Contact information:

Lily Cheng '06, clily@stanford.edu

Stanford University

Parasites and Pestilence: Infectious Public Health Challenges

Prof. Dr. Scott Smith, ssmith@stanford.edu

 

Above Image : Scanning electron micrograph showing spore groups and

ejected polar filaments from the microsporidium Bohuslavia asterias.

Image from http://www.biol.lu.se/cellorgbiol/microsporidia/index.html

 

 

 

 

Introduction


Microsporidia are eukaryotic, unicellular organisms belonging to the phylum Microspora. All microspoidia are obligate, spore-forming, intracellular parasites that invade vertebrates and invertebrates. A characteristic feature of microsporidia is the polar tube or polar filament found in the spore used to infiltrate host cells. They are widely distributed in nature with over 1200 species characterized. However, microsporidia have only recently been documented to parasite humans, and more research is needed to understand this emerging infectious disease. Infection in humans, Microsporidiosis, is primarily found in patients with compromised immune systems, especially those infected with Human Immunodeficiency Virus (HIV) or have undergone organ transplants.  Some species, however, have also been known to parasite those with health immune systems. Beyond the human realm, Microsporidia are important parasites in fisheries, veterinary medicines and pest management.

 

 

 

Classification and taxonomy


The scientific classification of Microsporidia has evolved through time with growing scientific research in the area, and the specifics are still currently debated. Initially thought to be a protozoan (kingdom Protista), recent studies using DNA techniques indicate phylum Microspora should be classified under the Fungi kingdom or at least as a sister kingdom to Fungi. The class, order and family within the Microspora phylum are also frequently revised and debated (traditional taxomony given at the family level bloew). Traditionally, species were identified by observing the physical characteristics of the spore, life cycle and relationship with the host cell. However, recent scientific studies using genetic tools (namely ribosomal RNA sequencing) have challenged this approach and suggest genetic markers a more correct method for scientific classification. More research is still needed to better understand the origins of microspora and of individual species.  Despite all this, there are now over 1200 species identified in 143 genera. Currently, at least 14 species in 8 genera are known to infect humans.

 

 

Family

 

Genera

 

Species

Nosematidea

Brachiola

B. algerae, B. vesicularum

Encephalitozoonidea

Encephalitozoon

E. cuniculi, E. hellem, E. intestinalis (syn. Septata intestinalis).

Enterocytozoonidea

Enterocytozoon

Enterocytozoon bieneusi,

Microsporidea

Microsporidium

M. ceylonensis, M. africanum

Nosematidea

Nosema

N. ocularum, N. connori (syn. B connori)

Pleistophoridea

Pleistophora

Sp.

Pleistophoridea

Trachipleistophora

T. hominis, T. anthropophthera,

Nosematidea

Vittaforma

Vittaforma corneae (syn. Nosema corneum)

 

Synonyms for microsporidia include: microspora, microsporan, microsporidea.

 

 

 

 

History of Discovery


The Phylum Microspora was discovered in the late 1800s, but the first human case was described only in 1959 in a Japanese child. The rise in microsporidiosis is associated with the arrival and spread of HIV; microsporidiasis is primarily found in patients with AIDS or are otherwise immuno-compromised (like organ transplant patients). However, at least three species of Nosema and one of Brachiola have been documented in immuno-competent patients. Microsporidia are considered casual, accidental or opportunistic agents in humans.

 

 

 

 

Agent Morphology


Microsporidia are primitive eukaryotes with well defined nuclei and plasma membrane but lack some typical organelles found in more typical eukaryotes mainly mitochondria, stacked golgi and peroxisumes. Microsporidia spores are all round and oblong and those associated with human infection tend to be about 1-4 µm in size (an important feature for diagnosis as some species are often mistaken for bacteria). All have a characteristic coiled polar tube, tubule or filament, layered polarplast, a posterior vacuole (thought to function as golgi) and a protective exospore made of proteins and chitin. Chitin is responsible for the sporesŐ high environmental resistance. Below are two diagrams (3D and 2 D) illustrating typical microsporidia spores.

 

 

    

Taken from: http://www.palaeos.com/Eukarya/Units/Microsporidia/Microsporidia.000.html

 

 

 

During infection, the cell membranes of the spore (also called the sporoplasm) is injected into a host cell though osmosis pressure.  After the infection, the microsporidium relies on the host cell for energy and begins to multiply inside the host cell cytoplasm. Species differ in their relationships with host cell; some species alter host cell functioning to induce more nutrient absorption and cell growth to accommodate the agent. Microsporidia may reproduce sexually or asexually. In asexual reproduction, nuclear division takes place and form one or more pairs of nuclei, and cellular division may isolate the nuclei or pair them in a diplokaryon arrangement. Sexual reproduction is not well understood but it thought to involve autogamous fusion and reorganization of genetic material.

 

 

Life Cycle

 

The life cycle of microsporidia varies between species, but can be generalized in the following diagram.

 

 

Taken from http://www.dpd.cdc.gov/dpdx/HTML/Microsporidiosis.html

 

 

 

First, (1) the environmentally resistant, infectious spore is ingested or otherwise contracted. This environmental stimulus activates germination in the spore, releasing the polar tubule through eversion.  (2) The spore then inject the polar tubule into a host cell and (3) release their sporoplasms into host cells. At this stage, the sporoplasms usually become meronts, cells with loosely organized organelles enclosed in a simple plasma membrane.  (4) The microsporidia meronts then multiply either in contact with the host cytoplasm (as with E. bieneusi) or within a parasitophorous vacuole (in E. interstinalis). (5) They then undergo sporogony to further divide and form sporoblasts, preparing the cell for thick exospore layers characteristic of mature spores. The spores can either be dispersed freely within the host cell cytoplasm or packaged in sporophorous vesicles (or pansporoblast membranes). This characteristic can be taxonomically important in distinguishing between species. Finally, (6) when spores completely fill the host cell, the plasma membrane is affected and releases the spores to its surroundings. The spores may then infect other surrounding cells, be transported to new sites within the host, or be excreted in feces or urine to infect other hosts.

 

The exact incubation period for microsporam spores is unknown, but the spores are considered extremely resistant and thus assumed to persist in the environment for long periods of time. 

 

 

 

Reservoirs and Vectors

 

Microsporidia spores are ubiquitous and are capable of infecting any animal cell including those of insects, fish, mammals and even other parasites! They are most commonly found to infect anthropods. Many of the 14 species infecting humans are also found in a number of wild and domesticated mammals (e.g. rabbits, mice, puppies, kittens, etc.). In fact, the first case of E. cuniculi was described in rabbits in 1923. There are no vectors for microsporidiosis in the standard sense (infected insects may infect humans if they are ingested, but this is neither necessary nor ubiquitous).

 

 

 

 

 

Transmission


The transmission of microsporidia is still unclear, but the most common way is thought to involved inhaling, ingesting or otherwise contracting spores (for example ocular or sexually transmitted). Spores of microsporidia may also be transmitted in water as species of Encephalitozoon, Enterocytozoon and Vittaforma have been documented in water sources (Dowd et al. 1998). Significant contact with infected animals may also transmit the disease (zoonoic infection) but cases are rare.

 

 

 

 

Clinical Presentation in Humans


Chronic diarrhea and wasting are the most common symptoms of microsporidiosis, but different species invade different sites including the cornea, binary track and muscles. Thus, the symptoms of microsporidiosis varies greatly depending on the site of infection.

 

o      In the intestinal or biliary tract, common symptoms include chronic diarrhea (often loose, watery and nonbloody), weight loss or wasting, abdominal pain, nausea, and vomiting.

o      Disseminated infection is characterized by symptoms of cholecystitis (inflammation of the gallbladder), renal failure, respiratory infection, headache, nasal congestion, ocular pain and sinus involvement.

o      Respitory infection may cause cough, dyspnea (labored breathing) and wheezing.

o      With ocular infection, symptoms range from foreign body sensations, eye pain, light sensitivity, redness, excessive tearing or blurred vision.

o      Those with urinary tract infections typically do not show symptoms.

o      Muscular infections cause general muscle weakness and pain.

o      Finally, infections of the brain or other nervous tissue cause seizures, headache and other symptoms depending the precise area of infection.

 

 

The following chart describes the clinical presentations of different microsporidia infections in humans.

 

 

Species

 

Clinical Presentation

B. algerae, B. vesicularum

Keratoconjunctivitis (inflammation of the eye), skin and deep muscle infection

E. cuniculi*, E. hellem *

Keratoconjunctivitis, respiratory and genitourinary tract infection, and disseminated infection

Enterocytozoon bieneusi*

Diarrhea, acalculous cholecystitis (inflammation of the gallbladder), and respiratory infection (rare)

E. intestinalis (syn. Septata intestinalis)*

GI infection, diarrhea, dissemination to ocular, genitourinary and respiratory tracts

M. ceylonensis, M. africanum

Cornea infection

N. ocularum, N. connori (syn. B connori)

Ocular infection

Vittaforma corneae (syn. Nosema corneum)

Ocular infection, urinary tract infection

Pleistophora Sp.

Muscular infection

T. hominis,

Muscular infection, stromal keratitis and disseminated infection

T. anthropophthera,

Disseminated infection

            Adapted from http://www.dpd.cdc.gov/dpdx/HTML/Microsporidiosis.htm

 

 

* The diagram below illustrates pictorially the sites of infection of these species.

 

 

        

                  Image taken rom http://www.dpd.cdc.gov/dpdx/HTML/Microsporidiosis.htm

 

 

 

 

 

Diagnostic Tests


The methods of diagnosis typically involve identifying spores in feces, urine, other bodily fluid or body tissues.  Transmission electron microscopy (TEM) is the gold standard for identifying specific species and diagnosing microsporidiosis but is often too expensive and time consuming. Alternatives include light microscopy using various stains including gram stains (microsporidia spores are gram-positive and stain dark violent and become readily visible under the microscope), modified trichrome stains (e.g. trichrome blue), Warthin-Starry silver stains, Giemsa, and chemofluorescent agents like Calcoflur. E. bieneusi measures between .8-1.5 µm while B. algerae, Encephalitozoon spp, V. corneae and Nosema spp. measure 1.5-4 µm under the microscope. Immunofluorscent assays (IFA) and molecular techniques are emerging methods for diagnosis.

 

 

 

 

Treatment, Therapy and Management


Treatment with albendazole is most common for all species and is coupled with topical Fumagillin for ocular infections (see table of key drug treatments below) However, most drug treatments will not fully eradicate the parasites. New, more effective drugs for microsporidiosis are still being discovered and tested. For example, Nikkomycin Z (NIK-Z), a drug that inhibits chitin synthesis, has been shown to be affective against a host of fungal pathogens. It proved successful in laboratory tests on Encephalitozoon species, but has yet to be tested in vivo.

 

Further management of the disease is often needed. In general, symptoms should be treated if possible. Patients with chronic or severe diarrhea should take care to replenish electrolytes and fluids regularly and maintain nutritional intakes.  

 

 

Drug Catagory

 

Drug

 

Treatment for

 

Dose

 

Precautions

Anthelmintics

Albendazole

Gastro, muscle, disseminated and ocular infections.

400mg PO bid for 2-4 weeks

Avoid pregnancy

Antibiotics

Fumagillin – Topical

 

 

 

 

 

Oral

 

Keratoconjunctivitis and ocular lesions (Encephalitozoon spp. B. algarae, E. hellum, E. cuniculi, V. corneae)

 

E. bieneusi

 

3 mg/ml drops 1 week topical use + management

 

 

 

 

Unknown

 

Not approved by FDA for microsporidiosis.

 

 

 

 

 

Thrombocytopenia

Antiprotozoals

Metronidazole

E. bieneusi and others.

500mg PO bid for 2 weeks.

 

Immunomodulatory

Thalidomide

Diarrhea when other drugs have failed

Unknown

Toxic, only as last resort.

Severe birth defects; avoid pregnancy.

 

 

 

 

 

Epidemiology


As mentioned, microsporidia are extremely widespread. They infect nearly every organism on earth from honey-bees and silkworm to mammals and birds. Relatively little is known about the epidemiology of microsporidia, as transmission and infection pathways are still somewhat unclear. Though active microsporidia spores have been found in water sources in developed and developing nations, microsporidiosis remains primarily a disease of HIV and AIDS patients. Microsporidia has been reported to infect 39% of AIDS patients with diarrhea and 30% of AIDS patients with Cryptosporidium.  Despite our relatively recent discovery of this pathogen, the infection among AIDS patients is remarkable and the parasite will be of growing importance in the future as HIV continues to spread and more research is undertaken to understand the role microsporidia play in the human health.  

Geography

 

Microsporidia has a worldwide distribution, affecting both developing and developed nations, but proper diagnosis remains difficult, especially in developing nations. Below is a map illustrating the few countries where microsporidiosis has been formally documented.

 

Map made by Lily Cheng, May 21th, 2006

 

 

 

Public Health and Prevention Strategies


In recent years, the US Environmental Protection Agency (EPA) has listed microsporidia in the EPA Candidate Contaminate List (CCL), deeming it an emerging water-borne pathogen needing monitorial attention. Filtrating water supplies remains the best preventative strategies available. Measurement and filtration techniques for microsporidia spores remain rudimentary and underdeveloped, though the scientific community is actively trying to amend this knowledge gap.

 

There are currently no vaccines available or being explored for microsporidia infection.  

 

Although microsporidia infection in humans mostly occur in patients with compromised immune systems, the further spread of AIDS worldwide increases our need to understand and manage microsporidia for the near future. As more research is done on this class of organisms, we find their prevalence increasing in human patients. This is indeed an emerging infectious disease.

 

 

 

 

 

Useful Web Links


CDC Website on Microsporidiosis         

 

E-medicine Website on Microsporidia

 

Microsporidia by The Microbial Biorealm

 

Website on Microsporidiosis for AIDS Patients

 

 

 

 

 

 

References



Bigliarid, Elisa; Bernuzzi, Anna Maria; Corona, Silvia; Gatti, Simonetta; Scaglia, Massimo; Sacchi, Luciano. 2000."In Vitro Efficacy of Nikkomycin Z against the Human Isolate of the Microsporidian Species Encephalitozoon hellem. " Antimicrob Agents Chemother, 44(11): 3012–3016., American Society for Microbiology.

 

Cama R.I., U.D. Parashar, D.N. Taylor, T. Hickey, D. Figueroa, Y.R. Ortega, S. Romero, J. Perez, C.R. Sterling, J.R. Gentsch, R.H. Gilman and R.I. Glass. Enteropathogens and other factors associated with severe disease in children with acute watery diarrhea in Lima, Peru. J. Infect. Dis., 179:1139-1144, 1999.

Canning, Elizabeth U.; Lom, Jiri. 1986. The Microsporidia of Vertebrates, Systematics of the Microsporidia, and Biology of the Microsporidia. London: Academic Press, 1986.

 

Chijide, Valda M. 28 Mar 2006. "Microsporidiosis." E-medicine by Web-MD. [website] Accessed May 2006.

 

Dowd, Scot E., Gerba, Charles P., Pepper, Ian L. "Confirmation of the Human-Pathogenic Microsporidia Enterocytozoon bieneusi, Encephalitozoon intestinalis, and Vittaforma corneae in Water." Applied Environmental Microbiology. 1998 64: 3332-3335

 

Illinois Natural History Survey,Systematics of Microsporidia, Center for Ecological Entomology.  [website] Accessed May 2006.

 

Joseph J, Vemuganti GK, Sharma S. Microsporidia: Emerging Ocular Pathogens. Indian J Med Microbiol [serial online] 2005 [cited 2006 May 21];23:80-91.

 

Larsson, Ronny. 12 May 2004. Cytology and taxonomy of the microsporidia. <Image: 1bohuslavis.jpg>[website] Accessed May 2006.

 

Miller, Jeff D. 1997. Microsporidia (Protozoa):
A Handbook of Biology and Research Techniques. [website] Accessed May 2006.

 

Whipple, Allison; Levine, Zeva, Damon-Moore, Laural; Kahrl, Ariel; Sacks, Hannah; Stulberg, Michael; Smith, Casey M.; Scogin, Shana. 2004.  "Microsporidia." The Microbial BioRealm. [website] Accessed May 2006.

White, Toby. 2006. "Microsporidia" The Palaeos: The Trace of Life on Earth. [website] Accessed May 2006.

 

Wolk, D.M., C.H. Johnson, E.W. Rice, M.M. Marshall, C.B. Plummer, and C.R. Sterling. A spore counting method and cell culture model for chlorine disinfection studies of the human Microsporidia, Encephalitozoon syn. Septata intestinalis. Appl. Environ. Microbiol., 66:1266-1273, 2000.