1. Significance
Protozoa are an extremely diverse group of unicellular organisms. Although there are many pathogenic species, only a few are considered problems for the water industry. Pathogenic protozoa are widely distributed in water supplies, are relatively resistant to inactivation by chemical disinfectants, and have caused numerous waterborne outbreaks. These organisms generally cause diarrhea or gastroenteritis of varying severity, although more serious consequences (including death) can occur. In the United States, 18% of drinking water-associated outbreaks between 1971 and 2006 were caused by protozoa.1 Of the 325 water-associated protozoan disease outbreaks reported worldwide, most were caused by Cryptosporidium spp. (51%) and Giardia duodenalis (41%).2 The remainder were caused by Entamoeba histolytica (2.8%), Cyclospora cayetanensis (1.8%), Toxoplasma gondii (0.9%), Cystoisospora belli (syn. Isospora belli) (0.9%), and Blastocystis hominis (0.6%). Balantidium coli, microsporidia (Encephalitozoon spp. and Enterocytozoon bieneusi), Acanthamoeba spp., and Naegleria fowleri were associated with 0.3% of outbreaks, but a definitive association between drinking water and disease outbreaks has yet to be established for some of these organisms. This group of organisms has a wide variety of morphological and physiological characteristics, and although some are relatively closely related (e.g., C. parvum, C. cayetanensis, and T. gondii), Giardia spp. are considered some of the most ancient extant eukaryotes, and microsporidia may be more closely related to fungi than to protozoa.3 Many parasitic protozoa form cyst, oocyst, or spore stages that allow them to survive in the environment and be resistant to some drinking water treatment practices. These pathogens are shed in the feces of infected humans and animals, thus following a fecal-oral route of transmission, except for the free-living Acanthamoeba and Naegleria. Some species of Cryptosporidium, Giardia, and T. gondii can be transmitted to humans from infected animals. Some are opportunistic pathogens, but most are frank pathogens infecting individuals regardless of their immune status, although the ultimate disease outcomes may vary.
During the 1970s, waterborne outbreaks due to Giardia duodenalis (syn. G. intestinalis, G. lamblia) were noted with increasing frequency, especially in communities using unfiltered surface water sources. Giardia cysts are common in environmental waters, and G. duodenalis has historically been the most frequently identified cause of waterborne disease outbreaks worldwide. Between 1995 and 2016, reports of giardiasis cases in the United States ranged from 14 623 to 27 778 cases per year,4 although the actual incidence was probably much higher. Between 2012 and 2017, there were 111 giardiasis outbreaks in the US, with 9 drinking water outbreaks associated with municipal systems or private wells.5 The United States has an estimated 260 000 cases of giardiasis each year, and the overall incidence of Giardia carriage may be as high as 2%.6 The relative contributions of waterborne, foodborne, and person-to-person transmission to nonepidemic giardiasis is unknown, but given that cases peak in late summer and fall and that the highest number of cases involve 0- to 5-year-olds, one significant pathway may be communal swimming areas (lakes, rivers, swimming pools, and water parks) heavily used by children in that age group.7 Cysts are infective immediately after being excreted from the infected host. Symptomatic infection can result in acute, chronic, or recurring bouts of diarrhea of varying severity. Mortality has rarely been reported, and effective drugs (e.g., metronidazole) are available.
In 1993, Cryptosporidium caused the largest waterborne disease outbreak in US history.8 Both drinking and recreational waterborne outbreaks have been reported. Infection with Cryptosporidium (cryptosporidiosis) can cause a severe diarrhea that is usually self-limiting in immune-competent individuals, but may be prolonged and life-threatening for immunocompromised individuals.9 Nitazoxanide is the only treatment for cryptosporidiosis approved by the US Food and Drug Administration, although its effectiveness is limited primarily to immune-competent individuals.10 Cryptosporidium is identified in 2.2% of all diarrhea cases in developed countries, although it is more common in children (7% of childhood diarrhea cases) and AIDS patients (average of 14%).11 Between 2010 and 2019, reports of cryptosporidiosis cases in the United States ranged from 8 008 to 13 979 per year.12
Cryptosporidium is particularly problematic for water utilities because it is common in surface waters and because the oocyst stage, which is found in environmental waters, is resistant to chlorine disinfection at the concentrations typically applied during drinking water treatment. Oocysts are infective immediately after being excreted from the infected host. The species of most concern in human infections (accounting for >95% of isolates recovered from humans) are Cryptosporidium parvum, which is found in a wide variety of animals, and Cryptosporidium hominis, which is primarily a human-specific pathogen.13,14 Cryptosporidium meleagridis accounts for approximately 1% of human infections. Other Cryptosporidium that are occasionally isolated from humans include C. felis, C. cuniculus, C. canis, C. andersoni, C. suis, C. muris, and various unnamed genotypes.13-21 As of 2020, 42 Cryptosporidium species have been described and are considered valid.22
Reports on the occurrence of Cryptosporidium spp. oocysts in untreated surface waters vary widely (4%-100%) but the Information Collection Rule (ICR) survey of 5838 untreated source waters throughout the United States reported an average occurrence of 6.8% with a mean concentration of 0.067 oocysts per liter.23 Except for one recent incident, only C. parvum and C. hominis have been identified as causes of waterborne and foodborne outbreaks of cryptosporidiosis.24-26 The only exception is a 2008 outbreak in England caused by a rabbit isolate, reclassified as C. cuniculus in 2010, that is closely related to C. hominis.27,28 The Long Term 2 Enhanced Surface Water Treatment Rule (LT2ESWTR) mandates that US water treatment plants monitor influents for Cryptosporidium.29
Three waterborne outbreaks caused by Toxoplasma gondii have been reported: one involving US soldiers exposed to contaminated creek water in Panama,30 one involving 100 acute cases associated with municipal drinking water in British Columbia, Canada,31 and a contaminated municipal cistern in Brazil.32 In the Canadian outbreak, the implicated water system used unfiltered chloramine-treated surface water. Toxoplasma requires intermediate and final (definitive) hosts to complete its life cycle. Felines are the only animals known to act as definitive hosts and to produce oocysts that are excreted in the feces. The oocysts require an external maturation period of 1 to 5 d to sporulate and become infective. Infection (toxoplasmosis) is usually asymptomatic in healthy adults but may result in flu-like symptoms or swollen glands. Pregnant women can pass the infection to their fetus, resulting in birth defects (e.g., intellectual disability, loss of vision and hearing) and sometimes death.33 Disease in immunocompromised individuals can be severe, widely disseminated, and result in brain lesions and death. Oocysts have been detected in both ground and surface waters.34,35
Cyclospora cayetanensis is an apicomplexan parasite closely related to Eimeria spp. It is increasingly recognized as a human intestinal pathogen worldwide and has been most commonly associated with consumption of contaminated produce (strawberries, raspberries, mesclun, and basil) in North America. The organism has been detected in water used as a source of drinking water,36 and one waterborne outbreak has been documented in the United States.37 Cyclospora cayetanensis is frequently detected in children in the developing world, as well as travelers to these regions. The organism produces oocysts that are shed in feces and require an external maturation period (5 to 14 d at 22 to 32 °C) to become infective. Infection is usually self-limiting in immunocompetent individuals but can cause diarrhea, abdominal cramps, nausea, fatigue, and weight loss, and it persists for several months.38 The disease can be effectively treated with trimethoprim-sulfamethoxazole.
Entamoeba histolytica is an anaerobic parasitic protozoan that causes amoebic dysentery. It is common in developing countries but rare in the United States; it is transmitted in areas with inadequate sanitation, where human feces contaminate drinking water and food. The symptoms of infection may be mild (loose stools, stomach pain, and stomach cramping) but the more severe form of disease is associated with fulminating diarrhea, bloody stools, weight loss, and fever. Rarely, E. histolytica trophozoites invade the liver, forming an abscess, and may spread to other parts of the body (e.g., the lungs or brain). It is a significant cause of morbidity and mortality in tropical regions of the world, causing an estimated 40 000 to 100 000 deaths each year.39 It is morphologically identical to but genetically and biochemically distinct from the nonpathogenic Entamoeba dispar, which is more common. Metronidazole treats infections relatively effectively. Two waterborne outbreaks in the United States have been attributed to E. histolytica.1 One was tentatively linked to a community drinking water well, but E. histolytica was not detected in the well water even though fecal samples contained E. histolytica, Campylobacter jejuni, and Giardia spp.
Microsporidia are small, spore-forming, obligate intracellular parasites that infect every major animal group and are recognized as the cause of gastroenteritis, renal disease, sinusitis, and keratitis in humans. Traditionally, they were considered protozoa but have been reclassified as fungi.3 There are more than 1200 species; the human pathogenic species are Enterocytozoon bieneusi, Encephalitozoon intestinalis, Encephalitozoon hellem, Encephalitozoon cuniculi, Trachipleistophora spp., Vittaforma corneae, Nosema spp., Microsporidium spp., and Brachiola vesicularum.40,41 A microsporidia intestinal infection is primarily characterized by malabsorption, caused by injury to the small intestine epithelium. If untreated, it can lead to death due to chronic malnutrition and dehydration. Many animals may serve as microsporidia reservoirs, but the transmission route to humans is not clearly understood. Because the spores are shed in feces, urine, respiratory secretions, and other body fluids, waterborne transmission is possible. However, only one water-associated outbreak has been reported.42 Two species of human-pathogenic microsporidia were detected in waters used to irrigate food crops that are traditionally eaten raw; however, there was no association with disease in this instance.43
Acanthamoeba are free-living amoebae (FLA) that can cause chronic encephalitis and keratitis.44,45 They are found worldwide in animal intestines, vegetative material, soil, dust, air, fresh water, marine water, sediments, sewage, compost, tap water, and bottled water.46,47 High numbers of amoebae are found in sediments and surface layers of fresh water lakes, corresponding to high-density bacterial populations. Acanthamoeba colonize hot tubs, chemical showers, eyewash fountains, drinking water fountains, dental units, dialysis units, swimming pools, air conditioning systems, humidifiers, and hot water systems, and their cysts are highly resistant to chlorine.48 Although Acanthamoeba spp. are the most prevalent of FLA, not all species or strains are pathogenic for humans. Acanthamoeba spp. including A. castellanii, A. culbertsoni, A. hatchetti, A. healyi, A. polyphaga, A. rhysodes, A. astronyxis, and A. divionensis have all been implicated in human infections.49 Portals of entry leading to human infection include the respiratory tract, skin ulceration, contact with corneal tissue, and wounds. Amoebae disseminate to various organs and the brain via the bloodstream. Contact lenses and contaminated contact lens solutions are highly associated with Acanthamoeba keratitis.50 Monitoring drinking water for amoebae is impractical because of the technical difficulty associated with recovering and identifying amoebae, and because no practical method is available to differentiate pathogenic from nonpathogenic strains. In addition, because nonwater exposures (e.g., household dust) far exceed drinking water exposure and because ingestion is not associated with encephalitis, enhanced water treatment rules are unlikely to reduce the incidence of disease. The organism was included on the US EPA first Contaminant Candidate List (CCL) but was removed after it was decided that no regulatory action was appropriate or necessary.51
Naegleria fowleri is a free-living amebic protozoan that is common in warm bodies of fresh water, hot springs, discharge from industrial plants, and poorly maintained swimming pools. The organism enters nasal passages during water activities and causes a rare but usually fatal infection of the central nervous system: primary amebic encephalitis (PAM). Between 1937 and 2018, there were 381 cases worldwide (182 confirmed, 89 probable, and 110 suspected).52 In the United States, between 2010 and 2019, there were 34 cases: 30 cases related to recreational water, 3 cases from nasal irrigation with contaminated tap water, and 1 case from contaminated tap water and the use of an outdoor water slide.53 Person-to-person transmission has not been documented and there is no effective therapy. One survey of US groundwaters reported that 16% of samples (n = 185) were positive for N. fowleri.54 In 2002, the deaths of 2 children were linked to N. fowleri in nondisinfected well water used for drinking water.55
A greater waterborne health risk than direct infection by FLA (e.g., Acanthamoeba spp. and Naegleria spp.) may be bacterial pathogens that can live and multiply inside FLA cells. Legionella pneumophila replicates inside the cysts and trophozoites of Acanthamoeba spp.,56 and Mycobacterium avium cells become more virulent after passing through environmental amoebae.50 Pathogenic Chlamydiales have been detected as endosymbionts of environmental and clinical isolates of Acanthamoeba spp.58 At least 30 groups or species of bacteria—many of them pathogens—can infect amoebae. The amoebae may protect such bacteria from disinfection during drinking water treatment and provide a safe haven for proliferation in the distribution system.