Microsporidium - Microsporidia (Encephalitozoon cuniculi, Encephalitozoon hellem, Encephalitozoon intestinalis, Enterocytozoon bieneusi and other species) - Ubiquitous fungi, opportunistic pathogens for immunocompromised patients: Molecular diagnosis (PCR) and species identification (sequencing)
Microsporidia are a group of spore-forming unicellular parasitic fungi. Initially they were considered protozoa or protists, but currently they are considered fungi. The existence of more than 1,500 species is admitted, but it is considered that there could be more than one million. Approximately 10% of the species infect vertebrates, but all groups of animals are admitted to harbor microsporidia. Each of the recognized microsporidia species in general infects a host species or a group of related species. Several species, mostly opportunistic, also infect humans.
Microsporidia produce highly resistant spores, capable of surviving outside their host for several years. The spore morphology was considered useful for differentiating species microscopically. The spores of most species are oval or pyriform, but some are elongated or spherical.
The spore is protected by a wall, which consists of three layers: a dense external exospora; a medium, broad and seemingly unstructured endospora, containing chitin; and a thin internal plasma membrane. Within that rigid and resistant cover is the sporoplasm that houses the nucleus and the cell cytoplasm, all of which is transferred to the host cell in the infection process. In most cases, they have two closely associated nuclei, which form a diplocarion.
The anterior half of the spore contains an apparatus similar to a harpoon with a long, filament-shaped polar filament, which is wound in the posterior half of the spore. The anterior part of the polar filament is surrounded by a sheet of membranes that forms the polaroplast. Behind the polar filament, there is a posterior vacuole.
The spore is activated to start the infection process by changes in pH and in the cation/anion concentration ratio when entering the digestive tract. The activation process is followed by edema of the polaroplast and the posterior vacuole, which causes a significant increase in pressure within the rigid walls of the spore. That pressure is due to the rapid entry of water through the aquaporin channels of the plasma membrane.
The process of sporoblast entry into the cell is very similar to an intracellular injection, in which the syringe would be constituted by the rigid wall of the spore, the needle is formed by the polar tube, and the plunger would be the pressure generated within the spore by various mechanisms.
Once inside the host cell, two key phases occur in its development process. The first, a proliferative state of binary or multiple fission and a second state or sporogony, in which the spore develops and matures. During the proliferative phase, karyokinesis occurs repeatedly before cell division occurs, resulting in rounded forms of multinucleated plasmodium as in E. bieneusi or tape-like multinucleated cells as in E. intestinalis. These phases can occur freely in the cytoplasm as in E. bieneusi, or inside a parasitopic vesicle in the case of E. intestinalis.
The first genus of Microsporidium described, Nosema, was initially placed by Nägeli in the group of Schizomycetes fungi along with some bacteria and yeasts. For some time, microsporidia were considered very primitive eukaryotes, placed in the group of Cnidospora protozoa. Subsequently, especially due to the lack of mitochondria, they were placed together with other protozoa, in the group of Archezoa protozoa. In addition, spore-forming organisms in general have a complex system of reproduction, both sexual and asexual. Today, microsporidia are placed inside fungi or as a fungal-related group with a common ancestor.
Currently there is sufficient genetic, structural and metabolic evidence to place them within the fungal kingdom. Some authors suggest that they share a common ancestor with Zygomycetes and perhaps, with Mucorales.
The phylum Microsporidia comprises 150 genera that group approximately 1500 known species, 14 of them described as producing human disease.
The species that affect humans are distributed in 7 genera (Enterocytozoon, Encephalitozoon, Anncaliia, Nosema, Pleistophora, Trachipleistophora and Vitaforma) and unclassified microsporidia (collectively called Microsporidium).
Main species of pathogenic Microsporidia of animals
Urinary tract infection, symptomatic and asymptomatic intestinal infection hepatitis, peritonitis, encephalitis, urethritis, prostatitis, nephritis, sinusitis, keratoconjunctivitis, cystitis, diarrhea, cellulitis, disseminated infection. Hosts: Mammals (rodents, rabbits, carnivorous primates).
Systemic infection, keratoconjunctivitis, sinusitis, pneumonitis, nephritis, prostatitis, urethritis, cystitis, diarrhea. Host: Birds of the genus Psitasis.
Cholangiopathy, cholangitis, acalculous cholecystitis, diarrhea, intestinal perforation, nephritis, keratoconjunctivitis. Host: Mammals (donkeys, dogs, pigs, cattle, goats, primates).
Diarrhea, attrition syndrome, rhinitis, bronchitis, sinusitis, cholangiopathy, acalculous cholangitis, cholecystitis. Hosts: Mammals (pigs, primates, dogs, cats, etc.) and birds.
Microsporidiosis is an emerging and opportunistic infection. Members of that group most often affect immunocompromised people (eg. HIV/AIDS), children, the elderly or individuals who wear contact lenses.
The first human case was in 1959 in a child with encephalitis, but since then it has been described related to a large group of clinical syndromes. Eight genera of microsporidia have been reported as human disease producers: Nosema, Vittaforma, Brachiola, Pleistophora, Trachipleistophora, Encephalitozoon (including the reclassified genus Septata), Enterocytozoon and Anncalii (previously known as Brachiola). Additionally, there is a group generically called microsporidia, which includes other species that have limited information even today (Sprague and Vavra).
In the host intestine, the spore germinates, it accumulates osmotic pressure until its rigid wall breaks at its thinnest point at the apex. The posterior vacuole swells, forcing the polar filament to rapidly expel the infectious content in the cytoplasm of the potential host. Simultaneously, the filament material is rearranged to form a tube that functions as a hypodermic needle and penetrates the intestinal epithelium.
Once inside the host cell, a sporoplasm grows, which divides or forms a multinucleated plasmodium, before producing new spores. The life cycle varies considerably. Some have a simple asexual life cycle, while others have a complex life cycle that involves multiple hosts and both sexual and asexual reproduction. Different types of spores can be produced at different stages, probably with different functions, including self-infection (transmission within a single host).
In animals and humans, microsporidia often cause debilitating chronic diseases instead of lethal infections. Effects on the host include reduction of longevity, fertility, weight and overall vigor. Vertical transmission of microsporidia is frequently reported.
At least 14 species of microsporidia, distributed in eight genera, have been recognized as human pathogens.
Microsporidia can infect a variety of hosts, including hosts that are parasites. In that case, the microsporidian species is a hyperparasite, that is, a parasite of a parasite.
Microsporidiosis is common in HIV-infected patients with CD4 positive T cell counts below 100/mm3 and their spontaneous eradication can occur by the sole induction of immune reconstitution from high-efficiency antiretroviral therapy (TARVAE), which evidences the importance of the integrity of the cellular immune response in the protection against that microorganism. Experimental studies in murine models and ex-vivo studies in humans demonstrate the involvement of proinflammatory cytokines such as Interferon gamma-γ (IFN-γ), interleukin-12 (IL-12), and tumor necrosis factor alpha (TNF-alpha) in resistance to Encephalitozoon spp.
Human microsporidiosis is cosmopolitan with a prevalence that ranges between 1 and 50% depending on the geographic region, the diagnostic method, and the demographic characteristics of the population studied.
Microsporidiosis is an emerging infection in immunodeficient patients that is not exclusive to those infected with the HIV virus, it also affects recipients of organ transplants or patients undergoing immunosuppressive chemotherapy.
The sources of human infection by microsporidia are still uncertain. The spores of these microorganisms are released into the environment with feces, urine, and respiratory secretions. Infected animals or people, as well as water and food contaminated with spores appear to be the most frequent sources of infection. Person-person transmission occurs, and eye infection may be the result of self-inoculation, caused by contaminated hands.
Microsporidiosis of the gastrointestinal tract
The gastrointestinal tract is affected only by Enterocytozoon intestinalis and Enterocytozoon bieneusi. These microorganisms behave as opportunistic agents, especially in patients with Acquired Immune Deficiency Syndrome (AIDS) in whom, in addition to causing intestinal infection, they can affect other organs and systems. Infection of the gastrointestinal tract is the most frequent form of microsporidiosis, more than 90% is caused by the ingestion of Enterocytozoon bieneusi spores, and the remaining 10% is mainly due to Encephalitozoon intestinalis.
The presence of granulomatous hepatitis after infection with Encephalitozoon cuniculi has been described in HIV-infected patients. Enterocytozoon bieneusi and Encephalitozoon intestinalis infection of the biliary tract may lead to sclerosing cholangitis in AIDS patients. These observations suggest that the biliary epithelium may be the reservoir for recurrences of Enterocytozoon bieneusi.
Enterocytozoon bieneusi infection does not cause active enteritis or ulcerations, but causes flattening of the villi and hyperplasia of the crypts. The parasitic load is higher at the level of the distal duodenum and proximal jejunum and, although microorganisms can be found in the ileum, they are rarely found in the colon. The microorganism is located in the apical region of the enterocyte and the epithelial cells of the biliary and pancreatic tract. Spores are rarely found on the basal surface or on the lamina propria. Enterocytozoon bieneusi rarely spreads, unlike Encephalitizoon, which is common in the lamina propria and disseminated to visceral organs.
Infection is associated with increased intraepithelial lymphocytes and epithelial disruption. At the apex of the villi, the cells are lost causing the denudation that is characteristic of Enterocytozoon bieneusi infection.
The infection is associated with malabsorption due to decreased mucosal surface and immaturity of the epithelial cells of the villi.
Encephalitozoon intestinalis is invasive and spores are commonly found on the apical and basal side of the enterocyte, as well as in cells of the lamina propria, including fibroblasts, endothelial cells, and macrophages, this pattern is typical of other members of the genus Encephalitozoon and reflects its ability to spread to visceral organs after ingestion of their spores. Dissemination can result in intestinal air necrosis, with clinical manifestations resembling an acute abdomen.
Encephalitozoon cuniculi has been related to cases of peritonitis, in the autopsy it has been possible to appreciate masses at the level of the omentum with focal necrosis, non-granulomatous inflammation, and the presence of spores of this Microsporidium.
Microsporidiosis in immunocompetent people
Both Enterocytozoon bieneusi and Encephalitozoon intestinalis have been associated with self-limited aqueous diarrhea in immunocompetent adults and children, particularly among people who reside or travel to tropical countries.
Microsporidiosis in immunocompromised patients
In the case of infection in AIDS patients, Microsporidium was recognized as an opportunistic pathogen causing diarrhea and attrition syndrome since 1985. Chronic diarrhea is the most prevalent clinical form of Microsporidium disease in the HIV patient. It is important to know that there is a increase in disseminated forms of microsporidiosis in HIV-infected patients.
Enterocytozoon bieneusi and Encephalitozoon intestinalis are the cause of chronic diarrhea and weakness syndrome, cholangiopathy and acalculous cholecystitis in HIV-infected patients or carriers of other types of immune compromised, particularly in patients with a T-cell count below 50 cells/mL.
Enterocytozoon bieneusi is considered one of the most important intestinal microorganisms associated with HIV infection, it is present in 5-30% of diarrhea not explained by another cause, the main clinical manifestations consist of: watery diarrhea without blood, anorexia, weight loss, and edema. Some patients experience intermittent diarrhea, and excrete spores of microsporidia in the absence of diarrhea. The stool is liquid or pasty, the diarrhea worsens with the ingestion of most foods, and often patients report abdominal pain or nausea and vomiting.
Laboratory tests and clinical manifestations provide evidence of intestinal malabsorption. Diarrhea is debilitating and weight loss leads to cachexia, which is an important cause or cofactor for death. One third of patients affected by intestinal microsporidiosis have coinfection with other intestinal pathogens.
Enterocytozoon bieneusi has been detected in the biliary tree and gallbladder of patients with cholangitis and acalculous cholecystitis.
Encephalitozoon intestinalis primarily causes diarrhea, subsequently it can spread in the biliary tract causing cholangitis and cholecystitis, unlike E. bieneusi, this microorganism can spread systemically, affecting the kidney or other organs.
The diagnosis of microsporidiosis has been based on the microscopic detection of microsporidial spores in infected secretions (mainly feces) or in tissue samples. Endoscopic biopsy is no more sensitive than stool examination due to the patch characteristics of the infected intestinal mucosa.
The laboratory should be alerted to the potential diagnosis, since the routine parasite examination does not detect Microsporidium spores. There is no blood or fecal leukocytes in Microsporidium infection, when any of these elements is present, the preparation by another microorganism should be suspected.
Stool examination under an optical microscope is the standard method for the diagnosis of gastrointestinal microsporidiosis. Species must be differentiated since microsporidia that spread like Encephalitozoon spp. they are sensitive to albendazole, while Enterocytozoon bieneusi is resistant. The definitive identification of the microsporidium that caused the infection was carried out by means of the ultrastructural examination using the electron microscope, but today it is carried out through molecular techniques (species-specific polymerase chain reaction [PCR]). If the stool test is negative in the context of chronic diarrhea (more than 2 months of evolution), endoscopy should be performed.
The difference between the size of the spores of Enterocytozoon and Encephalitozoon spp, allow to establish a tentative differential diagnosis between the genera, but this must be confirmed by molecular biology techniques.
The biopsy should be considered in all patients with chronic diarrhea of more than 2 months duration and examination of stool and urine negative. In this group of patients endoscopy demonstrates Microsporidium in 30% of individuals. If Microsporidium invades the lamina propria, urine tests should be repeated since Encephalitozoon spp. t is the most likely causative agent.
Due to the increase in microsporidiosis information among relatively immunocompetent individuals, diagnostic serological methods have been developed, with the aim of detecting subclinical infections in individuals that can transmit germs to at-risk patients. Serology can also detect who is at risk of reactivation of infection, under conditions of immunocompromise.
Microsporidia are currently underdiagnosed, due to its small size and the experience required by the microscopist in the laboratory diagnosis. In clinical samples suspected of microsporidia, PCR inhibitors are frequently found that may confuse the interpretation of the results and in addition microsporidiosis is not routinely included in the differential diagnosis of diarrhea, urine samples are not evaluated for microsporidia. as a potential cause of systemic infection.
Recommended tests for diagnosis:
The diagnosis has been based on microscopic identification, antibody detection or molecular diagnostic methods (PCR). Molecular methods (PCR), offer the advantage of providing a fast and specific diagnosis.
The diagnosis has been based on a combination of signs of immune deficiency with detection of spores in affected tissues, or in feces stained with fluorescent dyes, together with elevated antibody titres. Histopathology has been the standard diagnostic method using immunohistochemical methods to establish the diagnosis of granulomatous encephalitis along with interstitial nephritis.
It is important to differentiate Encephalitozoon from Enterocytozoon because the treatments are different (Encephalitozoon can be treated with albendazole and Enterocytozoon with fumagiline).
For such reasons, molecular methods (PCR) are currently recommended.
Tests performed in IVAMI:
• Molecular diagnosis (PCR) of Microsporidium.
• Species identification (sequencing).
• Samples of other affected tissues.
Storage and sending of the sample:
• Refrigerated (preferred) for less than 2 days.
• Frozen: more than 2 days (only for molecular diagnostic tests).
• Molecular diagnosis (PCR): 24 to 48 hours.
• Species identification (sequencing): 4 to 5 working days.
Cost of the test:
• Species identification (sequencing): Consult to firstname.lastname@example.org.