Correction: Haegeman et al. Looking beyond Virus Detection in RNA Sequencing Data: Lessons Learned from a Community-Based Effort to Detect Cellular Plant Pathogens and Pests. Plants 2023, 12, 2139.

In the original publication [...].

Detection, Characterization, and Distribution of the First Case of Pepino Mosaic Virus in Aotearoa New Zealand.

Tomato (Solanum lycopersicum L., family Solanaceae) represents one of the most economically valuable horticultural crops worldwide. Tomato production is affected by numerous emerging plant viruses. We identified, for the first time in New Zealand (NZ), Pepino mosaic virus (PepMV) in greenhouse grown tomato crops using a combination of methods from electron microscopy and herbaceous indexing to RT-qPCR and high-throughput sequencing. Phylogenetic and genomic analysis of a near-complete PepMV genome determined that the detected strain belonged to the mild form of the CH2 lineage of the virus. Subsequently, a delimiting survey of PepMV was conducted, and PepMV was detected at four additional locations. PCR-derived sequences obtained from samples collected from different greenhouses and from herbaceous indicator plants were identical to the original sequence. Since PepMV has never been reported in NZ before, seed pathways are speculated to be the most likely source of entry into the country.

Looking beyond Virus Detection in RNA Sequencing Data: Lessons Learned from a Community-Based Effort to Detect Cellular Plant Pathogens and Pests.

High-throughput sequencing (HTS), more specifically RNA sequencing of plant tissues, has become an indispensable tool for plant virologists to detect and identify plant viruses. During the data analysis step, plant virologists typically compare the obtained sequences to reference virus databases. In this way, they are neglecting sequences without homologies to viruses, which usually represent the majority of sequencing reads. We hypothesized that traces of other pathogens might be detected in this unused sequence data. In the present study, our goal was to investigate whether total RNA-seq data, as generated for plant virus detection, is also suitable for the detection of other plant pathogens and pests. As proof of concept, we first analyzed RNA-seq datasets of plant materials with confirmed infections by cellular pathogens in order to check whether these non-viral pathogens could be easily detected in the data. Next, we set up a community effort to re-analyze existing Illumina RNA-seq datasets used for virus detection to check for the potential presence of non-viral pathogens or pests. In total, 101 datasets from 15 participants derived from 51 different plant species were re-analyzed, of which 37 were selected for subsequent in-depth analyses. In 29 of the 37 selected samples (78%), we found convincing traces of non-viral plant pathogens or pests. The organisms most frequently detected in this way were fungi (15/37 datasets), followed by insects (13/37) and mites (9/37). The presence of some of the detected pathogens was confirmed by independent (q)PCRs analyses. After communicating the results, 6 out of the 15 participants indicated that they were unaware of the possible presence of these pathogens in their sample(s). All participants indicated that they would broaden the scope of their bioinformatic analyses in future studies and thus check for the presence of non-viral pathogens. In conclusion, we show that it is possible to detect non-viral pathogens or pests from total RNA-seq datasets, in this case primarily fungi, insects, and mites. With this study, we hope to raise awareness among plant virologists that their data might be useful for fellow plant pathologists in other disciplines (mycology, entomology, bacteriology) as well.

Solid implantable devices for sustained drug delivery.

Implantable drug delivery systems (IDDS) are an attractive alternative to conventional drug administration routes. Oral and injectable drug administration are the most common routes for drug delivery providing peaks of drug concentrations in blood after administration followed by concentration decay after a few hours. Therefore, constant drug administration is required to keep drug levels within the therapeutic window of the drug. Moreover, oral drug delivery presents alternative challenges due to drug degradation within the gastrointestinal tract or first pass metabolism. IDDS can be used to provide sustained drug delivery for prolonged periods of time. The use of this type of systems is especially interesting for the treatment of chronic conditions where patient adherence to conventional treatments can be challenging. These systems are normally used for systemic drug delivery. However, IDDS can be used for localised administration to maximise the amount of drug delivered within the active site while reducing systemic exposure. This review will cover current applications of IDDS focusing on the materials used to prepare this type of systems and the main therapeutic areas of application.

Realising respiratory microbiomic meta-analyses: time for a standardised framework.

In microbiome fields of study, meta-analyses have proven to be a valuable tool for identifying the technical drivers of variation among studies and results of investigations in several diseases, such as those of the gut and sinuses. Meta-analyses also represent a powerful and efficient approach to leverage existing scientific data to both reaffirm existing findings and generate new hypotheses within the field. However, there are currently limited data in other fields, such as the paediatric respiratory tract, where extension of original data becomes even more critical due to samples often being difficult to obtain and process for a range of both technical and ethical reasons. Performing such analyses in an evolving field comes with challenges related to data accessibility and heterogeneity. This is particularly the case in paediatric respiratory microbiomics - a field in which best microbiome-related practices are not yet firmly established, clinical heterogeneity abounds and ethical challenges can complicate sharing of patient data. Having recently conducted a large-scale, individual participant data meta-analysis of the paediatric respiratory microbiota (n = 2624 children from 20 studies), we discuss here some of the unique barriers facing these studies and open and invite a dialogue towards future opportunities. Video Abstract.

New Virus Diagnostic Approaches to Ensuring the Ongoing Plant Biosecurity of Aotearoa New Zealand.

To protect New Zealand's unique ecosystems and primary industries, imported plant materials must be constantly monitored at the border for high-threat pathogens. Techniques adopted for this purpose must be robust, accurate, rapid, and sufficiently agile to respond to new and emerging threats. Polymerase chain reaction (PCR), especially real-time PCR, remains an essential diagnostic tool but it is now being complemented by high-throughput sequencing using both Oxford Nanopore and Illumina technologies, allowing unbiased screening of whole populations. The demand for and value of Point-of-Use (PoU) technologies, which allow for in situ screening, are also increasing. Isothermal PoU molecular diagnostics based on recombinase polymerase amplification (RPA) and loop-mediated amplification (LAMP) do not require expensive equipment and can reach PCR-comparable levels of sensitivity. Recent advances in PoU technologies offer opportunities for increased specificity, accuracy, and sensitivities which makes them suitable for wider utilization by frontline or border staff. National and international activities and initiatives are adopted to improve both the plant virus biosecurity infrastructure and the integration, development, and harmonization of new virus diagnostic technologies.

Development and Validation of a Bioinformatic Workflow for the Rapid Detection of Viruses in Biosecurity.

The field of biosecurity has greatly benefited from the widespread adoption of high-throughput sequencing technologies, for its ability to deeply query plant and animal samples for pathogens for which no tests exist. However, the bioinformatics analysis tools designed for rapid analysis of these sequencing datasets are not developed with this application in mind, limiting the ability of diagnosticians to standardise their workflows using published tool kits. We sought to assess previously published bioinformatic tools for their ability to identify plant- and animal-infecting viruses while distinguishing from the host genetic material. We discovered that many of the current generation of virus-detection pipelines are not adequate for this task, being outperformed by more generic classification tools. We created synthetic MinION and HiSeq libraries simulating plant and animal infections of economically important viruses and assessed a series of tools for their suitability for rapid and accurate detection of infection, and further tested the top performing tools against the VIROMOCK Challenge dataset to ensure that our findings were reproducible when compared with international standards. Our work demonstrated that several methods provide sensitive and specific detection of agriculturally important viruses in a timely manner and provides a key piece of ground truthing for method development in this space.

Injectable Depot Forming Thermoresponsive Hydrogel for Sustained Intrascleral Delivery of Sunitinib Using Hollow Microneedles.

Purpose: Age-related macular degeneration is a vision-threatening disorder affecting the posterior segment of the eye. Drug delivery to the posterior segment is challenging owing to the complex anatomical and physiological structure, necessitating monthly injections of antivascular endothelial growth factors. Thermoresponsive hydrogels provide sustained drug delivery and ease of injection, due to their sol-gel transition. Poly (N-isopropyl acrylamide) (PNIPAAm) is a widely researched thermoresponsive hydrogel; however, insufficient wet strength and a wide mesh network make it inept for the entrapment of small molecules. Methods: A novel approach of grafting PNIPAAm with chitosan is exploited. A chitosan concentration altered in 10%, 30%, and 50% compared to PNIPAAm is investigated for entrapment of a small-molecular weight, hydrophilic drug, sunitinib (SUN), a multiple tyrosine kinase receptor inhibitor. Furthermore, these hydrogels were characterized using 1H-NMR, FTIR, differential scanning calorimetry (DSC), and thermogravimetric analysis for chemical characterization and viscosity, swellability, syringeability, degradation, and In-vitro permeation using Franz-diffusion cell. Results: In-vitro drug release kinetics suggested that the release of SUN could be controlled with the percentage of chitosan grafting; however, gel strength (3%-5% w/v) of 30% Cs-g-PNIPAAm did not significantly affect percentage drug release. Sustained release of SUN was observed for 1 month. In-vitro permeation studies on porcine sclera suggested that a thermoresponsive gel of chitosan grafted PNIPAAm (Cs-g-PNIPAAm) was able to sustain the drug release by 40%, compared to SUN solution. Conclusions: The study indicates that the synthesized Cs-g-NIPAAm hydrogel has the potential to serve as a tailorable injectable platform for intrascleral drug delivery applications.

Characterising clinical Staphylococcus aureus isolates from the sinuses of patients with chronic rhinosinusitis.

The role of Staphylococcus aureus in the pathogenesis of the chronic sinonasal disease chronic rhinosinusitis (CRS), has not been definitively established. Comparative analyses of S. aureus isolates from CRS with those from control participants may offer insight into a possible pathogenic link between this organism and CRS. The intra- and inter-subject S. aureus strain-level diversity in the sinuses of patients with and without CRS were compared in this cross-sectional study. In total, 100 patients (CRS = 64, control = 36) were screened for S. aureus carriage. The overall carriage prevalence of S. aureus in this cohort was 24% (CRS n = 13, control n = 11). Cultured S. aureus isolates from 18 participants were strain-typed using spa gene sequencing. The bacterial community composition of the middle meatus was assessed using amplicon sequencing targeting the V3V4 hypervariable region of the bacterial 16S rRNA gene. S. aureus isolates cultured from patients were grown in co-culture with the commensal bacterium Dolosigranulum pigrum and characterised. All participants harboured a single S. aureus strain and no trend in disease-specific strain-level diversity was observed. Bacterial community analyses revealed a significant negative correlation in the relative abundances of S. aureus and D. pigrum sequences, suggesting an antagonistic interaction between these organisms. Co-cultivation experiments with these bacteria, however, did not confirm this interaction in vitro. We saw no significant associations of CRS disease with S. aureus strain types. The functional role that S. aureus occupies in CRS likely depends on other factors such as variations in gene expression and interactions with other members of the sinus bacterial community.

Microbial river-to-sea continuum: gradients in benthic and planktonic diversity, osmoregulation and nutrient cycling.

BACKGROUND: Coastal aquatic ecosystems include chemically distinct, but highly interconnected environments. Across a freshwater-to-marine transect, aquatic communities are exposed to large variations in salinity and nutrient availability as tidal cycles create periodic fluctuations in local conditions. These factors are predicted to strongly influence the resident microbial community structure and functioning, and alter the structure of aquatic food webs and biogeochemical cycles. Nevertheless, little is known about the spatial distribution of metabolic properties across salinity gradients, and no study has simultaneously surveyed the sediment and water environments. Here, we determined patterns and drivers of benthic and planktonic prokaryotic and microeukaryotic community assembly across a river and tidal lagoon system by collecting sediments and planktonic biomass at nine shallow subtidal sites in the summer. Genomic and transcriptomic analyses, alongside a suite of complementary geochemical data, were used to determine patterns in the distribution of taxa, mechanisms of salt tolerance, and nutrient cycling. RESULTS: Taxonomic and metabolic profiles related to salt tolerance and nutrient cycling of the aquatic microbiome were found to decrease in similarity with increasing salinity, and distinct trends in diversity were observed between the water column and sediment. Non-saline and saline communities adopted divergent strategies for osmoregulation, with an increase in osmoregulation-related transcript expression as salinity increased in the water column due to lineage-specific adaptations to salt tolerance. Results indicated a transition from phosphate limitation in freshwater habitats to nutrient-rich conditions in the brackish zone, where distinct carbon, nitrogen and sulfur cycling processes dominated. Phosphorus acquisition-related activity was highest in the freshwater zone, along with dissimilatory nitrate reduction to ammonium in freshwater sediment. Activity associated with denitrification, sulfur metabolism and photosynthesis were instead highest in the brackish zone, where photosynthesis was dominated by distinct microeukaryotes in water (Cryptophyta) and sediment (diatoms). Despite microeukaryotes and archaea being rare relative to bacteria, results indicate that they contributed more to photosynthesis and ammonia oxidation, respectively. CONCLUSIONS: Our study demonstrates clear freshwater-saline and sediment-water ecosystem boundaries in an interconnected coastal aquatic system and provides a framework for understanding the relative importance of salinity, planktonic-versus-benthic habitats and nutrient availability in shaping aquatic microbial metabolic processes, particularly in tidal lagoon systems. Video abstract.

Application of Oxford Nanopore Technology to Plant Virus Detection.

The adoption of Oxford Nanopore Technologies (ONT) sequencing as a tool in plant virology has been relatively slow despite its promise in more recent years to yield large quantities of long nucleotide sequences in real time without the need for prior amplification. The portability of the MinION and Flongle platforms combined with lowering costs and continued improvements in read accuracy make ONT an attractive method for both low- and high-scale virus diagnostics. Here, we provide a detailed step-by-step protocol using the ONT Flongle platform that we have developed for the routine application on a range of symptomatic post-entry quarantine and domestic surveillance plant samples. The aim of this methods paper is to highlight ONT's feasibility as a valuable component to the diagnostician's toolkit and to hopefully stimulate other laboratories towards the eventual goal of integrating high-throughput sequencing technologies as validated plant virus diagnostic methods in their own right.

Termite gas emissions select for hydrogenotrophic microbial communities in termite mounds.

Organoheterotrophs are the dominant bacteria in most soils worldwide. While many of these bacteria can subsist on atmospheric hydrogen (H2), levels of this gas are generally insufficient to sustain hydrogenotrophic growth. In contrast, bacteria residing within soil-derived termite mounds are exposed to high fluxes of H2 due to fermentative production within termite guts. Here, we show through community, metagenomic, and biogeochemical profiling that termite emissions select for a community dominated by diverse hydrogenotrophic Actinobacteriota and Dormibacterota. Based on metagenomic short reads and derived genomes, uptake hydrogenase and chemosynthetic RuBisCO genes were significantly enriched in mounds compared to surrounding soils. In situ and ex situ measurements confirmed that high- and low-affinity H2-oxidizing bacteria were highly active in the mounds, such that they efficiently consumed all termite-derived H2 emissions and served as net sinks of atmospheric H2 Concordant findings were observed across the mounds of three different Australian termite species, with termite activity strongly predicting H2 oxidation rates (R2 = 0.82). Cell-specific power calculations confirmed the potential for hydrogenotrophic growth in the mounds with most termite activity. In contrast, while methane is produced at similar rates to H2 by termites, mounds contained few methanotrophs and were net sources of methane. Altogether, these findings provide further evidence of a highly responsive terrestrial sink for H2 but not methane and suggest H2 availability shapes composition and activity of microbial communities. They also reveal a unique arthropod-bacteria interaction dependent on H2 transfer between host-associated and free-living microbial communities.

Kinetic and Structural Characterization of the First B3 Metallo-ß-Lactamase with an Active-Site Glutamic Acid.

The structural diversity in metallo-ß-lactamases (MBLs), especially in the vicinity of the active site, has been a major hurdle in the development of clinically effective inhibitors. Representatives from three variants of the B3 MBL subclass, containing either the canonical HHH/DHH active-site motif (present in the majority of MBLs in this subclass) or the QHH/DHH (B3-Q) or HRH/DQK (B3-RQK) variations, were reported previously. Here, we describe the structure and kinetic properties of the first example (SIE-1) of a fourth variant containing the EHH/DHH active-site motif (B3-E). SIE-1 was identified in the hexachlorocyclohexane-degrading bacterium Sphingobium indicum, and kinetic analyses demonstrate that although it is active against a wide range of antibiotics, its efficiency is lower than that of other B3 MBLs but has increased efficiency toward cephalosporins relative to other ß-lactam substrates. The overall fold of SIE-1 is characteristic of the MBLs; the notable variation is observed in the Zn1 site due to the replacement of the canonical His116 by a glutamate. The unusual preference of SIE-1 for cephalosporins and its occurrence in a widespread environmental organism suggest the scope for increased MBL-mediated ß-lactam resistance. Thus, it is relevant to include SIE-1 in MBL inhibitor design studies to widen the therapeutic scope of much needed antiresistance drugs.

A standardized archaeal taxonomy for the Genome Taxonomy Database.

The accrual of genomic data from both cultured and uncultured microorganisms provides new opportunities to develop systematic taxonomies based on evolutionary relationships. Previously, we established a bacterial taxonomy through the Genome Taxonomy Database. Here, we propose a standardized archaeal taxonomy that is derived from a 122-concatenated-protein phylogeny that resolves polyphyletic groups and normalizes ranks based on relative evolutionary divergence. The resulting archaeal taxonomy, which forms part of the Genome Taxonomy Database, is stable for a range of phylogenetic variables including marker gene selection, inference methods, corrections for rate heterogeneity and compositional bias, tree rooting scenarios and expansion of the genome database. Rank normalization is shown to robustly correct for substitution rates varying up to 30-fold using simulated datasets. Taxonomic curation follows the rules of the International Code of Nomenclature of Prokaryotes while taking into account proposals to formally recognize the rank of phylum and to use genome sequences as type material. This taxonomy is based on 2,392 archaeal genomes, 93.3% of which required one or more changes to their existing taxonomy, mainly owing to incomplete classification. We identify 16 archaeal phyla and reclassify 3 major monophyletic units from the former Euryarchaeota and one phylum that unites the Thaumarchaeota-Aigarchaeota-Crenarchaeota-Korarchaeota (TACK) superphylum into a single phylum.

HPLC method for levothyroxine quantification in long-acting drug delivery systems. Validation and evaluation of bovine serum albumin as levothyroxine stabilizer.

Deficiency of thyroid hormones (hypothyroidism) is treated with oral levothyroxine (LEVO). However, the effectiveness of oral administration is highly dependent on the co-administration of food and other drugs. This factor, in combination with the chronic nature of this condition, mean that there are concerns with patient compliance. Development of long acting formulations to treat hypothyroidism could potentially solve this problem. However, LEVO instability in solution could be problematic. In order to develop long acting LEVO delivery systems in vitro drug release experiments should be carried out. However, short term LEVO stability in aqueous solution will prevent this. BSA was used as a stabiliser for LEVO; extending the stability of the drug in aqueous solutions from a few hours to 2 weeks. In order to achieve this, the required concentration of the protein was 0.1% w/v. Subsequently, an HPLC method capable of separating LEVO from the protein was developed and validated following ICH guidelines. The analysis was carried out using a reverse phase HPLC method on an Agilent 1220 Infinity II LC system. The column used to achieve separation was a Zorbax Eclipse plus C18 (95 Špore size, 250 mm length x 4.6 mm internal diameter; 5 µm particle size). The mobile phase used was composed of acetonitrile and 0.1% trifluoroacetic acid at a ratio of 50:50% v/v. UV detection of LEVO sodium was carried out at 225 nm. The retention time for the drug was 6.6 minutes. The method showed a limit of detection of 0.03 µg/mL and a limit of quantification of 0.09 µg/mL. Finally, this method was used to evaluate the release from implants containing 20% w/w of LEVO. These devices were prepared using a solvent casting method with poly(caprolactone) and LEVO. These devices showed an initial burst release over the first 3 days. Subsequently, they were capable of providing a linear release rate over the following 25 days.

Chemosynthetic and photosynthetic bacteria contribute differentially to primary production across a steep desert aridity gradient.

Desert soils harbour diverse communities of aerobic bacteria despite lacking substantial organic carbon inputs from vegetation. A major question is therefore how these communities maintain their biodiversity and biomass in these resource-limiting ecosystems. Here, we investigated desert topsoils and biological soil crusts collected along an aridity gradient traversing four climatic regions (sub-humid, semi-arid, arid, and hyper-arid). Metagenomic analysis indicated these communities vary in their capacity to use sunlight, organic compounds, and inorganic compounds as energy sources. Thermoleophilia, Actinobacteria, and Acidimicrobiia were the most abundant and prevalent bacterial classes across the aridity gradient in both topsoils and biocrusts. Contrary to the classical view that these taxa are obligate organoheterotrophs, genome-resolved analysis suggested they are metabolically flexible, with the capacity to also use atmospheric H2 to support aerobic respiration and often carbon fixation. In contrast, Cyanobacteria were patchily distributed and only abundant in certain biocrusts. Activity measurements profiled how aerobic H2 oxidation, chemosynthetic CO2 fixation, and photosynthesis varied with aridity. Cell-specific rates of atmospheric H2 consumption increased 143-fold along the aridity gradient, correlating with increased abundance of high-affinity hydrogenases. Photosynthetic and chemosynthetic primary production co-occurred throughout the gradient, with photosynthesis dominant in biocrusts and chemosynthesis dominant in arid and hyper-arid soils. Altogether, these findings suggest that the major bacterial lineages inhabiting hot deserts use different strategies for energy and carbon acquisition depending on resource availability. Moreover, they highlight the previously overlooked roles of Actinobacteriota as abundant primary producers and trace gases as critical energy sources supporting productivity and resilience of desert ecosystems.

Long-acting nanoparticle-loaded bilayer microneedles for protein delivery to the posterior segment of the eye.

Treatment of neovascular ocular diseases involves intravitreal injections of therapeutic proteins using conventional hypodermic needles every 4-6 weeks. Due to the chronic nature of these diseases, these injections will be administrated to patients for the rest of their lives and their frequent nature can potentially pose a risk of sight-threatening complications and poor patient compliance. Therefore, we propose to develop nanoparticle (NP)-loaded bilayer dissolving microneedle (MN) arrays, to sustain delivery of protein drugs in a minimally invasive manner. In this research, a model protein, ovalbumin (OVA)-encapsulated PLGA NPs were prepared and optimised using a water-in-oil-in-water (W/O/W) double emulsion method. The impact of stabilisers and primary sonication time on the stability of encapsulated OVA was evaluated using an enzyme-linked immunosorbent assay (ELISA). Results showed that the lower primary sonication time was capable of sustaining release (77 days at 28.5% OVA loading) and improving the OVA bioactivity. The optimised NPs were then incorporated into a polymeric matrix to fabricate bilayer MNs and specifically concentrated into MN tips by high-speed centrifugation. Optimised bilayer MNs exhibited good mechanical and insertion properties and rapid dissolution kinetics (less than 3 min) in excised porcine sclera. Importantly, ex vivo transscleral distribution studies conducted using a multiphoton microscope confirmed the important function of MN arrays in the localisation of proteins and NPs in the scleral tissue. Furthermore, the polymers selected to prepare bilayer MNs and OVA NPs were determined to be biocompatible with retinal cells (ARPE-19). This delivery approach could potentially sustain the release of encapsulated proteins for more than two months and effectively bypass the scleral barrier, leading to a promising therapy for treating neovascular ocular diseases.

Metabolic flexibility allows bacterial habitat generalists to become dominant in a frequently disturbed ecosystem.

Ecological theory suggests that habitat disturbance differentially influences distributions of habitat generalist and specialist species. While well-established for macroorganisms, this theory has rarely been explored for microorganisms. Here we tested these principles in permeable (sandy) sediments, ecosystems with much spatiotemporal variation in resource availability and physicochemical conditions. Microbial community composition and function were profiled in intertidal and subtidal sediments using 16S rRNA gene amplicon sequencing and metagenomics, yielding 135 metagenome-assembled genomes. Community composition and metabolic traits modestly varied with sediment depth and sampling date. Several taxa were highly abundant and prevalent in all samples, including within the orders Woeseiales and Flavobacteriales, and classified as habitat generalists; genome reconstructions indicate these taxa are highly metabolically flexible facultative anaerobes and adapt to resource variability by using different electron donors and acceptors. In contrast, obligately anaerobic taxa such as sulfate reducers and candidate lineage MBNT15 were less abundant overall and only thrived in more stable deeper sediments. We substantiated these findings by measuring three metabolic processes in these sediments; whereas the habitat generalist-associated processes of sulfide oxidation and fermentation occurred rapidly at all depths, the specialist-associated process of sulfate reduction was restricted to deeper sediments. A manipulative experiment also confirmed habitat generalists outcompete specialist taxa during simulated habitat disturbance. Together, these findings show metabolically flexible habitat generalists become dominant in highly dynamic environments, whereas metabolically constrained specialists are restricted to narrower niches. Thus, an ecological theory describing distribution patterns for macroorganisms likely extends to microorganisms. Such findings have broad ecological and biogeochemical ramifications.

Bacterial Signatures of Paediatric Respiratory Disease: An Individual Participant Data Meta-Analysis.

Introduction: The airway microbiota has been linked to specific paediatric respiratory diseases, but studies are often small. It remains unclear whether particular bacteria are associated with a given disease, or if a more general, non-specific microbiota association with disease exists, as suggested for the gut. We investigated overarching patterns of bacterial association with acute and chronic paediatric respiratory disease in an individual participant data (IPD) meta-analysis of 16S rRNA gene sequences from published respiratory microbiota studies. Methods: We obtained raw microbiota data from public repositories or via communication with corresponding authors. Cross-sectional analyses of the paediatric (<18 years) microbiota in acute and chronic respiratory conditions, with >10 case subjects were included. Sequence data were processed using a uniform bioinformatics pipeline, removing a potentially substantial source of variation. Microbiota differences across diagnoses were assessed using alpha- and beta-diversity approaches, machine learning, and biomarker analyses. Results: We ultimately included 20 studies containing individual data from 2624 children. Disease was associated with lower bacterial diversity in nasal and lower airway samples and higher relative abundances of specific nasal taxa including Streptococcus and Haemophilus. Machine learning success in assigning samples to diagnostic groupings varied with anatomical site, with positive predictive value and sensitivity ranging from 43 to 100 and 8 to 99%, respectively. Conclusion: IPD meta-analysis of the respiratory microbiota across multiple diseases allowed identification of a non-specific disease association which cannot be recognised by studying a single disease. Whilst imperfect, machine learning offers promise as a potential additional tool to aid clinical diagnosis.