Microsporidia Outbreak Linked to Water
By Kelly A. Reynolds, Ph.D.

Could microsporidia be the Cryptosporidium of the new millenium? This is the current question water quality researchers and public health officials are trying to answer.

Recently, clinical researchers from the Hepatogastroenterology and AIDS Unit in Lyon, France, identified microsporidia as a causative agent in a waterborne outbreak during the summer of 1995.1 During this incident, 200 persons were infected; however, no fecal contamination of water was evident and no source was found before the outbreak ended. An epidemiological study of conditions surrounding the outbreak and individual risk factors showed that the major factor associated with a diagnosis of microsporidiosis during the outbreak was living in an area corresponding to one of the three water distribution subsystems of the town. Other factors in a microsporidiosis diagnosis were human immunodeficiency virus (HIV) infection, male homosexuality, low CD4 cell counts and diarrhea.

The French research group has been surveying patient stool for assessment of microsporidia from 1993 to 1996. Among the 1,454 persons submitting stool samples to the reference laboratory, 338 tested positive for microsporidia. The immunocompetence of the individuals seemed to be a factor, since 261 persons were infected with HIV and 16 were transplant patients.

According to the above survey, microsporidiosis appears to be an endemic disease in HIV-positive persons (prevalence: 0.1 percent) and a sporadic disease in HIV-negative persons (prevalence: less than 1 out of a million). Other studies have documented the prevalence rate of microsporidia in AIDS patients to be as high as 29.3 percent.2 One can only wonder how many cases are being missed due to the fact that microsporidia aren't routinely tested for in water supplies or stool samples.

What are microsporidia?
The phylum Microspora consists of approximately 80 genera and over 700 species. They are collectively referred to as microsporidia, and the disease they produce is termed microsporidiosis. Microsporidia are important pathogens of both beneficial and pest insects. In fact, researchers are investigating these single-celled creatures as biological control agents for mosquitoes, grasshoppers and many other insect pests, particularly those that attack crops. Historically, however, they've caused significant economic problems for the silkworm, honeybee and commercial fishing industries.

Although most microsporidia infect fish or insects, they've also been identified with disease in mice, rabbits, foxes, dogs and humans. Human microsporidia pathogens also appear to have animal reservoirs including pigs.3 Several studies have documented microsporidiosis in immunocompromised individuals.4,5 Six genera have been reported as potentially harmful to humans including: 1) Encephalitozoon sp. (Encephalitozoon [Septata] intestinalis, Encephalitozoon cuniculi, Encephalitozoon hellem); 2) Enterocytozoon bieneusi; 3) Pleistophora sp.; 4) Vittaforma cornea; 5) Nosema sp. (Nosema connori and Nosema ocularum); and 6) Trachipleistophora hominis.

Daphnia magnaDaphnia pulex

Infected by: • White bacterial disease • Flabelliforma magnivora (Microsporidia) • Microsporidium nov. sp. 3 • Larssonia sp. (Microsporidia)

Considered to be a group of emerging pathogenic protozoa, microsporidia are small0.5-to-1.5 microns (µm)obligate intracellular parasites, meaning that they must invade a host to multiply. Their environmental and infectious form is a spore. After being ingested, and in the proximity of susceptible cells, microsporidia penetrate the cell and begin their life cycle of infection. Although most frequently associated with gastrointestinal disease, these organisms may also affect multiple bodily systems including the central nervous system, ocular systems, liver and skeletal muscles causing a variety of diseases such as diarrhea, hepatitis, peritonitis, keratoconjuctivitis, sinusitis, renal failure, myositis, gall bladder infection and blindness.6,7

Microsporidia are too small to be seen by a light microscope but they may be visualized by electron microscopya much more labor intensive and costly means of analysis. They can also be grown in laboratory cell cultures but require a minimum of 28 days for visual effects.8 These detection limitations have added to the problem of surveying water supplies for microsporidia. With no known therapy able to completely eradicate the parasite, prevention of infection is imperative.

Not a new pathogen
As a whole, microsporidia are thought to be ancient members of the evolutionary tree.9 First identified in 1857, microsporidia have long been recognized as a cause of disease in many non-human hosts. They're geographically widespread and have been documented as disease agents in most continents including North and South America, Europe, Asia, Africa and Australia. The only continent not associated with microsporidia infections is Antarctica.10

Reports of possible human microsporidia cases appeared as early as 1924, conclusive evidence for human infection wasn't available until 1959, when a 9-year-old Japanese boy showed symptoms of recurrent fever, loss of consciousness, headache and convulsions.11 No further cases were reported until 1973, when the first human case proven by autopsy was published involving a 4-month-old boy with severe diarrhea.12 Autopsy revealed microsporidia present in the lungs, stomach, small and large bowels, kidneys, adrenal glands, myocardium, liver and diaphragm. Sporadic cases were documented over the next two to three decades, especially in immunocompromised individuals and habitants of the tropics; but microsporidia hasn't received much attention from the water industry.

Drinking water significance
Listed in the top 10 on the USEPA's pathogen priority list for health and analytical methods research,13 microsporidia are a group of human pathogenic protozoa that have been found in treated wastewater, surface water and groundwater. They've also been listed by the Centers for Disease Control (CDC) and recognized by the National Institute of Health as important emerging pathogens. Lack of standard methods for their detection has in part delayed survey of these organisms in potential waterborne routes. Therefore, little is known about their survival, environmental transport and fate, or response to conventional drinking water treatments.

Found in feces and urine of infected individuals, human pathogenic microsporidia may find their way into drinking water sources. Microsporidia are protozoa of special interest because of their small size (1-to-5 microns), relative to Cryptosporidium oocysts (5-to-7 µm) or Giardia (6-to-10 µm).

The small size of microsporidia may enable them to escape filtration and treatment and present a greater problem to groundwater supplies, since they're thought to move more effectively through the subsurface environment. Although data is limited, the structure and similarity of microsporidia to other protozoan pathogens (Cryptosporidia and Giardia) suggests they're potentially resistant to disinfection.

The early warning signs of microsporidia as a human pathogen shouldn't be ignored, as they were in only recent history for Cryptosporidium. Sporadic cases and even small Crypto outbreaks were virtually ignored until the largest waterborne outbreak in the U.S. occurred in Milwaukee in 1993, causing 400,000 infections and the death of more than 100 drinking water consumers. Currently, Cryptosporidium is arguably recognized as the most problematic pathogen for the water treatment industry.

Conclusion
Although waterborne infections are one of the leading causes of human morbidity and mortality worldwide, in approximately 50 percent of identified waterborne outbreaks, no causative agent is identified, possibly due to lack of effective methods for detection.

Many questions remain as to the actual occurrence of microsporidia nationwide and the possibility of control with current treatment regulations. Only by committing resources to the understanding and prevention of microsporidia occurrence in drinking water can we begin to take a proactive stance regarding the control of this potentially significant human pathogen.

References
1. Cotte, L., et. al, "Waterborne outbreak of intestinal microsporidiosis in persons with and without human immunodeficiency virus infection," Journal of Infectious Disease, December 1999, 180: 2003-2008.

2. Canning, E.U., et. al, "Human microsporidioses: Site specificity, prevalence and species identification," AIDS, 7:S3-S7, 1993.

3. Deplazes, P., "Molecular epidemiology of Encephalitozoon cuniculi and first detection of Enterocytozoon bieneusi in fecal samples of pigs," Journal of Eukaryotic Microbiology, 43: 93S, 1996.

4. Kotler, D.P., "Gastrointestinal manifestations of immunodeficiency infection," Advances in Internal Medicine, 40: 197-241.

5. Van Gool, T., "High seroprevalence of Encephalitozoon species in immunocompetent subjects," Journal of Infectious Disease, 175: 1020-1024.

6. Atias, A., "Update on human microsporidiosis," Revista Medica de Chile, 123:762-772, 1995.

7. Hashimoto, T., and M. Hasegawa, "Origin and early evolution of eukaryotes inferred from the amino acid sequences of translation elongation factors alpha/Tu and 2/G," Advances in Biophysics, 32: 73-120, 1996.

8. Doultree, J.C., "In vitro growth of the microsporidian Septata intestinalis from an AIDS patient with disseminated illness," Journal of Clinical Microbiology, 1995.

9. Chukwuma, C., "Microsporidium in AIDS patientsa perspective," East African Medical Journal, 73: 72-75, 1996.

10. Curry, A., and E.U. Canning, "The microsporidia of vertebrates," Academic Press, New York, 1996.

11. Matsubayashi, H., T. Koike and T. Mikata, "A case of Encephalitozoon-like body infection in man," Archives in Pathology, 67: 181-187, 1959.

12. Margileth, A.M., A.J. Strano and R. Chandra, "Disseminated nosematosis in an immunologically compromised infant," Archives in Pathology, 95: 145-150, 1973.

13. U.S. Environmental Protection Agency, Mar. 2, 1998, Federal Register 63:10274-10287.

About the author Dr. Kelly A. Reynolds is a research scientist at the University of Arizona with a focus on the development of rapid methods for detecting human pathogenic viruses in drinking water. She is also a member of the WC&P Technical Review Committee.