Isolation and Identification of Nematode-Infecting Microsporidia.

Nematodes are naturally infected by the fungal-related pathogen microsporidia. These ubiquitous eukaryotic parasites are poorly understood, despite infecting most types of animals. Identifying novel species of microsporidia and studying them in an animal model can expedite our understanding of their infection biology and evolution. Nematodes present an excellent avenue for pursuing such work, as they are abundant in the environment and many species are easily culturable in the laboratory. The protocols presented here describe how to isolate bacterivorous nematodes from rotting substrates, screen them for microsporidia infection, and molecularly identify the nematode and microsporidia species. Additionally, we detail how to remove environmental contaminants and generate a spore preparation of microsporidia from infected samples. We also discuss potential pitfalls and provide suggestions on how to mitigate them. These protocols allow for the identification of novel microsporidia species, which can serve as an excellent starting point for genomic analysis, determination of host specificity, and infection characterization. © 2024 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Gathering samples Support Protocol 1: Generating 10× and 40× Escherichia coli OP50 and seeding NGM plates Basic Protocol 2: Microsporidia screening, testing for Caenorhabditis elegans susceptibility, and sample freezing Basic Protocol 3: DNA extraction, PCR amplification, and sequencing to identify nematode and microsporidia species Basic Protocol 4: Removal of contaminating microbes and preparation of microsporidia spores Support Protocol 2: Bleach-synchronizing nematodes.

High temperatures and low humidity promote the occurrence of microsporidians (Microsporidia) in mosquitoes (Culicidae).

BACKGROUND: In the context of climate change, a growing concern is that vector-pathogen or host-parasite interactions may be correlated with climatic factors, especially increasing temperatures. In the present study, we used a mosquito-microsporidian model to determine the impact of environmental factors such as temperature, humidity, wind and rainfall on the occurrence rates of opportunistic obligate microparasites (Microsporidia) in hosts from a family that includes important disease vectors (Culicidae). METHODS: In our study, 3000 adult mosquitoes collected from the field over 3 years were analysed. Mosquitoes and microsporidia were identified using PCR and sequencing of the hypervariable V5 region of the small subunit ribosomal RNA gene and a shortened fragment of the cytochrome c oxidase subunit I gene, respectively. RESULTS: DNA metabarcoding was used to identify nine mosquito species, all of which were hosts of 12 microsporidian species. The prevalence of microsporidian DNA across all mosquito samples was 34.6%. Microsporidian prevalence in mosquitoes was more frequent during warm months (> 19 °C; humidity < 65%), as was the co-occurrence of two or three microsporidian species in a single host individual. During warm months, microsporidian occurrence was noted 1.6-fold more often than during the cold periods. Among the microsporidians found in the mosquitoes, five (representing the genera Enterocytospora, Vairimorpha and Microsporidium) were positively correlated with an increase in temperature, whereas one (Hazardia sp.) was significantly correlated with a decrease in temperature. Threefold more microsporidian co-occurrences were recorded in the warm months than in the cold months. CONCLUSIONS: These results suggest that the susceptibility of mosquitoes to parasite occurrence is primarily determined by environmental conditions, such as, for example, temperatures > 19 °C and humidity not exceeding 62%. Collectively, our data provide a better understanding of the effects of the environment on microsporidian-mosquito interactions.

Overcoming research challenges: In vitro cultivation of Ameson portunus (Phylum Microsporidia).

Ameson portunus is an intracellular pathogen that infects marine crabs Portunus trituberculatus and Scylla paramamosain, causing significant economic losses. However, research into this important parasite has been limited due to the absence of an in vitro culture system. To address this challenge, we developed an in vitro cultivation model of A. portunus using RK13 cell line in this study. The fluorescent labeling assay indicated a high infection rate (∼60 %) on the first day post-infection and quantitative PCR (qPCR) detection demonstrated successful infection as early as six hours post-inoculation. Fluorescence in situ hybridization (FISH) and qPCR were used for the detection of A. portunus infected cells. The FISH probe we designed allowed detection of A. portunus in infected cells and qPCR assay provided accurate quantification of A. portunus in the samples. Transmission electron microscopy (TEM) images revealed that A. portunus could complete its entire life cycle and produce mature spores in RK13 cells. Additionally, we have identified novel life cycle characteristics during the development of A. portunus in RK 13 cells using TEM. These findings contribute to our understanding of new life cycle pathways of A. portunus. The establishment of an in vitro culture model for A. portunus is critical as it provides a valuable tool for understanding the molecular and immunological events that occur during infection. Furthermore, it will facilitate the development of effective treatment strategies for this intracellular pathogen.

First identification of Microsporidia MB in Anopheles coluzzii from Zinder City, Niger.

BACKGROUND: Malaria, a disease transmitted by Anopheles mosquitoes, is a major public health problem causing millions of deaths worldwide, mostly among children under the age of 5 years. Biotechnological interventions targeting parasite-vector interactions have shown that the microsporidian symbiont Microsporidia MB has the potential to disrupt and block Plasmodium transmission. METHODS: A prospective cross-sectional survey was conducted in Zinder City (Zinder), Niger, from August to September 2022, using the CDC light trap technique to collect adult mosquitoes belonging to the Anopheles gambiae complex. The survey focused on collecting mosquitoes from three neighborhoods of Zinder (Birni, Kangna and Garin Malan, located in communes I, II and IV, respectively). Collected mosquitoes were sorted and preserved in 70% ethanol. PCR was used to identify host species and detect the presence of Microsporidia MB and Plasmodium falciparum infection. RESULTS: Of the 257 Anopheles mosquitoes collected and identified by PCR, Anopheles coluzzii was the most prevalent species, accounting for 97.7% of the total. Microsporidia MB was exclusively detected in A. coluzzii, with a prevalence of 6.8% (17/251) among the samples. No significant difference in prevalence was found among the three neighborhoods. Only one An. coluzzii mosquito tested PCR-positive for P. falciparum. CONCLUSIONS: The results confirm the presence of Microsporidia MB in Anopheles mosquitoes in Zinder, Niger, indicating its potential use as a biotechnological intervention against malaria transmission. However, further studies are needed to determine the efficacy of Microsporidia MB to disrupt Plasmodium transmission as well as its impact on vector fitness.

MicroAnnot: A Dedicated Workflow for Accurate Microsporidian Genome Annotation.

With nearly 1700 species, Microsporidia represent a group of obligate intracellular eukaryotes with veterinary, economic and medical impacts. To help understand the biological functions of these microorganisms, complete genome sequencing is routinely used. Nevertheless, the proper prediction of their gene catalogue is challenging due to their taxon-specific evolutionary features. As innovative genome annotation strategies are needed to obtain a representative snapshot of the overall lifestyle of these parasites, the MicroAnnot tool, a dedicated workflow for microsporidian sequence annotation using data from curated databases of accurately annotated microsporidian genes, has been developed. Furthermore, specific modules have been implemented to perform small gene (<300 bp) and transposable element identification. Finally, functional annotation was performed using the signature-based InterProScan software. MicroAnnot's accuracy has been verified by the re-annotation of four microsporidian genomes for which structural annotation had previously been validated. With its comparative approach and transcriptional signal identification method, MicroAnnot provides an accurate prediction of translation initiation sites, an efficient identification of transposable elements, as well as high specificity and sensitivity for microsporidian genes, including those under 300 bp.

Insights into the differential molecular response of non-germinated and germinated spores of Ameson portunus in vitro by comparative transcriptome analysis.

Ameson portunus, the recently discovered causative agent of "toothpaste disease" of pond-cultured swimming crabs in China has caused enormous economic losses in aquaculture. Understanding the process of spore germination is helpful to elucidate the molecular mechanism of its invasion of host cells. Here, we obtained mature and germinating spores by isolation and purification and in vitro stimulation, respectively. Then, non-germinated and germinated spores were subjected to the comparative transcriptomic analysis to disclose differential molecular responses of these two stages. The highest germination rate, i.e., 71.45 %, was achieved in 0.01 mol/L KOH germination solution. There were 9,609 significantly differentially expressed genes (DEGs), with 685 up-regulated and 8,924 down-regulated DEGs. The up-regulated genes were significantly enriched in ribosome pathway, and the down-regulated genes were significantly enriched in various metabolic pathways, including carbohydrate metabolism, amino acid metabolism and other metabolism. The results suggested that spores require various carbohydrates and amino acids as energy to support their life activities during germination and synthesize large amounts of ribosomal proteins to provide sites for DNA replication, transcription, translation and protein synthesis of the spores of A. portunus within the host cells. Functional genes related to spore germination, such as protein phosphatase CheZ and aquaporin, were also analyzed. The analysis of transcriptome data and identification of functional genes will help to understand the process of spore germination and invasion.

Microsporidia (Encephalitozoon cuniculi) in Patients with Degenerative Hip and Knee Disease, Czech Republic.

Total joint arthroplasty is a commonly used surgical procedure in orthopedics. Revision surgeries are required in >10% of patients mainly because of prosthetic joint infection caused by bacteria or aseptic implant loosening caused by chronic inflammation. Encephalitozoon cuniculi is a microsporidium, an obligate intracellular parasite, capable of exploiting migrating proinflammatory immune cells for dissemination within the host. We used molecular detection methods to evaluate the incidence of E. cuniculi among patients who had total hip or knee arthroplasty revision. Out of 49 patients, E. cuniculi genotypes I, II, or III were confirmed in joint samples from 3 men and 2 women who had implant loosening. Understanding the risks associated with the presence of microsporidia in periprosthetic joint infections is essential for proper management of arthroplasty. Furthermore, E. cuniculi should be considered a potential contributing cause of joint inflammation and arthrosis.

Establishment of a TaqMan probe-based qPCR assay for detecting microsporidia Enterospora epinepheli in grouper.

Enterospora epinepheli is an intranuclear microsporidian parasite causing serious emaciative disease in hatchery-bred juvenile groupers (Epinephelus spp.). Rapid and sensitive detection is urgently needed as its chronic infection tends to cause emaciation as well as white faeces syndrome and results in fry mortality. This study established a TaqMan probe-based real-time quantitative PCR assays targeting the small subunit rRNA (SSU) gene of E. epinepheli. The relationship between the standard curve of cycle threshold (Ct) and the logarithmic starting quantity (SQ) was determined as Ct = -3.177 lg (SQ) + 38.397. The correlation coefficient (R2 ) was 0.999, and the amplification efficiency was 106.4%. The detection limit of the TaqMan probe-based qPCR assay was 1.0 × 101 copies/µL and that is 100 times sensitive than the traditional PCR method. There is no cross-reaction with other aquatic microsporidia such as Ecytonucleospora hepatopenaei, Nucleospora hippocampi, Potaspora sp., Ameson portunus. The intra-assay and inter-assay showed great repeatability and reproducibility. In addition, the test of clinical samples showed that this assay effectively detected E. epinepheli in the grouper's intestine tissue. The established TaqMan qPCR assays will be a valuable diagnostic tool for the epidemiological investigation as well as prevention and control of E. epinepheli.

Localization and tissue tropism of the symbiont Microsporidia MB in the germ line and somatic tissues of Anopheles arabiensis.

IMPORTANCE: Microsporidia MB is a symbiont with a strong malaria transmission-blocking phenotype in Anopheles arabiensis. It spreads in mosquito populations through mother-to-offspring and sexual transmission. The ability of Microsporidia MB to block Plasmodium transmission, together with its ability to spread within Anopheles populations and its avirulence to the host, makes it a very attractive candidate for developing a key strategy to stop malaria transmissions. Here, we report tissue tropism and localization patterns of Microsporidia MB, which are relevant to its transmission. We find that Microsporidia MB accumulates in Anopheles arabiensis tissues, linked to its sexual and vertical transmission. Its prevalence and intensity in the tissues over the mosquito life cycle suggest adaptation to maximize transmission and avirulence in Anopheles arabiensis. These findings provide the foundation for understanding the factors that may affect Microsporidia MB transmission efficiency. This will contribute to the development of strategies to maximize Microsporidia MB transmission to establish and sustain a high prevalence of the symbiont in Anopheles mosquito populations for malaria transmission blocking.

The global epidemiology of Microsporidia infection in birds: A systematic review and meta-analysis.

This study aimed to assess the global status and genetic diversity of Microsporidia infection in different birds. An online search was conducted in international databases from 1 January 1990 to 30 June 2022. A total of 34 articles (including 37 datasets) were included for the final meta-analysis. The pooled global prevalence of Microsporidia infection in birds was 14.6% (95% CI: 11.6-18.1). The highest prevalence of Microsporidia was found in wild waterfowl which was 54.5% (28.1-78.6). In terms of detection methods, the pooled prevalence was estimated to be 21.2% (95% CI: 12.1-34.4) and 13.4% (95% CI: 10.3-17.3) for using microscopic and molecular detection methods, respectively. Enterocytozoon bieneusi was the most common pathogen (24/31; 77.42% of the studies) according to PCR-based methods, and genotype D was the highest reported genotype (nine studies). In conclusion, designing strategies for the control and prevention of Microsporidia infection in birds should be recommended.

Increased virulence due to multiple infection in Daphnia leads to limited growth in 1 of 2 co-infecting microsporidian parasites.

Recent outbreaks of various infectious diseases have highlighted the ever-present need to understand the drivers of the outbreak and spread of disease. Although much of the research investigating diseases focuses on single infections, natural systems are dominated by multiple infections. These infections may occur simultaneously, but are often acquired sequentially, which may alter the outcome of infection. Using waterfleas (Daphnia magna) as a model organism, we examined the outcome of sequential and simultaneous multiple infections with 2 microsporidian parasites (Ordospora colligata and Hamiltosporidium tvaerminnensis) in a fully factorial design with 9 treatments and 30 replicates. We found no differences between simultaneous and sequential infections. However, H. tvaerminnensis fitness was impeded by multiple infection due to increased host mortality, which gave H. tvaerminnensis less time to grow. Host fecundity was also reduced across all treatments, but animals infected with O. colligata at a younger age produced the fewest offspring. As H. tvaerminnensis is both horizontally and vertically transmitted, this reduction in offspring may have further reduced H. tvaerminnensis fitness in co-infected treatments. Our findings suggest that in natural populations where both species co-occur, H. tvaerminnensis may evolve to higher levels of virulence following frequent co-infection by O. colligata.

Biogenesis, inheritance, and 3D ultrastructure of the microsporidian mitosome.

During the reductive evolution of obligate intracellular parasites called microsporidia, a tiny remnant mitochondrion (mitosome) lost its typical cristae, organellar genome, and most canonical functions. Here, we combine electron tomography, stereology, immunofluorescence microscopy, and bioinformatics to characterise mechanisms of growth, division, and inheritance of this minimal mitochondrion in two microsporidia species (grown within a mammalian RK13 culture-cell host). Mitosomes of Encephalitozoon cuniculi (2-12/cell) and Trachipleistophora hominis (14-18/nucleus) displayed incremental/non-phasic growth and division and were closely associated with an organelle identified as equivalent to the fungal microtubule-organising centre (microsporidian spindle pole body; mSPB). The mitosome-mSPB association was resistant to treatment with microtubule-depolymerising drugs nocodazole and albendazole. Dynamin inhibitors (dynasore and Mdivi-1) arrested mitosome division but not growth, whereas bioinformatics revealed putative dynamins Drp-1 and Vps-1, of which, Vps-1 rescued mitochondrial constriction in dynamin-deficient yeast (Schizosaccharomyces pombe). Thus, microsporidian mitosomes undergo incremental growth and dynamin-mediated division and are maintained through ordered inheritance, likely mediated via binding to the microsporidian centrosome (mSPB).

Differential molecular responses of hemolymph and hepatopancreas of swimming crab, Portunus trituberculatus, infected with Ameson portunus (Microsporidia).

Ameson portunus (Microsporidia) has caused serious economic losses to the aquaculture industry of swimming crab, Portunus trituberculatus. The hemolymph and hepatopancreas are the main immune organs of P. trituberculatus, and the main sites of A. portunus infection. Elucidating the response characteristics of hemolymph and hepatopancreas to microsporidian infection facilitates the development of microsporidiosis prevention and control strategy. This study performed comparative transcriptomic analysis of hemolymph (PTX/PTXA) and hepatopancreas (PTG/PTGA) of P. trituberculatus uninfected and infected with A. portunus. The results showed that there were 223 and 1309 differentially expressed genes (DEGs) in PTX/PTXA and PTG/PTGA, respectively. The lysosome pathway was significantly enriched after the invasion of the hemolymph by A. portunus. Also, immune-related genes were all significantly up-regulated in the hemolymph and hepatopancreas, suggesting that the invasion by A. portunus may activate host immune responses. Unlike hemolymph, antioxidant and detoxification-related genes were also significantly up-regulated in the hepatopancreas. Moreover, metabolism-related genes were significantly down-regulated in the hepatopancreas, suggesting that energy synthesis, resistance to pathogens, and regulation of oxidative stress were suppressed in the hepatopancreas. Hemolymph and hepatopancreas have similarity and tissue specificity to microsporidian infection. The differential genes and pathways identified in this study can provide references for the prevention and control of microsporidiosis.

Single-cell genomics reveals new rozellid lineages and supports their sister relationship to Microsporidia.

The phylum Rozellomycota has been proposed for a group of early-branching holomycotan lineages representing obligate parasites and hyperparasites of zoosporic fungi, oomycotes or phytoplankton. Given their predominantly intracellular lifestyle, rozellids are typically known from environmental ribosomal DNA data, except for the well-studied Rozella species. To date, the phylogenetic relationship between rozellids and microsporidians (Microsporidia) is not fully understood and most reliable hypotheses are based on phylogenomic analyses that incorporate the only publicly available rozellid genome of Rozella allomycis. Here, we provide genomic data of three new rozellid lineages obtained by single-cell sequencing from environmental samples and show with a phylogenomic approach that rozellids form a monophyletic group that is sister to microsporidians, corroborating the previously proposed phylum Rozellomycota. Whereas no mitochondrial genes coding for the respiratory Complex I could be found, we discovered a gene coding for a nucleotide phosphate transporter in one of the three draft genomes. The scattered absence of Complex I genes and scattered presence of nucleotide transporter genes across diverse microsporidian and rozellid lineages suggest that these adaptations to a parasitic lifestyle, which reduce the parasite's capability to synthesize ATP but enables it to steal ATP from its host, evolved independently in microsporidians and rozellids.

Exploring the highly reduced spliceosome of Pseudoloma neurophilia.

Spliceosomal introns evolved early in eukaryogenesis, originating from self-splicing group II introns that invaded the proto-eukaryotic genome1. Elements of these ribozymes, now called snRNAs (U1, U2, U4, U5, U6), were co-opted to excise these invasive elements. Prior to eukaryotic diversification, the spliceosome is predicted to have accumulated hundreds of proteins2. This early complexification has obscured our understanding of spliceosomal evolution. Reduced systems with few introns and tiny spliceosomes give insights into the plasticity of the splicing reaction and provide an opportunity to study the evolution of the spliceosome3,4. Microsporidia are intracellular parasites possessing extremely reduced genomes that have lost many, and in some instances all, introns5. In the purportedly intron-lacking genome of the microsporidian Pseudoloma neurophilia6, we identified two introns that are spliced at high levels. Furthermore, with only 14 predicted proteins, the P. neurophilia spliceosome could be the smallest known. Intriguingly, the few proteins retained are divergent compared to canonical orthologs. Even the central spliceosomal protein Prp8, which originated from the proteinaceous component of group II introns, is extremely divergent. This is unusual given that Prp8 is highly conserved across eukaryotes, including other microsporidia. All five P. neurophilia snRNAs are present, and all but U2 have diverged extensively, likely resulting from the loss of interacting proteins. Despite this divergence, U1 and U2 are predicted to pair with intron sequences more extensively than previously described. The P. neurophilia spliceosome is retained to splice a mere two introns and, with few proteins and reliance on RNA-RNA interactions, could function in a manner more reminiscent of presumed ancestral splicing.

Comparing Microsporidia-targeting primers for environmental DNA sequencing.

Metabarcoding is a powerful tool to detect classical, and well-known "long-branch" Microsporidia in environmental samples. Several primer pairs were developed to target these unique microbial parasites, the majority of which remain undetected when using general metabarcoding primers. Most of these Microsporidia-targeting primer pairs amplify fragments of different length of the small subunit ribosomal RNA (SSU-rRNA) gene. However, we lack a broad comparison of the efficacy of those primers. Here, we conducted in silico PCRs with three short-read (which amplify a few-hundred base pairs) and two long-read (which amplify over a thousand base pairs) metabarcoding primer pairs on a variety of publicly available Microsporidia sensu lato SSU-rRNA gene sequences to test which primers capture most of the Microsporidia diversity. Our results indicate that the primer pairs do result in slight differences in inferred richness. Furthermore, some of the reverse primers are also able to bind to microsporidian subtaxa beyond the classical Microsporidia, which include the metchnikovellidan Amphiamblys spp., the chytridiopsid Chytridiopsis typographi and the "short-branch" microsporidian Mitosporidium daphniae.

Title: Comparaison des amorces ciblant les Microsporidies pour le séquençage de l'ADN environnemental. Abstract: Le métabarcoding est un outil puissant pour détecter les microsporidies classiques et bien connues à « longues branches ¼ dans les échantillons environnementaux. Plusieurs paires d'amorces ont été développées pour cibler ces parasites microscopiques exceptionnels, dont la majorité restent indétectables lors de l'utilisation d'amorces générales de métabarcoding. La plupart de ces paires d'amorces ciblant les microsporidies amplifient des fragments de différentes longueurs du gène de la petite sous-unité de l'ARN ribosomal (SSU-rRNA). Cependant, nous manquons d'une comparaison générale de l'efficacité de ces amorces. Ici, pour tester quelles amorces capturent la plus grande partie de la diversité des microsporidies, nous avons réalisé des PCR in silico avec trois paires d'amorces de métabarcoding à lecture courte (qui amplifient quelques centaines de paires de bases) et deux paires d'amorces de métabarcoding à lecture longue (qui amplifient plus d'un millier de bases), sur une variété de séquences du gène SSU-rRNA de Microsporidia sensu lato accessibles au public. Nos résultats indiquent que les paires d'amorces entraînent de légères différences dans la richesse déduite. En outre, certaines des amorces inverses sont également capables de se lier à des sous-taxons de microsporidies au-delà des Microsporidia classiques, notamment les Metchnikovellidae Amphiamblys spp., le Chytridiopsida Chytridiopsis typographi et la microsporidie à « branches courtes ¼ Mitosporidium daphniae.

Development of a duplex PCR for the simultaneous detection of EHP and IHHNV and analysis of the correlation between these two pathogens.

Infectious hypodermal and hematopoietic necrosis virus (IHHNV) is one of the linearly single-stranded DNA viruses. Ecytonucleospora hepatopenaei (EHP) is an intracellular parasitic microsporidian. IHHNV and EHP are pathogens that have been widely prevalent in shrimp farming. Both of them are associated with growth retardation of the penaeid shrimp, which causes serious economic losses to shrimp farming. Shrimp can be co-infected with IHHNV and EHP. In this study, a rapid duplex polymerase chain reaction (PCR) was developed and optimized for the simultaneous detection of EHP and IHHNV. The detection limit of the duplex PCR could reach 1.5 × 102 copies for EHP and IHHNV. A total of 578 Litopenaeus vannamei samples were detected by the established duplex PCR detection method. The results suggested that 398 samples were infected with EHP, 362 samples were infected with IHHNV, and 265 samples were co-infected with EHP and IHHNV. The case-control analysis of the detected shrimp samples showed a certain synergistic effect between EHP and IHHNV.

Microsporidia.

In this Quick guide, Thomas Whelan and Naomi Fast introduce the microsporidia: obligate intracellular parasites with the most extremely reduced genomes known in eukaryotes.

Differences in the proliferation trend of 'Microsporidium' sp. PL03 in Culex pipiens and C. torrentium larvae.

Our study aimed to examine whether there are differences in the proliferation trend of microsporidia in mosquito larvae of the same genus (Culex spp.). DNA-barcoding and quantitative analyses were used to determine microsporidian rDNA copies in 'early' (L1 + L2) and 'late' (L3 + L4) Culex larvae in a natural population. In the study area, C. pipiens and C. torrentium larvae were infected by 'Microsporidium' sp. PL03 at similar levels. Infection by this microsporidian species probably elicits a notable immune response in C. pipiens, whereas in C. torrentium, it may evade or suppress the host immune response.

Metagenomic sequencing for investigation of a national keratoconjunctivitis outbreak, Israel, 2022.

BackgroundEpidemics of keratoconjunctivitis may involve various aetiological agents. Microsporidia are an uncommon difficult-to-diagnose cause of such outbreaks.AimDuring the third quarter of 2022, a keratoconjunctivitis outbreak was reported across Israel, related to common water exposure to the Sea of Galilee. We report a comprehensive diagnostic approach that identified Vittaforma corneae as the aetiology, serving as proof of concept for using real-time metagenomics for outbreak investigation.MethodsCorneal scraping samples from a clinical case were subjected to standard microbiological testing. Samples were tested by calcofluor white staining and metagenomic short-read sequencing. We analysed the metagenome for taxonomical assignment and isolation of metagenome-assembled genome (MAG). Targets for a novel PCR were identified, and the assay was applied to clinical and environmental samples and confirmed by long-read metagenomic sequencing.ResultsFluorescent microscopy was suggestive of microsporidiosis. The most abundant species (96.5%) on metagenomics analysis was V. corneae. Annotation of the MAG confirmed the species assignment. A unique PCR target in the microsporidian rRNA gene was identified and validated against the clinical sample. The assay and metagenomic sequencing confirmed V. corneae in an environmental sludge sample collected at the exposure site.ConclusionsThe real-time utilisation of metagenomics allowed species detection and development of diagnostic tools, which aided in outbreak source tracking and can be applied for future cases. Metagenomics allows a fully culture-independent investigation and is an important modality for public health microbiology.