[Advances in clinical diagnosis and treatment of microsporidial keratitis].

Microsporidia, a unicellular eukaryotic microorganism, poses a risk of infecting the eyes, precipitating microsporidia keratitis (MK). This condition typically manifests in two forms: microsporidian keratoconjunctivitis (MKC) or stromal keratitis (MSK). While MKC often resolves spontaneously, it can progress to MSK, a vision-threatening condition that, in severe instances, may lead to corneal perforation. Epidemiological studies reveal that MK is prevalent in Southeast Asia, particularly during the rainy season. Diagnosis encompasses a range of methods, including corneal scraping for microbiological analysis, PCR testing, and advanced imaging techniques such as AS-OCT and IVCM. Therapeutic approaches vary, with MKC typically managed through local and systemic drug therapy, while MSK may necessitate more aggressive interventions, including corneal transplantation. In China, MK case reports are scarce, and physicians still grapple with a lack of comprehensive understanding regarding its clinical presentation, diagnostic strategies, and treatment options. This deficit can lead to missed or misdiagnoses, as well as overtreatment. Consequently, this review endeavors to comprehensively outline the epidemiology, etiology, clinical features, diagnostic modalities, and therapeutic interventions for MK, thereby offering valuable insights and guidance for clinical practice.

A comparative analysis of blood and faecal-based laboratory methods in the diagnosis of extraintestinal microsporidia infection.

Diagnosis of extraintestinal microsporidiosis is always hampered due to non-specific symptoms and difficulty in diagnosis. This study aimed to compare the diagnostic utility of blood and faecal-based polymerase chain reaction (PCR) to detect microsporidiosis in immunocompromised patients. A total of 42 immunocompromised patients consisting of HIV-infected and chemotherapy-treated patients were enrolled. Paired faecal and blood samples were collected and subjected to PCR to detect Enterocytozoon bieneusi and Encephalitozoon spp. Faecal samples were microscopically screened for microsporidia spores. Overall, 42.9% (18/42) of patients were positive for microsporidiosis. Of this, 19.0% (8/42) and 4.8% (2/42) were positive by blood and stool PCR respectively. Meanwhile, 33.3% (14/42) of the faecal specimens were microscopically positive. Among the positive patients, 22.2% (4/18) had microsporidia confirmed by blood PCR and stool microscopy, suggestive of dissemination. Interestingly, the stool specimen in which microsporidia spores were detected via microscopy is not positive via PCR method. This highlights the limitation of the faecal-based detection method and the important use of blood samples for diagnosing extraintestinal microsporidiosis. Only E. bieneusi species were detected in all PCR-positive samples. This study highlights the diagnostic value of blood PCR in diagnosing extraintestinal microsporidiosis infections.

Paospora carinifang n. gen., n. sp. (Microsporidia: Spragueidae), a parasite of the ridgetail white prawn, Palaemon carinicauda.

A new microsporidian disease of the pond-reared ridgetail white prawn, Palaemon carinicauda, was found in China. Light microscopy, pathology, and scanning electron microscopy showed that the parasite infected the host's skeletal muscle tissue and formed spherical sporophorous vesicles (SPOVs). Electron microscopy revealed that its merogonic life stages developed in direct contact with the host cytoplasm. The sporogonic life stages underwent octosporoblastic sporogony with the formation of eight uninucleate spores in each SPOV. Fresh SPOVs were 5.4 ± 0.55 µm in diameter. The octospores were oval and measured 2.3 × 1.5 µm (fresh) and 1.96 × 1.17 µm (fixed). The isofilar polar filament was coiled with 9-10 turns and arranged in two rows. Phylogenetic analysis based on the SSU rRNA gene suggests that this microsporidium has close affinities with members of the genera Potaspora and Apotaspora, but represents an independent generic taxon. We therefore propose the establishment of a new genus and species (Paospora carinifang n. gen., n. sp.) within the family Spragueidae. We also propose a taxonomic revision to transfer Potaspora macrobrachium to this new genus and reclassify it as Paospora macrobrachium comb. nov.

Host-specific effects of a generalist parasite of mosquitoes.

Microsporidians are obligate parasites of many animals, including mosquitoes. Some microsporidians have been proposed as potential agents for the biological control of mosquitoes and the diseases they transmit due to their detrimental impact on larval survival and adult lifespan. To get a more complete picture of their potential use as agents of biological control, we measured the impact of Vavraia culicis on several life-history traits of Aedes aegypti and Anopheles gambiae. We measured the infection dynamics and clearance rate for the two species, and we assessed sexual dimorphism in infection dynamics within each species. Our results show differences in infection dynamics, with Ae. aegypti life-history traits being more affected during its aquatic stage and exhibiting higher clearance of the infection as adults. In contrast, An. gambiae was unable to clear the infection. Additionally, we found evidence of sexual dimorphism in parasite infection in An. gambiae, with males having a higher average parasite load. These findings shed light and improve our knowledge of the infection dynamics of V. culicis, a microsporidian parasite previously recognized as a potential control agent of malaria.

Microsporidian EnP1 alters host cell H2B monoubiquitination and prevents ferroptosis facilitating microsporidia survival.

Microsporidia are intracellular eukaryotic pathogens that pose a substantial threat to immunocompromised hosts. The way these pathogens manipulate host cells during infection remains poorly understood. Using a proximity biotinylation strategy we established that microsporidian EnP1 is a nucleus-targeted effector that modifies the host cell environment. EnP1's translocation to the host nucleus is meditated by nuclear localization signals (NLSs). In the nucleus, EnP1 interacts with host histone H2B. This interaction disrupts H2B monoubiquitination (H2Bub), subsequently impacting p53 expression. Crucially, this inhibition of p53 weakens its control over the downstream target gene SLC7A11, enhancing the host cell's resilience against ferroptosis during microsporidian infection. This favorable condition promotes the proliferation of microsporidia within the host cell. These findings shed light on the molecular mechanisms by which microsporidia modify their host cells to facilitate their survival.

Microsporidian coinfection reduces fitness of a fungal pathogen due to rapid host mortality.

Infection outcomes can be strongly context dependent, shifting a host-symbiont relationship along a parasitism-mutualism continuum. Numerous studies show that under stressful conditions, symbionts that are typically mutualistic can become parasitic. The reverse possibility, a parasite becoming mutualistic, has received much less study. We investigated whether the parasitic microsporidium Ordospora pajunii can become beneficial for its host Daphnia dentifera in the presence of the more virulent fungal pathogen Metschnikowia bicuspidata. We found that, even though infection with O. pajunii reduces the frequency of penetration of M. bicuspidata spores into the host body cavity, it does not improve the survival or reproduction of the host; conversely, coinfection increased the mortality of Daphnia. This shorter lifespan of coinfected hosts disrupted the life cycle of M. bicuspidata, greatly reducing its fitness. Thus, coinfection with both pathogens was detrimental to the host at the individual level but might be beneficial for the host population as a result of greatly reduced production of M. bicuspidata spores. If so, this would mean that O. pajunii outbreaks should delay or prevent M. bicuspidata outbreaks. In support of this, in an analysis of dynamics of naturally occurring outbreaks in two lakes where these pathogens co-occur, we found a time lag in occurrence between O. pajunii and M. bicuspidata, with M. bicuspidata epidemics only occurring after the collapse of O. pajunii epidemics. Thus, these results suggest that the interaction between co-occurring symbionts, and the net impact of a symbiont on a host, might be qualitatively different at different scales.IMPORTANCEUnderstanding the factors that modify infection probability and virulence is crucial for identifying the drivers of infection outbreaks and modeling disease epidemic progression, and increases our ability to control diseases and reduce the harm they cause. One factor that can strongly influence infection probability and virulence is the presence of other pathogens. However, while coexposures and coinfections are incredibly common, we still have only a limited understanding of how pathogen interactions alter infection outcomes or whether their impacts are scale dependent. We used a system of one host and two pathogens to show that sequential coinfection can have a tremendous impact on the host and the infecting pathogens and that the outcome of (co-)infection can be negative or positive depending on the focal organization level.

Effect of Microsporidia MB infection on the development and fitness of Anopheles arabiensis under different diet regimes.

BACKGROUND: Microsporidia MB (MB) is a naturally occurring symbiont of Anopheles and has recently been identified as having a potential to inhibit the transmission of Plasmodium in mosquitoes. MB intensity is high in mosquito gonads, with no fitness consequences for the mosquito, and is linked to horizontal (sexual) and vertical (transovarial) transmission from one mosquito to another. Maximising MB intensity and transmission is important for maintaining heavily infected mosquito colonies for experiments and ultimately for mosquito releases. We have investigated how diet affects the MB-Anopheles arabiensis symbiosis phenotypes, such as larval development and mortality, adult size and survival, as well as MB intensity in both larvae and adults. METHODS: F1 larvae of G0 females confirmed to be An. arabiensis and infected with MB were either combined (group lines [GLs]) or reared separately (isofemale lines [IMLs]) depending on the specific experiment. Four diet regimes (all mg/larva/day) were tested on F1 GLs: Tetramin 0.07, Tetramin 0.3, Gocat 0.3 and Cerelac 0.3. GLs reared on Tetramin 0.3 mg/larva/day were then fed either a 1% or 6% glucose diet to determine adult survival. Larvae of IMLs were fed Tetramin 0.07 mg and Tetramin 0.3 mg for larval experiments. The mosquitoes in the adult experiments with IMLs were reared on 1% or 6% glucose. RESULTS: Amongst the four larval diet regimes tested on An. arabiensis development in the presence of MB, the fastest larval development highest adult emergence, largest body size of mosquitoes, highest prevalence and highest density of MB occurred in those fed Tetramin 0.3 mg/larva/day. Although adult MB-positive mosquitoes fed on 6% glucose survived longer than MB-negative mosquitoes, there was no such effect for those fed on the 1% glucose diet. Development time, wing length and adult survival were not significantly different between MB-infected and uninfected An. arabiensis fed on the Tetramin 0.07 mg/larva/day diet, demonstrating that the MB-conferred fitness advantage was diet-dependent. CONCLUSIONS: Microsporidia MB does not adversely impact the development and fitness of An. arabiensis, even under limited dietary conditions. The diet regime of Tetramin 0.3 mg/larva/day + 6% glucose for adults is the superior diet for the mass rearing of MB-infected An. arabiensis mosquitoes. These results are important for rearing MB-infected An. arabiensis in the laboratory for experiments and the mass rearing required for field releases.

Nematocida displodere mechanosensitive ion channel of small conductance 2 assembles into a unique 6-channel super-structure in vitro.

Mechanosensitive ion channels play an essential role in reacting to environmental signals and sustaining cell integrity by facilitating ion flux across membranes. For obligate intracellular pathogens like microsporidia, adapting to changes in the host environment is crucial for survival and propagation. Despite representing a eukaryote of extreme genome reduction, microsporidia have expanded the gene family of mechanosensitive ion channels of small conductance (mscS) through repeated gene duplication and horizontal gene transfer. All microsporidian genomes characterized to date contain mscS genes of both eukaryotic and bacterial origin. Here, we investigated the cryo-electron microscopy structure of the bacterially derived mechanosensitive ion channel of small conductance 2 (MscS2) from Nematocida displodere, an intracellular pathogen of Caenorhabditis elegans. MscS2 is the most compact MscS-like channel known and assembles into a unique superstructure in vitro with six heptameric MscS2 channels. Individual MscS2 channels are oriented in a heterogeneous manner to one another, resembling an asymmetric, flexible six-way cross joint. Finally, we show that microsporidian MscS2 still forms a heptameric membrane channel, however the extreme compaction suggests a potential new function of this MscS-like protein.

Study on intestinal parasitic infections and gut microbiota in cancer patients at a tertiary teaching hospital in Malaysia.

Intestinal parasitic infections (IPIs) can lead to significant morbidity and mortality in cancer patients. While they are unlikely to cause severe disease and are self-limiting in healthy individuals, cancer patients are especially susceptible to opportunistic parasitic infections. The gut microbiota plays a crucial role in various aspects of health, including immune regulation and metabolic processes. Parasites occupy the same environment as bacteria in the gut. Recent research suggests intestinal parasites can disrupt the normal balance of the gut microbiota. However, there is limited understanding of this co-infection dynamic among cancer patients in Malaysia. A study was conducted to determine the prevalence and relationship between intestinal parasites and gut microbiota composition in cancer patients. Stool samples from 134 cancer patients undergoing active treatment or newly diagnosed were collected and examined for the presence of intestinal parasites and gut microbiota composition. The study also involved 17 healthy individuals for comparison and control. Sequencing with 16S RNA at the V3-V4 region was used to determine the gut microbial composition between infected and non-infected cancer patients and healthy control subjects. The overall prevalence of IPIs among cancer patients was found to be 32.8%. Microsporidia spp. Accounted for the highest percentage at 20.1%, followed by Entamoeba spp. (3.7%), Cryptosporidium spp. (3.0%), Cyclospora spp. (2.2%), and Ascaris lumbricoides (0.8%). None of the health control subjects tested positive for intestinal parasites. The sequencing data analysis revealed that the gut microbiota diversity and composition were significantly different in cancer patients than in healthy controls (p < 0.001). A significant dissimilarity was observed in the bacterial composition between parasite-infected and non-infected patients based on Bray-Curtis (p = 0.041) and Jaccard (p = 0.021) measurements. Bacteria from the genus Enterococcus were enriched in the parasite-infected groups, while Faecalibacterium prausnitzii reduced compared to non-infected and control groups. Further analysis between different IPIs and non-infected individuals demonstrated a noteworthy variation in Entamoeba-infected (unweighted UniFrac: p = 0.008), Cryptosporidium-infected (Bray-Curtis: p = 0.034) and microsporidia-infected (unweighted: p = 0.026; weighted: p = 0.019; Jaccard: p = 0.031) samples. No significant dissimilarity was observed between Cyclospora-infected groups and non-infected groups. Specifically, patients infected with Cryptosporidium and Entamoeba showed increased obligate anaerobic bacteria. Clostridiales were enriched with Entamoeba infections, whereas those from Coriobacteriales decreased. Bacteroidales and Clostridium were found in higher abundance in the gut microbiota with Cryptosporidium infection, while Bacillales decreased. Additionally, bacteria from the genus Enterococcus were enriched in microsporidia-infected patients. In contrast, bacteria from the Clostridiales order, Faecalibacterium, Parabacteroides, Collinsella, Ruminococcus, and Sporosarcina decreased compared to the non-infected groups. These findings underscore the importance of understanding and managing the interactions between intestinal parasites and gut microbiota for improved outcomes in cancer patients.

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.

A novel neurotropic microsporidium from the swamp guppy Micropoecilia picta from Grenada, West Indies.

A novel microsporidium was observed in wild swamp guppies Micropoecilia picta from Levera Pond within Levera National Park Grenada, West Indies. Initial observations indicated similarity with Pseudoloma neurophilia, an important pathogen in zebrafish Danio rerio. P. neurophilia exhibit broad host specifity, including members of the family Poecillidae, and both parasites infect the central nervous system. However, spore morphology and molecular phylogeny based on rDNA showed that the swamp guppy microsporidium (SGM) is distinct from P. neurophilia and related microsporidia (Microsporidium cerebralis and M. luceopercae). Spores of the SGM were smaller than others in the clade (3.6 µm long). Differences were also noted in histology; the SGM formed large aggregates of spores within neural tissues along with a high incidence of numerous smaller aggregates and single spores within the surface tissue along the ventricular spaces that extended submeninx, whereas P. neurophilia and M. cerebralis infect deep into the neuropile and cause associated lesions. Analysis of small subunit ribosomal DNA sequences showed that the SGM was <93% similar to these related microsporidia. Nevertheless, one of 2 commonly used PCR tests for P. neurophilia cross reacted with tissues infected with SGM. These data suggest that there could be other related microsporidia capable of infecting zebrafish and other laboratory fishes that are not being detected by these highly specific assays. Consequently, exclusive use of these PCR tests may not accurately diagnose other related microsporidia infecting animals in laboratory and ornamental fish facilities.

Phylogeny, morphology, virulence, ecology, and host range of Ordospora pajunii (Ordosporidae), a microsporidian symbiont of Daphnia spp.

The impacts of microsporidia on host individuals are frequently subtle and can be context dependent. A key example of the latter comes from a recently discovered microsporidian symbiont of Daphnia, the net impact of which was found to shift from negative to positive based on environmental context. Given this, we hypothesized low baseline virulence of the microsporidian; here, we investigated the impact of infection on hosts in controlled conditions and the absence of other stressors. We also investigated its phylogenetic position, ecology, and host range. The genetic data indicate that the symbiont is Ordospora pajunii, a newly described microsporidian parasite of Daphnia. We show that O. pajunii infection damages the gut, causing infected epithelial cells to lose microvilli and then rupture. The prevalence of this microsporidian could be high (up to 100% in the lab and 77% of adults in the field). Its overall virulence was low in most cases, but some genotypes suffered reduced survival and/or reproduction. Susceptibility and virulence were strongly host-genotype dependent. We found that North American O. pajunii were able to infect multiple Daphnia species, including the European species Daphnia longispina, as well as Ceriodaphnia spp. Given the low, often undetectable virulence of this microsporidian and potentially far-reaching consequences of infections for the host when interacting with other pathogens or food, this Daphnia-O. pajunii symbiosis emerges as a valuable system for studying the mechanisms of context-dependent shifts between mutualism and parasitism, as well as for understanding how symbionts might alter host interactions with resources. IMPORTANCE: The net outcome of symbiosis depends on the costs and benefits to each partner. Those can be context dependent, driving the potential for an interaction to change between parasitism and mutualism. Understanding the baseline fitness impact in an interaction can help us understand those shifts; for an organism that is generally parasitic, it should be easier for it to become a mutualist if its baseline virulence is relatively low. Recently, a microsporidian was found to become beneficial to its Daphnia hosts in certain ecological contexts, but little was known about the symbiont (including its species identity). Here, we identify it as the microsporidium Ordospora pajunii. Despite the parasitic nature of microsporidia, we found O. pajunii to be, at most, mildly virulent; this helps explain why it can shift toward mutualism in certain ecological contexts and helps establish O. pajunii is a valuable model for investigating shifts along the mutualism-parasitism continuum.

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.

Quantification of the dark fungal taxon Cryptomycota using qPCR.

Fungi are present in a wide variety of natural environments, and in the last years, various studies have shown that they are quite abundant in aquatic ecosystems. In addition, a whole new highly diverse phylum, the Cryptomycota, was discovered. Nevertheless, research on aquatic fungi and a detailed evaluation of their functions and distribution are still sparse. One of the main reasons is a limitation in reliable identification and quantification methods. To bridge part of the research gap, this study aims to implement a quantitative PCR method to detect and quantify the newly discovered phylum. We developed and validated a Cryptomycota-specific qPCR primer pair targeting the 5.8S region that detects the majority of Cryptomycota, but Microsporidia. The resulting amplicon is 102 bp long. We used different environmental samples to evaluate the primer pair, various fungal sequences as negative control and positive control sequences. Obtained amplicons were sequenced using Illumina, and the obtained ASVs were all classified as Cryptomycota. The qPCR method works reliably and specifically for the quantification of Cryptomycota in environmental samples.

Willingness to accept and participate in a Microsporidia MB-based mosquito release strategy: a community-based rapid assessment in western Kenya.

BACKGROUND: Microsporidia MB, an endosymbiont naturally found in Anopheles mosquitoes inhibits transmission of Plasmodium and is a promising candidate for a transmission-blocking strategy that may involve mosquito release. A rapid assessment was carried out to develop insight into sociodemographic factors, public health concerns, and malaria awareness, management, and prevention practices with the willingness to accept and participate in Microsporidia MB-based transmission-blocking strategy to develop an informed stakeholder engagement process. METHODS: The assessment consisted of a survey conducted in two communities in western Kenya that involved administering a questionnaire consisting of structured, semi-structured, and open questions to 8108 household heads. RESULTS: There was an overall high level of willingness to accept (81%) and participate in the implementation of the strategy (96%). Although the willingness to accept was similar in both communities, Ombeyi community was more willing to participate (OR 22, 95% CI 13-36). Women were less willing to accept (OR 0.8, 95% CI 0.7-0.9) compared to men due to fear of increased mosquito bites near homes. Household heads with incomplete primary education were more willing to accept (OR 1.6, 95% CI 01.2-2.2) compared to those educated to primary level or higher. Perceiving malaria as a moderate or low public health issue was also associated with a lower willingness to accept and participate. Experience of > 3 malaria cases in the family over the last six months and knowledge that malaria is transmitted by only mosquito bites, increased the willingness to accept but reduced the willingness to participate. Awareness of malaria control methods based on mosquitoes that cannot transmit malaria increases the willingness to participate. CONCLUSION: The study showed a high level of willingness to accept and participate in a Microsporidia MB-based strategy in the community, which is influenced by several factors such as community, disease risk perception, gender, education level, knowledge, and experience of malaria. Further research will need to focus on understanding the concerns of women, educated, and employed community members, and factors that contribute to the lower disease risk perception. This improved understanding will lead to the development of an effective communication strategy.

Keratic Precipitates: An Overlooked Sign of Microsporidial Stromal Keratitis?

PURPOSE: To elucidate a distinctive clinical feature in cases of microsporidial stromal keratitis (MSK). METHOD: A retrospective observational study of cases with a histopathological and/or microbiological diagnosis of MSK on corneal biopsy or host corneal button between 2016 and 2022 was conducted. RESULTS: Eighteen cases with a confirmed histopathological and/or microbiological diagnosis of MSK were detected. Careful review of slit-lamp photographs revealed the presence of pigmented keratic precipitates (KPs) beyond the area of stromal keratitis in five out of eighteen cases (27.7%). CONCLUSION: The presence of pigmented KPs beyond the area of lesion can alert the clinician to keep microsporidia as a differential cause for stromal keratitis. Management can be tailored accordingly for a better outcome.

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.

Staining blindly: an update on coloring techniques for fecal smears in parasitology: a scoping review.

Dye application for parasite highlighting in the Ova and Parasite exam is a common practice in parasitology diagnosis. Methods: A scoping review investigated how staining solutions interact with parasite structures. After screening 1334 papers, 35 met eligibility criteria. Results: Differentiating background from foreground in the fecal smear under light microscopy is the core of the research on this topic. Refractivity, unevenness of staining, size and temperature were explored to enhance staining protocols. Cryptosporidium spp. and Microsporidia were the main studied species. Conclusion: Studies on diagnostic efficacy outperform those that elucidate the physical-chemical interaction between dyes and parasites. An alternative approach involves technicians using computational tools to reduce subjectivity in fecal smear interpretation, deviating from conventional methods.

What is this article about? Coloring parasites during fecal exams has been widely used to find parasites in human feces. We searched for articles that could help us to answer the question: 'How do dyes give color to parasites?'. Then, we filtered the information from a total of 1334 articles to 35.What were the results? Cryptosporidium spp. and Microsporidia are microbes that can be seen only through a microscope. Researchers were interested in these two species in the last 40 years. Differentiating parasites from dirt on a glass slide is the main problem researchers are trying to solve. The way the light goes through parasites under a microscope, variation of staining, size and temperature of dyes have been explored to identify what gives better results in coloring protocols.What do the results of the study mean? Little is known about the chemical interaction between dyes and parasites. On the other hand, there are many studies on how good coloring methods are and comparing protocols. An alternative to the conventional approaches in staining parasites is the use of computational tools to reduce doubt in the exam interpretation by technicians.

FIVE YEARS OF PARTULA SNAIL PRE-EXPORT HEALTH SCREENING.

Between 2015 and 2019, a health screening was carried out annually on captive-bred Partula snails prior to export for reintroduction as part of an international effort to repopulate areas of French Polynesia, where the snails were extinct or critically endangered. In total, 129 separate tank populations of 12 different species were screened at ZSL London Zoo. Wet mounts and smears stained with modified Ziehl-Neelsen (MZN) of 535 fecal samples were examined, and 45% contained flagellated protozoa, and 35.5% had MZN-positive oocysts, measuring 3-5 µm in diameter. Smaller (2 µm) presumptive spores, MZN-positive bacilli, ciliated protozoa and nematodes were recorded less frequently. Fecal bacterial culture yielded mixed species, with a clear predominance of Myroides species (88.9% of samples). The MZN-positive oocysts (3-5 µm) were present in 6.5% of impression smears from the apices of 432 snails examined postmortem, plus acid-fast bacilli in a few cases, but no 2 µm spores. Mixed bacteria were cultured from coelomic swabs, with Myroides species again the most common (63.5%). Histologic examination was carried out on 292 snails. Autolysis affected almost 90% of those found dead but only 3.4% of euthanized snails. Histology commonly identified microsporidial sporocysts in the digestive gland and midgut epithelium of all but two species. Intracellular, extracytoplasmic Cryptosporidium-like organisms were also common in the midgut but were only observed when snails were fixed in 10% formalin (2017-2019), not ethanol. There were no clear pathologic changes associated with either organism. Pigmented hemocytic nodules were commonly observed, most frequently in the foot process; these were either age related or evidence of prior chronic inflammatory reaction and of low clinical significance. With no evidence of poor health and no significant organisms found, a total of 4,978 individuals representing 12 species were exported for reintroduction.