Zealand. Patient-derived nasopharyngeal samples were used to inoculate Vero cells and, three to four days later, a cytopathic effect was observed in seven viral cultures. Viral growth kinetics was characterized using Vero and VeroE6/TMPRSS2 cells. The identity of the viruses was verified by RT-qPCR, Western blot, indirect immunofluorescence assays, and electron microscopy. Whole-genome sequences were analyzed using two different yet complementary deep sequencing platforms (MiSeq/Illumina and Ion PGM™/Ion Torrent™), classifying the viruses as SARS-CoV-2 B.55, B.31, B.1, or B.1.369 based on the Pango Lineage nomenclature. All seven SARS-CoV-2 isolates were susceptible to remdesivir (EC50 values from 0.83 to 2.42 µM) and ß-D-N4-hydroxycytidine (molnupiravir, EC50 values from 0.96 to 1.15 µM) but not to favipiravir (>10 µM). Interestingly, four SARS-CoV-2 isolates, carrying the D614G substitution originally associated with increased transmissibility, were more susceptible (2.4-fold) to a commercial monoclonal antibody targeting the spike glycoprotein than the wild-type viruses. Altogether, this seminal work allowed for early access to SARS-CoV-2 isolates in New Zealand, paving the way for numerous clinical and scientific research projects in the country, including the development and validation of diagnostic assays, antiviral strategies, and a national COVID-19 vaccine development program.

Genetic evaluation of ESBL-producing Escherichia coli urinary isolates in Otago, New Zealand.

OBJECTIVES: The incidence of infections with ESBL-producing Escherichia coli (ESBL-Ec) in New Zealand is increasing. ESBL-Ec most commonly cause urinary tract infections and are seen in both community and hospitalized patients. The reason for the increasing incidence of ESBL-Ec infections is unknown. METHODS: In this study, 65 urinary ESBL-Ec isolates from the Otago region in 2015 were fully genetically characterized to understand the mechanisms of transmission. The ESBL gene, E. coli STs, plasmid types and genetic context (e.g. insertion sequences) of ESBL genes were determined by a combination of whole genome and plasmid sequencing. The phylogenetic relationships of the isolates were compared with ESBL-Ec isolates sequenced as part of the 2016 nationwide survey. RESULTS: Significant diversity of E. coli strains, plasmids, and the genetic context of ESBL genes was seen. However, there was evidence of common mobile genetic elements in unrelated ESBL-Ec. CONCLUSIONS: Multiple introductions of ESBL resistance genes or resistant bacterial strains with limited horizontal transmission of mobile genetic elements accounts for the increased incidence of ESBL-Ec in this low prevalence area. Future studies should investigate modes of transmission of ESBL-Ec in the Otago region.

Rapid Response to SARS-CoV-2 in Aotearoa New Zealand: Implementation of a Diagnostic Test and Characterization of the First COVID-19 Cases in the South Island.

It has been 20 months since we first heard of SARS-CoV-2, the novel coronavirus detected in the Hubei province, China, in December 2019, responsible for the ongoing COVID-19 pandemic. Since then, a myriad of studies aimed at understanding and controlling SARS-CoV-2 have been published at a pace that has outshined the original effort to combat HIV during the beginning of the AIDS epidemic. This massive response started by developing strategies to not only diagnose individual SARS-CoV-2 infections but to monitor the transmission, evolution, and global spread of this new virus. We currently have hundreds of commercial diagnostic tests; however, that was not the case in early 2020, when just a handful of protocols were available, and few whole-genome SARS-CoV-2 sequences had been described. It was mid-January 2020 when several District Health Boards across New Zealand started planning the implementation of diagnostic testing for this emerging virus. Here, we describe our experience implementing a molecular test to detect SARS-CoV-2 infection, adapting the RT-qPCR assay to be used in a random-access platform (Hologic Panther Fusion® System) in a clinical laboratory, and characterizing the first whole-genome SARS-CoV-2 sequences obtained in the South Island, right at the beginning of the SARS-CoV-2 outbreak in New Zealand. We expect that this work will help us and others prepare for the unequivocal risk of similar viral outbreaks in the future.

The Immune Response to SARS-CoV-2 and Variants of Concern.

At the end of 2019 a newly emerged betacoronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), was identified as the cause of an outbreak of severe pneumonia, subsequently termed COVID-19, in a number of patients in Wuhan, China. Subsequently, SARS-CoV-2 rapidly spread globally, resulting in a pandemic that has to date infected over 200 million individuals and resulted in more than 4.3 million deaths. While SARS-CoV-2 results in severe disease in 13.8%, with increasing frequency of severe disease with age, over 80% of infections are asymptomatic or mild. The immune response is an important determinant of outcome following SARS-CoV-2 infection. While B cell and T cell responses are associated with control of infection and protection against subsequent challenge with SARS-CoV-2, failure to control viral replication and the resulting hyperinflammation are associated with severe COVID-19. Towards the end of 2020, several variants of concern emerged that demonstrate increased transmissibility and/or evasion of immune responses from prior SARS-CoV-2 infection. This article reviews what is known about the humoral and cellular immune responses to SARS-CoV-2 and how mutation and structural/functional changes in the emerging variants of concern impact upon the immune protection from prior infection or vaccination.

Comprehensive analysis of SARS-CoV-2 antibody dynamics in New Zealand.

OBJECTIVES: Circulating antibodies are important markers of previous infection and immunity. Questions remain with respect to the durability and functionality of SARS-CoV-2 antibodies. This study explored antibody responses in recovered COVID-19 patients in a setting where the probability of re-exposure is effectively nil, owing to New Zealand's successful elimination strategy. METHODS: A triplex bead-based assay that detects antibody isotype (IgG, IgM and IgA) and subclass (IgG1, IgG2, IgG3 and IgG4) responses against Nucleocapsid (N) protein, the receptor binding domain (RBD) and Spike (S) protein of SARS-CoV-2 was developed. After establishing baseline levels with pre-pandemic control sera (n = 113), samples from PCR-confirmed COVID-19 patients with mild-moderate disease (n = 189) collected up to 8 months post-infection were examined. The relationship between antigen-specific antibodies and neutralising antibodies (NAbs) was explored with a surrogate neutralisation assay that quantifies inhibition of the RBD/hACE-2 interaction. RESULTS: While most individuals had broad isotype and subclass responses to each antigen shortly after infection, only RBD and S protein IgG, as well as NAbs, were relatively stable over the study period, with 99%, 96% and 90% of samples, respectively, having responses over baseline 4-8 months post-infection. Anti-RBD antibodies were strongly correlated with NAbs at all time points (Pearson's r ≥ 0.87), and feasibility of using finger prick sampling to accurately measure anti-RBD IgG was demonstrated. CONCLUSION: Antibodies to SARS-CoV-2 persist for up to 8 months following mild-to-moderate infection. This robust response can be attributed to the initial exposure without immune boosting given the lack of community transmission in our setting.

The Rcs stress response inversely controls surface and CRISPR-Cas adaptive immunity to discriminate plasmids and phages.

Bacteria harbour multiple innate defences and adaptive CRISPR-Cas systems that provide immunity against bacteriophages and mobile genetic elements. Although some bacteria modulate defences in response to population density, stress and metabolic state, a lack of high-throughput methods to systematically reveal regulators has hampered efforts to understand when and how immune strategies are deployed. We developed a robust approach called SorTn-seq, which combines saturation transposon mutagenesis, fluorescence-activated cell sorting and deep sequencing to characterize regulatory networks controlling CRISPR-Cas immunity in Serratia sp. ATCC 39006. We applied our technology to assess csm gene expression for ~300,000 mutants and uncovered multiple pathways regulating type III-A CRISPR-Cas expression. Mutation of igaA or mdoG activated the Rcs outer-membrane stress response, eliciting cell-surface-based innate immunity against diverse phages via the transcriptional regulators RcsB and RcsA. Activation of this Rcs phosphorelay concomitantly attenuated adaptive immunity by three distinct type I and III CRISPR-Cas systems. Rcs-mediated repression of CRISPR-Cas defence enabled increased acquisition and retention of plasmids. Dual downregulation of cell-surface receptors and adaptive immunity in response to stress by the Rcs pathway enables protection from phage infection without preventing the uptake of plasmids that may harbour beneficial traits.

Human liver-derived MAIT cells differ from blood MAIT cells in their metabolism and response to TCR-independent activation.

Mucosal associated invariant T (MAIT) cells are anti-microbial innate-like T cells that are abundant in blood and liver. MAIT cells express a semi-invariant T-cell receptor (TCR) that recognizes a pyrimidine ligand, derived from microbial riboflavin synthesis, bound to MR1. Both blood and liver derived (ld)-MAIT cells can be robustly stimulated via TCR or by cytokines produced during bacterial or viral infection. In this study, we compared the functional and transcriptomic response of human blood and ld-MAIT cells to TCR signals (Escherichia coli or the pyrimidine ligand) and cytokines (IL-12 + IL-18). While the response of blood and ld-MAIT cells to TCR signals were comparable, following cytokine stimulation ld-MAIT cells were more polyfunctional than blood MAIT cells. Transcriptomic analysis demonstrated different effector programmes of ld-MAIT cells with the two modes of activation, including the enrichment of a tissue repair signature in TCR-stimulated MAIT cells. Interestingly, we observed enhancement of IL-12 signaling and fatty acid metabolism in untreated ld-MAIT cells compared with blood MAIT cells. Additionally, MAIT cells from blood and liver were modulated similarly by TCR and cytokine signals. Therefore, we report that blood and ld-MAIT cells are fundamentally different but undergo conserved changes following activation via TCR or by cytokines.

Biological functions of MAIT cells in tissues.

Mucosal associated invariant T (MAIT) cells have a recognised innate-like capacity for antibacterial host defence, consequent on the specificity of their T cell receptor (TCR) for small molecule metabolites produced by a range of prokaryotic and fungal species, their effector memory phenotype, and their expression of cytotoxic molecules. However, recent studies have identified at least two other important functions of MAIT cells in antiviral immunity and in tissue homeostasis and repair. Each are related to distinct transcriptional programmes, which are activated differentially according to the specific immune context. Here we discuss these diverse functions, we review the evidence for the newly identified role of MAIT cells in promoting tissue repair, and we discuss emerging data pointing to the future directions of MAIT cell research including roles in cancer, in antiviral immunity and recent studies in the immune response to SARS-CoV-2 infection. Overall these studies have made us aware of the potential for pleiotropic roles of MAIT cells and related cell populations in micee and humans, and have created a simple and attractive new paradigm for regulation in barrier tissues, where antigen and tissue damage are sensed, integrated and interpreted.

Uncoupling Molecular Testing for SARS-CoV-2 From International Supply Chains.

The rapid global rise of COVID-19 from late 2019 caught major manufacturers of RT-qPCR reagents by surprise and threw into sharp focus the heavy reliance of molecular diagnostic providers on a handful of reagent suppliers. In addition, lockdown and transport bans, necessarily imposed to contain disease spread, put pressure on global supply lines with freight volumes severely restricted. These issues were acutely felt in New Zealand, an island nation located at the end of most supply lines. This led New Zealand scientists to pose the hypothetical question: in a doomsday scenario where access to COVID-19 RT-qPCR reagents became unavailable, would New Zealand possess the expertise and infrastructure to make its own reagents onshore? In this work we describe a review of New Zealand's COVID-19 test requirements, bring together local experts and resources to make all reagents for the RT-qPCR process, and create a COVID-19 diagnostic assay referred to as HomeBrew (HB) RT-qPCR from onshore synthesized components. This one-step RT-qPCR assay was evaluated using clinical samples and shown to be comparable to a commercial COVID-19 assay. Through this work we show New Zealand has both the expertise and, with sufficient lead time and forward planning, infrastructure capacity to meet reagent supply challenges if they were ever to emerge.

Appropriateness of trimethoprim as empiric treatment for cystitis in 15-55 year-old women: an audit.

AIM: To assess whether trimethoprim remains an appropriate empiric treatment for uncomplicated cystitis in women 15-55 years old. METHODS: General practitioners in Auckland, Nelson-Marlborough, Otago and Southland were invited to participate in this audit of current practice. Participating general practitioners were asked to submit urine to the laboratory for microscopy and culture from any woman aged 15-55 years presenting with uncomplicated cystitis. Urine samples submitted as part of the audit were identified by a "copy to" code. Data on laboratory results were extracted from the laboratory information system. RESULTS: Data were collected from June 2016 to August 2018. Four hundred and eighty-one samples were submitted, of which 340 (70.7%) met the inclusion criteria of the audit. A urinary pathogen was identified in 181 (53.2%) specimens, of which 148 (81.8%) were E. coli, 13 (7.2%) other coliforms and 20 (11.0%) Staphylococcus saprophyticus. Of the E. coli isolates, 109 of 148 (73.6%, 95% CI 66.6-80.7) were susceptible to trimethoprim, 144 of 144 (100%, 95% CI 100-100) to nitrofurantoin and 143 of 148 (96.6%, 95% CI 93.7-99.5) to cefalexin. Of the urinary pathogens, 139 of 185 (75.1%, 95% CI 68.9-81.4) were susceptible to trimethoprim, 164 of 177 tested (92.7%, 95% CI 88.8-96.5) to nitrofurantoin and 166 of 178 tested (93.3%, 95% CI 89.6-96.9) to cefalexin. Overall, a uropathogen resistant to trimethoprim was detected in 13.5%, to nitrofurantoin in 3.8%, and to cefalexin in 3.5% of samples tested. CONCLUSION: Similar rates of resistance to trimethoprim were seen in women 15-55 years old presenting with cystitis compared with unselected samples submitted from the general community. Given the high rates of resistance, trimethoprim is no longer appropriate as an empiric treatment option for cystitis in this group. Nitrofurantoin or cefalexin are appropriate alternative empiric treatment options. Given the current recommendation that a urine sample should not be submitted to the laboratory from women with uncomplicated cystitis, ongoing audits will be required to ensure that empiric treatment recommendations remain appropriate.

One Health Aotearoa: a transdisciplinary initiative to improve human, animal and environmental health in New Zealand.

There is increased recognition that complex health challenges at the human-animal-environmental interface require a transdisciplinary, "whole-of-society" approach. This philosophy is particularly pertinent in Aotearoa-New Zealand because of the country's relatively isolated island ecosystem, economic reliance on agriculture and its intensification, and existing indigenous worldview that emphasises holism and interconnectivity between humans, animals and the environment. In New Zealand, the One Health Aotearoa (OHA) alliance was established in order to better connect researchers and to address a growing number of infectious diseases challenges. The emphasis of OHA is to bring together and facilitate interactions between people from diverse disciplines, link to stakeholders and communities, and engage with policy-makers, government operational agencies, and funders, thus providing a holistic and integrative systems-thinking approach to address priority questions and achieve desired outcomes in One Health. The initial focus of OHA has been on infectious diseases, but there is increasing recognition of the potential benefits of the alliance to address broader complex issues. Greater involvement and overlap of the environmental sciences, human and animal health sciences, social science, and indigenous kaupapa Maori research is particularly critical for ensuring its success within the New Zealand context. Given the economic and cultural importance of New Zealand's "clean, green" image, a One Health approach that draws strongly on the environmental sciences makes particular sense. Furthermore, as the global environment becomes increasingly stressed by anthropogenic pressures our research may hold potential solutions for similar challenges elsewhere.