Microsporidial Keratitis: Need for Increased Awareness

Abstract

Since the devastation of the European silk worm industry in the 19th century, microsporidia have been recognized as important organisms. An enormous literature is available on their biology, phylogeny, classification, disease profile, diagnosis, and treatment; however, it is only recently that ophthalmologists have begun to take note of these organisms. The last two decades have seen several publications related to ocular microsporidiosis, in particular those forms affecting the cornea. Both immunocompetent and immunocompromised patients are at risk of developing corneal infections that may range from self limiting mild keratoconjunctivitis to severe stromal keratitis recalcitrant to medical treatment. Exposure to soil, muddy water, and minor trauma are possible risk factors. Although reliable prevalence data are lacking, recent studies indicate a high prevalence of microsporidial keratoconjunctivitis in the rainy season, especially in India and other countries with similar climates. For instance, a high prevalence has been documented in Singapore. We bring together the information available on ocular microsporidiosis.

Introduction

Microsporidia are diverse group of obligate intracellular parasites closely related to fungi. They first came to the attention of scientists when Carl Wilhelm von Nägeli spoke about a strange parasite associated with European silkworm disease on September 21, 1857, in a meeting at Bonn.³⁸ He called the organism Nosema bombycis. Long before microsporidia were described in human disease they were well known as pathogens of invertebrates and fish. They are normal flora in the intestine of immunocompetent individuals,⁹⁵ but the need to investigate these organisms increased with the discovery of microsporidian infections, especially gastrointestinal, in immunocompromised humans in the 1980s and 1990s.¹²⁸ The awareness of this organism among ophthalmologists was low until the 1990s when several cases of microsporidial keratoconjunctivitis associated with human immunodeficiency virus (HIV) infection were reported. In the first decade of this century there has been an upsurge of reports from various parts of the world in microsporidial infections of the eye, especially in immunocompetent individuals. We focus on the ocular infections associated with microsporidia.

Microsporidia exists as a single, highly organized spore that range in size from 1–40 μm.¹¹⁸ A normal unit membrane and two rigid extracellular walls bind the spore. Within the spore membrane is the sporoplasm, which is the infective material of microsporidia. The sporoplasm contains a single nucleus or two nuclei arranged as a diplokaryon, cytoplasm enriched with ribosomes, golgi apparatus, and the polaroplast.⁶³ Mitochondria were thought to be absent;¹²³ however, mitosome (mitochondrial remnant) has been demonstrated116, 125, 131The polaroplast is a large organization of membranes occupying the anterior part of the spore. The most obvious organelle associated with infection is the polar filament or the polar tube (diameter 0.1–0.2 μm). The polar filament is attached at the apex of the spore via an umbrella-shaped structure, called the anchoring disk, from which it extends to the posterior of the spore. The number of coils, their arrangement, and even the angle of helical tilt are conserved and diagnostic for a particular species.¹⁰⁹ The polar filament terminates at the posterior vacuole. Under appropriate conditions in a suitable host, the polar filament is discharged. Although discharge of the polar tube is well recognized, the mechanism of cell penetration is unclear. It remains uncertain whether the polar tubule functions like a hypodermic needle to inject sporoplasm into the host cytoplasm or whether it enters a new host by phagocytosis. Also, there is no evidence to suggest receptor-mediated endocytosis. Internalization of the microsporidian spores without discharge of their polar tubules occurs in cell cultures. Inside the host cell there are two distinct phases in the development of microsporidia: a proliferative phase (merogony) and a sporogonic phase (sporogony). In merogony, the sporoplasms are released from the spores and become meronts by repeated binary fissions. Meronts develop into sporonts, characterized by a dense surface coat. Sporonts multiply by multiple fissions and divide into sporoblasts that finally develop into mature spores.⁵

Microsporidiosis in humans occurs worldwide, with prevalence rates between 0% and 50% depending on the region, method of diagnosis, and demographic characteristics of the population.²¹ In regions of South America, Africa, and Asia where antiretroviral therapies are not readily accessible, microsporidiosis has been consistently identified in patients with acquired immunodeficiency syndrome (AIDS) and additional risk factors that include poor sanitary conditions and exposure to animals.4, 11, 79, 105 Microsporidiosis continues to be increasingly recognized in non-HIV-infected persons such as travellers, children, the elderly, and organ transplant recipients.2, 71, 94, 117, 129 The source of most microsporidia infections is still uncertain, but the genotypes that infect humans have now been identified in animals, suggesting that microsporidiosis is a zoonotic disease.⁸¹ Microsporidia may spread through contact with water sources and is an Environment Protection Agency microbial contaminant candidate of concern for waterborne transmission.²¹ There also appears to be foodborne transmission as a consequence of contaminated irrigation water, and organisms have been identified on lettuce, parsley, and strawberries in Costa Rica.⁸ Dairy cows' milk was shown to be positive for microsporidia, suggesting transmission through milk.⁷³

The hypothesis that resistance to microsporidiosis depends upon functional T lymphocytes is based on the greater severity of disease in AIDS patients with declining CD4⁺ T-cell levels and the development of lethal experimental microsporidia infections in mice depleted of CD4⁺ and CD8⁺ T cells.67, 92 Recent studies on experimental microsporidiosis in murine models and ex vivo human studies demonstrated the importance of the proinflammatory (Th1) cytokines such as interferon (IFN)-γ, tumor necrosis factor (TNF)-α, and interleukin (IL)-12, along with a role for nitric oxide, in resistance to Encephalitozoon spp.34, 104 CD8⁺ intraepithelial lymphocytes were observed to increase rapidly after oral administration of E. cuniculi to mice. These cells appeared to participate in proinflammatory responses via IFN-γ production and cytotoxic activity and also contributed to immune regulation via IL-10 secretion.⁹²

The number of microsporidian species is estimated to be from 1,000 to 1,500, which is likely to increase with inclusion of new hosts. Microsporidia were largely unknown as human pathogens before the HIV era. Currently, seven genera are associated with human disease: Enterocytozoon, Encephalitozoon (Septata), Nosema, Vittaforma, Pleistophora, Trachipleistophora, Anncaliia (Brachiola) and unclassified microsporidial organisms (Microsporidia). Stenhaus was the first to study electron microscopic features of the micrsporidian spores.³⁸ Traditionally, the classification of microsporidia in humans depended on transmission electron microscopy (TEM)¹¹⁰ The presence of a polar tubule classifies an organism as a member of the phylum Microspora.⁷⁴ Species differentiation using ultrastructural examinations is usually possible, but two human microsporidia (Encephalitozoon cuniculi and Encephalitozoon hellem) are similar even at the ultrastructural level. The fine structure of the spores with the unique coiled polar tube, the nature of host–parasite interface, and the method of division are criteria for diagnosis and species differentiation of microsporidia (Table 1).

An alternative classification is based on molecular sequencing. Sequence data from GenBank (SSU rRNA) has been analyzed and published in 2005 describing three classes based on habitat: Aquasporidia, Marinosporidia and Terresporidia.¹²⁴ There is as yet no agreement on this classification.⁷⁰

Encephalitozoon cuniculi, Encephalitozoon hellem, and Encephalitozoon intestinalis are three of the four most common human microsporidian parasites. In addition to humans, E. cuniculi and E. intestinalis have been found in various mammals such as rabbits, rats, mice, horses, foxes, cats, dogs, musk rats, leopards, and baboons, and E. hellem infection is common in some birds. As a result, intra-species genotyping can differentiate Encephalitozoon parasites from various hosts. Genes with repetitive sequences such as the PTP and SWP-I can be good targets for genotype analysis.¹³³ Sequence comparison of the PTP gene divided E. cuniculi into three genotypes in congruence with internal transcribed spacer (ITS) analysis. The advantage with PTP gene is the length polymorphism, which allows the development of simple polymerase chain reaction (PCR)-based genotyping tools. Genotype III has a deletion of one copy of the 78-bp central repeats, enabling it to be differentiated from genotypes I and II by electrophoresis of PCR products. In addition, genotypes I and II can be differentiated from each other by restriction fragment length polymorphism analysis, which provides a mechanism for confirmation and circumvents the need for DNA sequencing. Similar results were observed at the SWP-I gene. Length polymorphism originating from variations of repeat numbers also existed among the three E. cuniculi genotypes. This length variation was further seen within some genotypes and between different copies of the SWP-I gene, which divided the genotypes I and III into several sub genotypes.¹³³

PCR and sequence analysis of the PTP gene in E. hellem also support the use of repetitive sequences as genotyping targets. Four genotypes of E. hellem were found in the 24 isolates analyzed at this genetic locus. This typing resolution is much higher than the one produced by sequence analysis of the ITS, which yielded two genotypes. In fact, the typing resolution at the ITS locus was even lower than the sequence analysis of SSU rRNA, which divided the isolates into three genotypes. As in E. cuniculi, the PTP gene had an additional advantage of having length polymorphism, which enabled the differentiation of the E. hellem genotypes by electrophoresis of PCR products without restriction digestion or sequence analysis. No genetic heterogeneity was detected in E. intestinalis in the study by Xiao et al.¹³³ The SSU rRNA, ITS, and PTP-I genes analyzed, however, had no repetitive sequences in E. intestinalis. Results of this study indicated the existence of extensive genetic diversity in E. cuniculi and E. hellem isolates from humans. This genetic diversity was previously underestimated by the analysis of ITS sequence, but now can be assessed easily by the analysis of the repetitive region of the SWP-I or PTP gene. Genetic targets with repetitive sequences are needed for genotype analysis of E. intestinalis.¹³³Analyses of the single ITS of the rRNA genes have revealed that there is considerable genetic variation within Enterocytozoon bieneusi isolates of human and animal origins, and more than 50 genotypes have so far been described based on subtle differences within this 243-bp sequence.⁸¹ No other genetic markers are available for this group. However, ITS-based classification of isolates of E. cuniculi and E. hellem has largely been confirmed by data for other genetic loci.⁸¹ Nevertheless, additional independent markers for E.bieneusi are highly desirable in order to clarify the genetic structure of the parasite's populations. Five different ITS genotypes of E. bieneusi infecting humans have been confirmed in independent studies, and another 12 were discovered in single studies, with one study accounting for eight of these novel genotypes.¹¹² With the development of simple PCR-based genotyping tools, more extensive epidemiologic studies and characterizations of large number of isolates from humans and animals will enable the evaluation of the significance of the genetic diversity, the role of animals in human infection, and the transmission dynamics of microsporidiosis.

Although microsporidia contain unique infective organelles, they lack several structures that are usually considered hallmarks of eukaryotic life, such as typical mitochondria, peroxisomes, and centrioles. They also possess several seemingly “prokaryotic” characteristics, such as 70S ribosomes, tiny genomes, and a fused 5.8S and 28S rRNA. For these reasons, microsporidia were long thought to be a primitive or ancestral eukaryotic lineage that diverged from the universal eukaryotic ancestor before the gain of the α-proteobacterial endosymbiont, which eventually became the mitochondrion.¹⁰ This hypothesis placed microsporidia into Kingdom Archezoa, a group of organisms defined by their lack of mitochondria.¹⁰ Initially, molecular data seemed to support the inclusion of microsporidia in the Archezoa. Notably, analyses of ribosomal RNA¹²³, elongation factor 1-alpha (EF1-α), and elongation factor 2 (EF-2) sequences⁶² placed microsporidia at the base of the eukaryotic tree prior to evolution of mitochondria. Later, α- and β-tubulin phylogenies26, 64, 65 indicated a close relationship to fungi, in stark contradiction to the data supporting microsporidia as members of the Archezoa. Additional analyses conducted on mitochondrial Hsp70,45, 50, 98 TATA-box binding protein,²⁸ the largest subunit of RNA polymerase II (RPB1),⁴⁹ and pyruvate dehydrogenase subunits E1α and β²⁷ bolstered the proposed microsporidia–fungi relationship. Tanabe's EF1-α analyses¹¹³ were in concordance with early work, placing microsporidia near the base of the eukaryotic tree, whereas his RPB1 analysis placed microsporidia at the base of an animal/fungal clade. Keeling's analyses⁶⁶ placed microsporidia within the zygomycete clade.

A combined analysis reveals that branch lengths are much more conservative and consistent across fungi; however, one could still argue that the microsporidia and the ascomycetes and basidiomycetes do possess the longer branches in the tree. Nevertheless, the bootstrap support for the sisterhood of microsporidia and ascomycetes and basidiomycetes is fairly strong.⁴⁷ Although the validity of EF-1α as a phylogenetic marker to assess microsporidian relationships has seriously been called into question, Tanabe et al proposed that indel information in EF-1α could shed light on the relationship between microsporidia and fungi.¹¹³ They identified a two amino acid deletion that is present in all examined fungi, but absent in microsporidia. This implies that microsporidia and fungi are only related as sisters. Although there are currently only three EF-1α sequences from microsporidia in the NCBI database (from Antonospora locustae, E. cuniculi, and Glugea plecoglossi), they do represent a large fraction of microsporidian diversity. On its own, the indel data might not be too compelling; however, it is consistent with results of this study where the sisterhood of microsporidia and fungi could not be statistically rejected by analysis of either dataset.⁴⁷ When more EF-1α sequences become available, particularly from microsporidia considered to be basal, the usefulness of the indel as a phylogenetic marker can be more effectively evaluated. Meanwhile, using genome-wide analysis of synteny, Lee et al have shown that microsporidia are true fungi that descended from zygomycete ancestor. They also suggest that the microsporidia may have a genetically controlled sexual cycle.⁷²