Amebae and ciliated protozoa as causal agents of waterborne zoonotic disease

Review

Amebae and ciliated protozoa as causal agents of

waterborne zoonotic disease

Frederick L. Schustera,*, Govinda S. Visvesvarab

aCalifornia Department of Health Services, Viral and Rickettsial Disease Laboratory,

850 Marina Bay Parkway, Richmond, CA 94804, USAbCenters for Disease Control and Prevention, Division of Parasitic Diseases,

4770 Buford Highway, NE, Atlanta, GA, USA

Abstract

The roles free-living amebae and the parasitic protozoa Entamoeba histolytica and Balantidium

coli play as agents of waterborne zoonotic diseases are examined. The free-living soil and water

amebae Naegleria fowleri, Acanthamoeba spp., and Balamuthia mandrillaris are recognized

etiologic agents of mostly fatal amebic encephalitides in humans and other animals, with immu-

nocompromised and immunocompetent hosts among the victims. Acanthamoeba spp. are also agents

of amebic keratitis. Infection is through the respiratory tract, breaks in the skin, or by uptake of water

into the nostrils, with spread to the central nervous system. E. histolytica and B. coli are parasitic

protozoa that cause amebic dysentery and balantidiasis, respectively. Both intestinal infections are

spread via a fecal–oral route, with cysts as the infective stage. Although the amebic encephalitides

can be acquired by contact with water, they are not, strictly speaking, waterborne diseases and are not

transmitted to humans from animals. Non-human primates and swine are reservoirs for E. histolytica

and B. coli, and the diseases they cause are acquired from cysts, usually in sewage-contaminated

water. Amebic dysentery and balantidiasis are examples of zoonotic waterborne infections, though

human-to-human transmission can occur. The epidemiology of the diseases is examined, as are

diagnostic procedures, anti-microbial interventions, and the influence of globalization, climate

change, and technological advances on their spread.

Published by Elsevier B.V.

Keywords: Free-living amebae; Amebic encephalitis; Amebic keratitis; Amebic dysentery; Balantidiasis;

Acanthamoeba; Balamuthia; Naegleria fowleri; Entamoeba histolytica; Balantidium coli

www.elsevier.com/locate/vetpar

Veterinary Parasitology 126 (2004) 91–120

* Corresponding author. Tel.: +1 510 307 8651; fax: +1 510 307 8599.

E-mail address: [email protected] (F.L. Schuster).

0304-4017/$ – see front matter. Published by Elsevier B.V.

doi:10.1016/j.vetpar.2004.09.019

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 92

1.1. Current status of taxonomy. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 93

2. Life cycles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 94

2.1. Environmental persistence and infectivity . . . . . . . . . . . . . . . . . . . . . . . . . . 96

3. Infection and disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 96

3.1. Central nervous system . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 98

3.2. Corneal surface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 99

3.3. Nasopharyngeal and cutaneous infections . . . . . . . . . . . . . . . . . . . . . . . . . . 99

3.4. Intestinal infections . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 99

4. Epidemiology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 100

4.1. Free-living amebae . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 100

4.2. Entamoeba histolytica and commensal amebae . . . . . . . . . . . . . . . . . . . . . . 104

4.3. Balantidium coli . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 105

4.4. Developed and developing regions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 106

5. Diagnostic techniques. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 108

5.1. Recovery and identification of free-living amebae . . . . . . . . . . . . . . . . . . . . 108

5.2. Recovery and identification of Entamoeba histolytica and Balantidium coli . . 108

6. Control and management . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 110

6.1. The environment and free-living amebae . . . . . . . . . . . . . . . . . . . . . . . . . . 110

6.2. Transmission of Entamoeba histolytica and Balantidium coli . . . . . . . . . . . . 111

6.3. Treatment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111

7. Future trends . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 112

7.1. Globalization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 112

7.2. Climate change . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 114

7.3. Modern technology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 115

8. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 116

Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 116

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 117

1. Introduction

The organisms dealt with in this section run the gamut from free-living to parasitic.

Among the free-living representatives are the amebae Acanthamoeba, Naegleria, and

Balamuthia. The parasitic forms include Entamoeba histolytica and Balantidium coli. The

free-living amebae have been described as facultative parasites, opportunistic pathogens,

and amphizoic amebae (Page, 1974), the latter term implying ability to live within and

F.L. Schuster, G.S. Visvesvara / Veterinary Parasitology 126 (2004) 91–12092

outside of a host. Their role as pathogens was recognized since the 1960’s, while

Entamoeba (�1870) and Balantidium (�1860) have been known for more than a century

as etiologic agents of human disease. Because of their medical and economic impact on

human populations, E. histolytica, and to a lesser extent B. coli, have been the subjects of

intensive studies on transmission, possible zoonotic associations, and anti-microbial

therapy.

A parasite is an organism that lives at the expense of and causes damage to its host,

and which cannot survive for long in the absence of the host. Technically, the free-living

amebae are not parasites, although often described as such in the literature. Nevertheless,

they have made a niche for themselves in the field of parasitology, acquiring some

degree of notoriety while doing so. This has come about because the diseases caused

by these amebae—particularly the encephalitides—are mostly fatal, difficult to

diagnose premortem, and lack a well-defined optimal anti-microbial therapy. Several

factors over the past two decades have resulted in increased interest in these opportunistic

amebae: (1) the HIV/AIDS epidemic that led to increased numbers of cases of

Acanthamoeba and Balamuthia encephalitides; (2) the popularity of soft contact lenses that

became the vehicle for Acanthamoeba to reach the corneal surface to cause keratitis; and

(3) increased leisure time and affluence allowing vacationing in areas where there are warm

lakes and hot springs, exposing people to Naegleria fowleri, the cause of meningoence-

phalitis.

1.1. Current status of taxonomy

Taxonomy of the Protozoa has long been based upon morphologic criteria. In recent

years, morphology has taken a back seat to the use of molecular techniques as the moving

force in taxonomy, often with the resultant abandonment of comfortable assumptions about

relationships, and the recognition of new species and even genera based on sequencing the

16S and 18S rDNA genes. Once members of a single phylum, the major protozoan types

are now placed in their own different phyletic categories. The amebae dealt with in this

report represent a polyphyletic assemblage, encompassing a number of different

taxonomic Orders. We follow, in part, Page’s taxonomic scheme for the amebae (Page,

1988).

� Phylum Rhizopoda

Class Heterolobosea

Order Schizopyrenida

Family Vahlkampfiidae

Naegleria fowleri

Class Lobosea

Order Acanthopodida

Family Acanthamoebidae

Acanthamoeba spp., Balamuthia mandrillaris

Of uncertain ordinal relationship

Family Entamoebidae

Entamoeba histolytica, E. dispar, E. coli, E. hartmanni, E. moshkovskii

F.L. Schuster, G.S. Visvesvara / Veterinary Parasitology 126 (2004) 91–120 93

(Note: B. mandrillaris, originally described as a leptomyxid ameba, is included in the

Family Acanthamoebidae, based on data from 16S rRNA gene sequencing (Amaral Zettler

et al., 2000; Booton et al., 2003)).

The one ciliate dealt with in this paper is the only one parasitizing humans. The

taxonomic scheme presented here is from Lynn and Small (2000).

� Phylum Ciliophora

Class Litostomatea

Order Vestibuliferida

Family Balantidiidae

Balantidium coli

2. Life cycles

The general pattern of life cycles for the protozoa covered in this section is a trophic or

feeding stage, alternating with a resting, resistant cyst stage. There are no intermediate

hosts in the life cycles and, with the exception of B. coli, sexual reproduction is not known

to occur. None of these organisms requires vectors for transmission, an exception being E.

histolytica which can be carried by some insects as mechanical vectors.

Naegleria and Acanthamoeba, as free-living trophic amebae, present in soil or water,

feed primarily on bacteria. Balamuthia appears to feed on other Protozoa, probably other

amebae, found in the soil. Naegleria, in addition to the ameboid and cystic stages, has a

flagellated stage that arises from the trophic ameboid stage. The flagellate typically neither

feeds nor divides and is transitory, ultimately reverting to the ameboid stage. However,

species of Naegleria have been identified on the basis of sequencing data in which the

ability to flagellate has been lost, or in which the flagellate undergoes division (De

Jonckheere, 2002). The cysts of these amebae, which develop as growth conditions become

unfavorable (lack of food, waste product accumulation, desiccation), survive in nature until

growth conditions improve. The ameba cysts are enclosed by a wall that may be two or

three layers thick, and may or may not have pores through which the excysting ameba can

exit. They are all obligate aerobes but some can tolerate anaerobic conditions for short time

intervals. All have been grown in culture, either in the presence of bacteria as a food source

(xenically, from xeno-, G., stranger, or in this case, presence of another organism), or in the

absence of any other organisms (axenically) in a variety of enriched nutrient media

(Schuster, 2002). The trophic and cystic forms of Acanthamoeba are compared in Figs. 1

and 2, and the ameboid and flagellated stages of Naegleria are seen in Figs. 3 and 4.

The parasites E. histolytica and B. coli, similarly have trophic and cystic stages. The cyst

stage for both organisms is the transmissive stage in the life-cycle. In E. histolytica, a

precyst stage is recognized as intermediate between the trophozoite and the mature cyst.

The latter is quadrinucleate, with rod-like ribonucleoprotein elements called chromatoid

bodies and glycogen reserves, both of which disappear as the cyst ages. The cyst

germinates in the host’s small intestine to give rise to a quadrinucleate ameba that, by

nuclear and cytoplasmic divisions, produces eight small amebae that eventually localize in

the colon and caecum. Cysts are essential for transmission since the trophozoite cannot

survive passage through the stomach with its low pH. Cysts of E. histolytica are not as


highly resistant to harsh environmental stresses as those of free-living amebae, but

comparative studies of survival have not been performed. E. histolyctica is an anaerobe but

can tolerate trace amounts of oxygen for short time periods, perhaps through association

with intestinal bacteria that scavenge oxygen (Ravdin, 1986). As with other anaerobic

protozoa, mitochondria are lacking although the amebae retain a mitochondrial vestige, the

mitosome, as well as genes of mitochondrial origin (Bakatselou et al., 2003). The ameba is

cultured both xenically and axenically in a variety of enriched media (Diamond and Clark,

2002).

Balantidium spp. are known only as commensals or parasites found in the intestinal tract

of various animal species; none is free-living. The trophic B. coli is covered with uniformly

arranged rows of cilia propelling it through the viscous mass of undigested food and

bacteria passing through the colon (Fig. 5). The name of the organism derives from its

pouch-like shape (balanti-, G., bag or sac). The organism has a cytostome through which

debris, bacteria and other particulate material are ingested and pass into food vacuoles. As

with other ciliates, there is a micro- and macronucleus, and two contractile vacuoles that


Figs. 1–4. Photomicrographs of stained or unstained wet-mounts of amebae. (1) Acanthamoeba trophozoites seen

in an unstained wet-mount preparation with differential interference contrast optics. Note the characteristic

projecting pseudopods, called acanthapodia, over the ameba surface (arrow). Contractile vacuoles (V) and nuclei

(N) can be seen in two of the amebae. Bar represents 10 mm. (2) Unstained cysts of a large species of

Acanthamoeba (A. comandoni) are seen in a wet-mount preparation with differential interference contrast optics.

The cyst wall is made up of two layers, endocyst and ectocyst. The stellate form of the endocyst (I) is evident, as is

the outer wall or ectocyst (O) enclosing the entire structure. Pores (ostioles) are located at the junctures of the two

walls (arrow). Bar represents 10 mm. (3) Naegleria fowleri amebae stained with trichrome. The nucleus and its

dark staining nucleolus are clearly seen (N). Bar represents 10 mm. (4) The flagellate stage of N. fowleri is seen in

this stained wet-mount preparation. The shape of the organism is ovoid, with two (sometimes four) flagella seen at

the anterior end of the organism. The nucleus is at the anterior end of the flagellate, in close association with the

flagellar apparatus. Bar represents 5 mm.

serve as osmoregulatory organelles. Conjugation has been observed in cultures (Zaman,

1978). The organism is the largest among protozoan parasites or commensals found in

humans, measuring up to 150 mm in length. Since the trophic ciliate cannot survive passage

through the stomach, the cyst with its protective wall is the infective stage (Fig. 6).

Encystment of trophic ciliates in the intestine is initiated during departure from the host’s

colon. Even though the ciliate is found in the same anaerobic environment as E. histolytica,

it is an aerobe. The organism can be grown in vitro in xenic cultures, but has not been grown

axenically (Diamond and Clark, 2002).

2.1. Environmental persistence and infectivity

The trophic protozoon, whether it is the ciliate, or ameba, is poorly equipped to survive

environmental conditions for prolonged periods of time. This is particularly true of

Entamoeba and Balantidium, in which the infective stage is the cyst. For the free-living

amebae, the infective stage can be either the trophic or cystic stages. Because of its wall and

the dormancy of the enclosed organism, the cyst allows survival during periods unfavorable

for growth. Under laboratory conditions, Acanthamoeba cysts have remained viable �20

years (Mazur et al., 1995). Accurate data on longevity of cysts in nature is lacking, but cyst

viability depends to a great extent on environmental conditions. Cysts of the free-living

amebae are better suited to survive prolonged desiccation and other environmental stresses,

while the cysts of Entamoeba and Balantidium are more fragile and temperature sensitive,

and remain viable only in a moist environment for limited time periods (see below).

3. Infection and disease

A summary of the disease-related properties of the organisms can be found in Table 1.


Figs. 5–6. Light microgrpahs of Balantidium coli from human stool. (5) Trophozoite with fringe of cilia evident

on its surface. Internal details are obscured by the density of the ciliate contents, although a contractile vacuole can

be seen at posterior end of the organism, and the outline of the macronucleus is also evident. Bar represents 10 mm.

(6) Cyst surrounded by a wall. The outline of the macronucleus can be seen in the cytoplasm. Bar represents

10 mm.

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Table 1

Diseases caused by free-living amebae, Entamoeba histolytica, and Balantidium coli: sources of infection, portals of entry, and affected organs

Parasite Disease Major site(s) in host Portal of entry Source of infection

Acanthamoeba spp. Acanthamoeba encephalitis

(acanthamebiasis)

Brain, skin, nasopharynx, lungs Breaks in skin;

respiratory tract

Soil, water

Acanthamoeba keratitis Cornea Corneal surface

Balamuthia

mandrillaris

Balamuthia encephalitis

(balamuthiasis)

Brain, skin, nasopharynx, lungs Breaks in skin;

respiratory tract

Soil, watera

Naegleria fowleri Naegleria meningoencephalitis

(naegleriasis)

CNS Nasal passages Recreational waters (lakes,

pools, hot springs, etc.)

Entamoeba histolytica Amebic dysentery (amebiasis) Colon, liver, lung, brain,

cutaneous lesions

Oral route Feces-contaminated

water or food

Balantidium coli Dysentery (balantidiasis) Colon Oral route Feces-contaminated

water or food

a Potential for transmission but not reported.

3.1. Central nervous system

Prominent among the infections of the brain and spinal cord are the encephalitides

caused by Acanthamoeba spp. and N. fowleri. The disease caused by Naegleria is termed

primary amebic meningoencephalitis (PAM) and is acquired during swimming or other

water-related activities (Fig. 7). Naegleria as trophic amebae (or cysts) are carried into the

nostrils along with water and migrate from the nasal epithelial surface along the olfactory

nerves across the cribriform plate to the brain (Martinez et al., 1973). Once there, they

attack the olfactory and frontal lobes, as well as the base of the brain, the brainstem, and

cerebellum, and are found in a purulent exudate in the subarachnoid space and in the

cerebrospinal fluid (CSF). PAM is a fulminating infection. Onset occurs 1–2 days after

exposure to the amebas, and death occurs in 7–10 days following infection.

Acanthamoeba (Figs. 8 and 9) and Balamuthia (Fig. 10) are responsible for

granulomatous amebic encephalitis (GAE), usually without meningeal involvement

(Martinez, 1985; Martinez and Visvesvara, 1997, 2001). The name reflects the host’s


Figs. 7–10. Sections of human brain infected with amebae, as seen by light microscopy. (7) Concentrations of

Naegleria fowleri seen in a tangential section of the perivascular space. The wall of the blood vessel is not seen in

the section (cf. Fig. 8). Note the typical vesicular nucleus (N) of the trophic amebae (H&E stain). Bar represents

40 mm. (8) Acanthamoeba castellanii stained with H&E. Large numbers of trophic amebae are seen in the

perivascular region. The adjacent blood vessel (B) can be seen. The patient had a kidney transplant and was treated

with steroids. Bar represents 35 mm. (9) Trichrome stain of section of blood vessel wall showing large numbers of

encysted A. castellanii (C), identified by the enclosing cyst wall. Unlike Naegleria, Acanthamoeba encysts in host

tissue. Bar represents 35 mm. (10) Balamuthia mandrillaris trophic amebae (A) in an H&E-stained tissue section

from an experimentally infected immunocopromised mouse inoculated intranasally with about 1000 trophic

amebae and cysts. Note the large size of the amebae, as compared to Naegleria and Acanthamoeba. Bar represents

70 mm.

granulomatous response to the presence of the amebae. Portals of entry for both organisms

are either through breaks in the skin that become contaminated with soil, or as cysts taken

into the respiratory tract as dust or airborne soil particles. No association between

recreational water activity and infection has been reported, but this is a possible route of

infection. From the point of entry, the amebae spread hematogenously to the central

nervous system (CNS) where they cause GAE. Areas of the brain affected include the

cerebrum, cerebellum, and brainstem, but amebae are generally not found in the CSF.

Cutaneous or nasopharyngeal lesions are also produced, depending on the portal of entry

(see below). GAE is typically a chronic, insidious infection that can take weeks to years

after infection before becoming clinically apparent. E. histolytica is also found, though

rarely, in brain abscesses following hematogenous dissemination from other sites in the

body (Albach and Booden, 1978).

3.2. Corneal surface

Acanthamoeba spp. are also the causal agents of amebic keratitis, occurring as the result

of corneal trauma or, more often, improper care of contact lenses. Amebae present in water

can either directly infect the cornea, or can be carried to the cornea from the contact lens

storage case on the lens surface. In contact lens wearers, the source of amebae is typically

non-sterile tap or distilled water used in preparation of homemade saline solutions for lens

care. In corneal trauma, amebae are inoculated directly to the corneal surface by injury

with, to cite two actual cases, a stalk of hay or a cinder blown into the eye (Jones et al.,

1975; Ma et al., 1981). Usually only one eye is affected. The disease is characterized by a

ring-shaped stromal infiltrate, corneal ulceration, photophobia and pain, and is often

mistaken for viral keratitis, which delays effective treatment. Once established in the

corneal stroma, the amebae are difficult to eradicate. Apparent recovery from the disease

through anti-microbial treatment can be followed by recurrence when amebae encysted in

the stroma reactivate following therapy. Keratitis victims may require one or more corneal

transplants to repair damage or to reduce the parasite load in the eye. In a worst-case

scenario, enucleation has been necessary to provide relief, although the availability of

effective anti-microbials has made this unlikely.

3.3. Nasopharyngeal and cutaneous infections

These are due to Acanthamoeba and Balamuthia and develop when cysts or trophic

amebae are introduced into breaks in the skin or into the nasal passages. These infections

can remain localized, but usually amebae disseminate to other locations in the body,

particularly the CNS, and there is no sharp demarcation between these infections and

encephalitis.

3.4. Intestinal infections

Intestinal infections are generally spread by the classic fecal–oral route. Infection

results from ingestion of cysts in water or food; trophic organisms cannot survive passage

through the stomach, except in cases of low stomach acidity.


E. histolytica is responsible for amebic dysentery (Albach and Booden, 1978; Haque et

al., 2003; Ravdin, 1986). Once reaching the colon, the invasive amebae attack the intestinal

epithelial surface, causing flask-shaped ulcerations in the wall and producing a blood- and

mucus-flecked loose stool. In severe amebiasis cases, the intestinal wall can perforate

releasing amebae into the peritoneal cavity. Amebae can also disseminate from the

intestinal tract via the hepatic portal system to form discrete abscesses in the liver, lungs, or

brain. Cutaneous amebiasis, though rare, has been reported, often presenting as lesions in

the anogenital area, and related to passage of amebae from the intestinal tract (Magana-

Garcıa and Arista-Viveros, 1993). Primary cutaneous amebiasis has also been reported, in

which lesions form in regions not contiguous with the intestinal tract (Parshad et al., 2002).

A commensal variant of E. histolytica, now recognized as a distinct species E. dispar, is

found in the gut but is non-invasive (Diamond and Clark, 1993), and may be the basis for

large numbers of asymptomatic infections in humans.

In humans, E. histolytica is the sole parasitic ameba of the gut. Other amebae found

in the intestinal tract, which are present as commensals, are E. coli, E. hartmanni,

E. moshkovskii, Endolimax nana, Iodamoeba butschlii and, as already noted, E. dispar.

Dientamoeba fragilis, once regarded as an ameba, is now known to be a trichomonad

flagellate which, while retaining intracellular vestiges of a flagellar apparatus, lacks basal

bodies and flagella (Brugerolle and Lee, 2000).

B. coli is associated with infections in humans and in pigs, the reservoir for human

balantidiasis. In the colon, the organisms produce ulcerations resembling those produced

by Entamoeba, which can become secondarily infected by intestinal bacteria, or vice versa.

The ciliates are often found in clusters or ‘‘nests’’ in the ulcers or the mucosal surface

(Levine, 1961). In addition to the colon, spread can occur, though rarely, to the peritoneal

cavity (Zaman, 1978).

4. Epidemiology

4.1. Free-living amebae

The number of infections caused by free-living amebae is relatively small, given their

ubiquitous global distribution (Table 2). Reported cases number in the hundreds or, for

amebic keratitis, in the low thousands. The amebae are present in soil and water and, as

such, it is virtually impossible to avoid contacting them. Several studies have detected anti-

ameba antibodies in surveyed human populations as evidence of contact with these amebae

(see below). Not all of the amebae encountered in the environment, however, have the

potential for causing disease.

Acanthamoeba spp. is the most common and widespread ameba found in the

environment and is almost invariably present in any soil sample plated for isolation of

amebae (Page, 1988). They have been isolated from fresh, brackish, and salt waters, soil

samples ranging from the Antarctic to arid desert-like areas, sewage dump sites, as well as

from the home environment in garden and flowerpot soils, water taps, humidifiers, home

aquaria, etc. The amebae have been recovered from laboratory eye wash stations, dental

irrigation systems, and from hospital plumbing and hydrotherapy pools, posing a threat to


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Table 2

Epidemiological considerations of diseases caused by waterborne amebae and Balantidium coli

Disease Estimated number of human cases Distribution and special habitats Disease in non-human hosts

Acanthamoeba encephalitis �200 cases (1960’s to �2000) World-wide; soil and water Wild and domestic animals

(dogs, horse, Indian buffalo,

kangaroo, bull, monkeys, ovines)

Acanthamoeba keratitis >3000 cases (1980’s to �2000) Keratitis not reported

Balamuthia encephalitis �100 cases (1990 to �2000) World-wide; soil and

probably water

Apes, horse, sheep, dogs

Naegleria meningo-

encephalitis

�200 cases (1960’s to �2000) World-wide; naturally warm or

thermally polluted waters

Cattle, tapir

Amebic dysentery �500 million infected,

with �100,000 deaths/year

Tropical and subtropical regions;

developing nations

Monkeys, apes, dogs

Balantidium dysentery Incidence �1%, based on

surveys of stool samples

Tropical and subtropical regions;

developing nations

Pigs, monkeys, apes

immunocompromised patients. Acanthamoeba spp. and Naegleria have been isolated from

nasal passages of humans, particularly during periods of high winds carrying dust, as

occurs during the harmattan in western Africa (Abraham and Lawande, 1982). They occur

as laboratory contaminants, appearing in tissue cultures, and have been misidentified as

‘‘transformed’’ tissue culture cells. They have been isolated from human and animal stool

samples, and their presence can be due to either their having passed through the host’s

intestinal tract as cysts which germinate in the stool, or as a result of secondary

(environmental) contamination of the stool sample. Acanthamoeba and other soil amebae

are regarded as coprophilic organisms (copros-, G., dung; philo-, G., liking), exploiting

environments rich in organic materials and bacteria, their natural food.

Approximately 17 species of Acanthamoeba have been described based upon phenotypic

characteristics such as size and cyst morphology (Page, 1988). Sequencing of 18S rDNA has

been the basis of more recent descriptions, with isolates falling into twelve (T1–T12) different

lineages containing either single species or complexes of species (Stothard et al., 1998).

Species that have been most often associated with human systemic infections are A. polyphaga

(T4), A. castellanii (T4), A. culbertsoni (T10), A. hatchetti (T11), and A. healyi (T12). Most

clinical isolates of Acanthamoeba are thermotolerant, growing at temperatures of�37 8C, but

there are also thermotolerant species that are non-pathogenic. Surprisingly, some

Acanthamoeba isolates from clinical cases do not grow well at 37 8C, but require a lower

temperature (30 8C) for optimal growth (Schuster and Visvesvara, 1998). In addition to human

cases, Acanthamoeba GAE has been reported from sheep, dogs, a kangaroo, a monkey, a horse,

and a bull (Greene, 1998; Kadlec, 1978; Martinez, 1985). Acanthamoeba spp. have also been

isolated from poikilotherms (fish and reptiles) (Dykova et al., 1999; Sesma and Ramos, 1989).

Individuals at risk for systemic Acanthamoeba infections include those who are

immunocompromised or immunodeficient. These infections paralleled to a great extent the

AIDS epidemic in the United States and elsewhere. With the development and use of more

effective therapies for treatment, GAE in HIV/AIDS patients has virtually disappeared, but

still remains a risk for organ transplant patients or those who are being administered anti-

inflammatory steroids (Steinberg et al., 2002).

Not all Acanthamoeba spp. are pathogenic. While it can be assumed that an ameba

isolated from brain or cutaneous lesions is a pathogen, the same assumption cannot be

made for those amebae isolated from the environment. The gold standard for assessing

pathogenicity is the ability to produce encephalitis in the mouse model, following

intranasal instillation of a suspension of amebae (Martinez et al., 1973, 1975). In addition

to infected brain tissue, amebae can also be found in the lungs of experimentally infected

mice (Fig. 11).

Acanthamoeba spp. isolated from air conditioning cooling towers and hospital

plumbing systems have been shown to harbor Legionella and Legionella-like bacteria.

Legionella spp. are fastidious in their growth requirements and it was something of a puzzle

that these bacteria should flourish in air conditioning cooling towers and plumbing pipes

where nutrients were limiting. The suggestion that Legionella could proliferate in amebae

found in the same water (Rowbotham, 1980) led to studies demonstrating the ability of

these bacteria to parasitize amebae, particularly Acanthamoeba, just as they invade and

destroy macrophages in the infected human host (Gao and Kwaik, 2000; Newsome et al.,

1998). Bacteria from the lysed amebae are released in aerosols, to be carried through the


ventilating system. The implication of this relationship is that the bacteria that cause

legionellosis can survive and proliferate in environments otherwise unsuitable for their

growth, causing disease in immunodepressed populations, such as hospital patients.

Acanthamoeba keratitis (AK) is caused by a much wider variety of Acanthamoeba spp.,

most of which are part of the T4 rDNA complex (Stothard et al., 1998). Thermotolerance is not

as critical for these organisms found at the corneal surface, which is slightly below normal

mammalian body temperature, as it is for those with systemic sites of infection. Corneal

trauma or improper care of contact lenses (daily wear or extended-wear soft lens types) are the

causes of AK, the former being more common among males and patients �50 years, and the

latter among females (Stehr-Green et al., 1989). Tap or distilled waters, used in preparation of

homemade saline solutions for lens care, are often the source of the amebae, which can then

bind to the lens, the lens case, and ultimately to the corneal surface. Data from 1989 indicated

that 64% of contact lens wearers with AK had prepared saline solutions from salt tablets using

distilled water (Stehr-Green et al., 1989). Typically, individuals developing AK are otherwise

in good health, with no evidence of immunodeficiency.

No naturally occurring AK cases have ever been described in animals, and attempts at

developing an animal model to study AK have met with mixed results. In an in vitro study

comparing attachment of amebae to corneas from a variety of animal eyes, attachment

occurred to human, pig, and hamster corneas but not to those of mice, rats, horses, dogs,

chickens, and guinea pigs (Niederkorn et al., 1992).

Balamuthia, another agent that causes GAE, is found in soil and perhaps water, but is

difficult to isolate from nature (Schuster et al., 2003). There have been about 100 cases of


Fig. 11. Transmission electron micrograph showing three trophic Acanthamoeba castellanii (A) in lung tissue of

an experimentally infected mouse inoculated intranasally with about 1000 amebae. Bar represents 10 mm.

GAE in humans reported in the literature since the first descriptions of this opportunistic

pathogen (Visvesvara et al., 1990, 1993). Given the difficulty in recognizing the pathogen,

it is likely that many cases go unreported. All clinical isolates of this organism are members

of a single species, as determined by DNA sequencing of clinical isolates (Booton et al.,

2003). Infection comes about as the result of inhalation of airborne soil particles, as well as

contamination of breaks in the skin with soil. Early descriptions of Balamuthia GAE were

in HIV/AIDS patients and others with impaired immune systems, such as the elderly, and

drug and alcohol abusers (Visvesvara et al., 1990). More recent cases have been from

immunocompetent children. The disease has also been reported in non-human primates

in zoos (Canfield et al., 1997; Rideout et al., 1997), dogs (Visvesvara, unpublished

observations), a sheep (Fuentealba et al., 1992), and a horse (Kinde et al., 1998). A lowland

gorilla at a zoo died of meningoencephalitis that was initially attributed to Acanthamoeba

(Anderson et al., 1986), but the ameba was subsequently identified as Balamuthia

(Visvesvara, unpublished observation).

N. fowleri is the causal agent of PAM. Although there are >30 species of Naegleria as

determined from sequencing data (De Jonckheere, 2002, 2004), N. fowleri is the only

species that has been recovered from clinical cases. Two other species, N. australiensis and

N. italica, are pathogenic in the mouse model of disease, but have never been associated

with any human cases. As for Acanthamoeba, thermotolerant species of Naegleria are

known (e.g. N. lovaniensis), but are not pathogenic for humans. Most victims of PAM are

children or young adults in good health with a history of swimming in naturally warm or

thermally polluted waters, where growth of the ameba is favored. In Baja California

(Mexico), irrigation canals were the source of infections in children who had been

swimming in the canal waters. In the United States, most PAM cases have been reported

from the southern tier of the country, with Florida, Virginia, Texas, and California

having relatively large percentages of victims (Martinez, 1985). Thermal effluents from

factories and power plants have also promoted growth of the pathogen, and cooling ponds

of nuclear power plants are monitored for the presence of N. fowleri (Reveiller et al., 2003).

Amebae have been isolated from sun-warmed domestic water supplies in both the United

States and Australia, but risk of infection from drinking water is minimal. More likely,

infection in these cases resulted from aspiration of water into the nostrils while washing.

Because of the short prodromal period, the association between infection and water-related

activities is readily apparent. The occurrence of the disease among young persons suggests

that diving, horseplay, prolonged immersion in water, and aspirating mud stirred up from

the bottom of lakes and ponds, are factors that promote infection and disease. In addition to

humans, PAM has been reported from cattle, and from a tapir (Daft et al., 1999; Lozano-

Alarcon et al., 1997). Willaertia sp., a free-living ameba closely related to N. fowleri,

was identified in the stomach wall of a dog with gastric ulcers and adenocarcinoma

(Steele et al., 1997)

4.2. Entamoeba histolytica and commensal amebae

E. histolytica is the only ameba parasitic in the human intestine. Once regarded as a

single species with pathogenic and non-pathogenic variants, the non-pathogenic form is

now recognized as a different species, E. dispar (Diamond and Clark, 1993). The latter


species is associated with individuals who are infected with Entamoeba, but are

asymptomatic for amebic dysentery. The picture is further complicated by another

intestinal ameba, E. hartmanni, which is morphologically similar to E. histolytica, but of

smaller size (3–10 mm versus 20–30 mm for E. histolytica) and non-pathogenic.

An estimated 500 million humans are infected with the parasite, but only 10–20%

exhibit dysenteric disease, the prevalence varying with different geographic locations

(Trager, 1986). Spread of E. histolytica is by the fecal–oral route, and water and food

contaminated with fecal waste containing cysts are the major vehicles of infection. Anal

intercourse practiced among gay males has also been recognized as a means of

transmission (Judson, 1984). Food handlers are another possible source of infection, as are

mechanical vectors such as flies and cockroaches. Dogs that have developed amebiasis by

ingestion of human feces, can be a potential source of human infections, though the reverse

is more likely (Barr, 1998; Botero, 1972). However, E. histolytica in the dog rarely encysts,

and trophic amebae in dog stools are not infective (Barr, 1998; Eyles et al., 1954). In the

mammalian host, ingested cysts germinate in the small intestine and are carried to the

colon, where the amebae attack the epithelial lining. Cysteine proteases are probably a

virulence determinant of the amebae (Bruchhaus et al., 2003). In cases of dysentery with

accompanying diarrhea, numerous trophic amebae, often containing ingested red blood

cells, are found in the unformed or loose stool. In asymptomatic cases, the formed stool

contains cysts whose development parallels the removal of water from the stool as it passes

to the rectal area. Trophic amebae in unformed stools do not encyst, nor are they infective

to others. The cyst, by virtue of its protective wall, is the prime infective stage in the ameba

life-cycle. The presence of non-pathogenic commensals in the gut seen upon examination

of stool samples can lead to erroneous laboratory diagnoses.

Cysts of Entamoeba can remain viable for 2–4 weeks outside of the host provided they

are in a moist or wet environment. Desiccation and extreme temperatures can render cysts

non-viable. Amebic dysentery is primarily a disease of tropical or sub-tropical areas of the

world, where climatic conditions favor cyst survival and sewage is apt to contaminate the

drinking water supply. But sporadic outbreaks of the disease have occurred throughout the

world, regardless of temperatures, and one such textbook example occurred in Chicago

during the 1933 World’s Fair with over 1000 cases (Albach and Booden, 1978). In addition

to humans, E. histolytica is found in a large number of non-human primates and, less

often, in dogs (Barr, 1998; Eyles et al., 1954). Another species, E. polecki, found in pigs

has been reported from humans but is not highly infective for humans (Levine, 1961).

E. moshkovskii, an ameba morphologically similar to E. histolytica but non-pathogenic,

has been isolated from sewage in several parts of the world and, recently, has been reported

in 21% of Bangladeshi children sampled as a non-invasive commensal (Ali et al., 2003).

Optimal temperature for growth of the organism is 25 8C, rather than 37 8C, and it is

hardier than E. histolytica. Other amebae that are found in the human gut as commensals

are also transmitted by the fecal–oral route.

4.3. Balantidium coli

This protozoon is the only ciliate known to cause infections in humans. Some fifty

species have been described, often on size differences, and the validity of these species is


unconfirmed. B. coli is spread by the fecal–oral route from pig-to-human and from

human-to-human, the latter mode of transmission occurring in institutionalized

populations (mental hospitals, orphanages, prisons). Balantidiasis is a disease of

tropical and sub-tropical regions, and the Philippines is cited as an endemic area (Zaman,

1978). Pigs, which harbor the ciliate, are typically the source of human infections,

although species-to-species transfer requires adaptation of the parasite. The ciliates in

pigs are non-invasive and non-pathogenic. Transmission occurs when swine are in close

contact with humans, and there is a lack of adequate sanitary facilities for sewage

treatment and disposal. The health of the host can be a factor, since individuals who are

malnourished, suffering from concurrent infections, or alcoholism are at greater risk of

developing balantidiasis. Nevertheless, the disease is uncommon in humans, and the

number of symptomatic cases of balantidiasis in the world is probably in the hundreds of

thousands.

Cysts can remain viable for weeks in pig feces, particularly if kept moist and away

from direct sunlight. Trophic ciliates can survive for as long as 10 days in the environment

(Zaman, 1978). Infection occurs when fecal material from swine contaminates

drinking water or food. Handling of pig intestine can also be a mode of transmission.

Once ingested, cysts give rise to active ciliates in the colon. These ciliates feed upon

bacteria and debris in the gut, but also release enzymes (hyaluronidase) that attack the

mucosal surface, producing flask-shaped ulcers in the wall of the colon and causing

diarrheic stools with blood and mucus. Secondary infection of the colonic lesions by

bacteria can worsen the clinical picture. Table 3 summarizes the epidemiological factors

involved in these protozoal diseases, along with their potential for zoonotic and waterborne

transmissions.

4.4. Developed and developing regions

Amebic dysentery and balantidiasis are a reflection of poor sanitation and inadequate

protection of the water supply from sewage contamination. Their prevalence, particularly

amebiasis, is high in developing countries. Although they occur in developed regions, their

appearance is sporadic and infection is most likely due to travel outside of developed areas

of North America, Australasia, Japan, and Western Europe.

Paradoxically, the situation is reversed in the case of diseases caused by free-living

amebae. They are reported mostly from developed regions with only sporadic reports from

the developing areas of the world. This anomaly may be due, however, not to the actual

incidence of the diseases but rather to the availability of diagnostic procedures and medical

care. Difficult enough to diagnose in developed nations, it is highly likely that amebic

encephlatides go undetected and undiagnosed in regions of Africa and Southeast Asia.

There are reports of encephalitis cases from South America (Recavarran-Arce et al., 1999)

and Thailand (Sangruchi et al., 1994), but these present only an incomplete picture of what

might actually exist. India has had a large number of keratitis cases associated with use of

contact lenses. Given the enormity of the HIV/AIDS epidemic and its large population of

immunodeficient victims, there are almost certainly opportunistic infections caused by

amebae that go unidentified in many developing nations.


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Table 3

Opportunistic and parasitic diseases: populations at risk, and potential for zoonotic and waterborne transmission

Disease Populations at risk Zoonotic potential/animal source Potential as waterborne infection

Acanthamoeba encephalitis Immunocompromised hosts

(AIDS and transplant patients,

those with concurrent diseases)

Not zoonotic Minor

Acanthamoeba keratitis Contact lens users Not zoonotic When unsterilized water is used

for preparation of ophthalmic

solutions; immersion in hot tubs

Balamuthia encephalitis Immunocompromised and

immunocompetent hosts

Not zoonotic Minor

Naegleria meningo-

encephalitis

Apparently healthy children,

young adults, active in water

Not zoonotic Most infections contracted by

water-related activities

Amebic dysentery Travelers to developing countries Zoonotic/non-human

primates, dogs

Sewage-polluted water and

vegetables

Balantidium dysentery Rural or agrarian populations;

institutionalized groups

Zoonotic/pigs,

non-human primates

Sewage-polluted water; pig feces

5. Diagnostic techniques

5.1. Recovery and identification of free-living amebae

Infections caused by the free-living amebae are difficult to diagnose, and most are

recognized on autopsy or necropsy. Often, computed tomography and magnetic resonance

imaging of the brain of encephalitis patients indicate space-occupying lesions that lead to

biopsies and diagnoses. With infections caused by Acanthamoeba and Balamuthia, diagnoses

are made by finding the amebae in tissue sections stained with hematoxylin-eosin (H&E), or

by indirect immunofluorescence (IIF) staining using rabbit anti-ameba sera (Visvesvara et al.,

1993). The latter technique is preferred as a diagnostic tool because of its specificity in

identification of the etiologic agent. Few laboratories, however, are equipped to perform IIF

staining, the Centers for Disease Control and Prevention in Atlanta being one. Serological

screening of patients with suspect encephalitides can detect anti-ameba antibodies in

suspected Acanthamoeba and Balamuthia infections (Visvesvara et al., 1990, 1993). Because

these are chronic infections, there is time for an antibody response which can be detected by

indirect immunofluorescent staining. The polymerase chain reaction (PCR) has been used to

detect ameba DNA in tissue and CSF samples, but the technique is still in its early

developmental stages and requires more testing before its reliability can be certified.

Cutaneous infections by Acanthamoeba and Balamuthia are diagnosed using the same

techniques as for CNS infections. Because of their insidious nature and the difficulty in

antemortem recognition, the prognosis for recovery is poor and most cases are fatal.

In Acanthamoeba keratitis there is usually a strong association between contact lens

wear or corneal trauma and the occurrence of disease. Because of the pain associated with

the infection, victims are likely to seek medical help soon after symptoms develop. Cysts of

infecting amebae can be recognized in corneal scrapings that have been stained with

calcofluor white, a fluorescent stain that binds to polysaccharide polymers in the

Acanthamoeba cyst wall (Wilhelmus et al., 1986). The amebae can also be cultured onto

non-nutrient agar with bacteria for identification (Visvesvara, 1999; Schuster, 2002).

Naegleria meningoencephalitis, as mentioned above, is almost always associated

with swimming or other water activity. Initial diagnosis is made by finding trophic amebae

in the CSF following lumbar puncture. Naegleria amebae can be recognized under the

microscope by their characteristic limacine movement, with an anterior eruptive

pseudopod (Visvesvara, 1999). Because of its fulminant nature, early diagnosis is

essential, and recovery from the infection is rare, most cases being fatal. Here, too, staining

with H&E or IIF of brain tissue is helpful in confirming a diagnosis of PAM, but this is

usually done postmortem. Because Naegleria infections are often fatal within a 2-week

period, there is little or no antibody response to the presence of the amebae, and serological

procedures are of little avail. Microscopic observation of fresh CSF for active amebae is the

best opportunity for an on-the-spot identification.

5.2. Recovery and identification of Entamoeba histolytica and Balantidium coli

E. histolytica is recovered from dysenteric stools as trophic amebae. During fulminant

disease, trophozoites do not encyst before being expelled with the unformed stool, and can


be seen by microscopic examination of stool samples (Fig. 12). In asymptomatic cases

or in carriers, the cyst is the stage that is found in the stool sample. The presence of cysts

of commensal amebae from the gut can be the cause of confusion or misdiagnosis, but

there are distinct differences between cysts of E. histolytica and those of E. coli,

Endolimax nana, and Iodamoeba butschlii that technicians in diagnostic laboratories

are trained to recognize (Leber and Novak, 1999). E. histolytica can be differentiated

from the non-pathogenic E. dispar and other commensal amebae in dysenteric stools

by the presence of ingested erythrocytes in the former (Tanyuksel and Petri, 2003).

Other manifestations of Entamoeba infections such as liver, lung, or brain abscesses

can be detected by sonographic or radiological imaging and, in cases where amebae have

invaded tissues, serology can be of use. PCR, ELISA, indirect hemagglutination assays are

used for laboratory detection of Entamoeba (Gonin and Trudel, 2003; Tanyuksel and Petri,

2003).

Balantidium infections are most often diagnosed on finding active ciliates or cysts in

stool samples of patients. History of contact with swine may also be indicative of infection.

Infection can occur among people directly involved in raising pigs (farmers), in treatment

of pigs for infections (veterinarians), and in handling of pig organs (slaughter house

personnel). Infection can occur if pork becomes contaminated with pig intestinal contents

or feces and is not adequately cooked before being consumed.

For all the above parasites, culturing is an additional and confirmatory means of

identification although, in many cases, culturing of organisms from clinical samples is apt

to be time- and labor-intensive and is not recommended for rapid diagnosis. Cultivation

techniques and appropriate media for Entamoeba and Balantidium are described in

Diamond and Clark (2002), and for free-living amebae, in Schuster (2002) and Visvesvara

(1999). Many of the organisms require complex media (with limited shelf lives) and growth

conditions that might be available only in reference or research laboratories.


Fig. 12. Photomicrograph of Entamoeba histolytica, trophozoite (T) and cyst (C), seen in a trichrome-stained

fecal smear. Four nuclei can be seen in the cyst, as can a large dark-staining chromatoid body. A single nucleus is

evident in the trophic ameba. Bar represents 20 mm.

6. Control and management

6.1. The environment and free-living amebae

Avoidance of the free-living amebae is virtually impossible since they are ubiquitous

in the environment, as evidenced by the presence of antibodies in screened human

populations (Cerva, 1989; Cursons et al., 1980; Marciano-Cabral et al., 1987; Huang et al.,

1999). Domestic and wild mammals, which might be expected to have closer contacts with

soil than humans, also exhibit antibodies against amebae (Cerva, 1981; Kollars and

Wilhelm, 1996). It is not clear, however, if these antibodies provide immunological

protection against infection. It is also not clear if victims of naegleriasis, which generally

occurs in immunocompetent humans, might have some inapparent immunological defi-

ciency that renders them vulnerable to infections.

Some precautions can be taken to minimize contact with these opportunistic pathogens.

Immunodeficient patients are well advised to guard against exposure to airborne dust and

soil particles that might carry cysts of opportunistic amebae to the respiratory system, or

contamination of open wounds or sores with soil. N. fowleri, which has a clear association

with swimming and other activities that allow water to enter the nasal passages, is easier to

guard against. Natural or artificially warm waters, if they are not properly chlorinated,

could harbor Naegleria amebae. Maintaining effective levels of chlorine in recreational

waters is achievable in swimming pools and spas, but less so in the case of lakes and ponds.

However, adequate chlorination is not necessarily protective, as was seen in a retrospective

diagnosis of 16 PAM fatalities associated with a swimming pool in Czechoslovakia (Cerva

and Novak, 1968; Kadlec et al., 1980). In Florida, which has had and continues to have

sporadic cases of PAM associated with swimming in its warm lakes, the chance of infection

has been estimated to be about 1:2.6 million exposures (Wellings, 1977). Numerous

persons have been exposed to the amebae by swimming in the Florida waters, yet the

number of PAM cases remains negligible. In a survey covering a 1-year period in the

United States, four cases (6.8%) of Naegleria meningoencephalitis were found in 59

recreational waterborne disease outbreaks involving >2000 persons (Lee et al., 2002).

There is no evidence to indicate that infections by free-living amebae have any zoonotic

basis. Non-human animals also can develop these infections, but there is no likelihood of

their being able to pass the infection along to humans, either directly or indirectly through

pollution of drinking water or contamination of crop foods. Although free-living amebae

can be recovered from stool samples, their presence is not indicative of infection or disease,

and is more likely an indication of their being coprophiles.

In Acanthamoeba keratitis, the domestic water supply in the victim’s home, used in

preparation of homemade saline solutions for contact lens care, is the most likely source of

the amebae. Using molecular techniques for characterization of amebae, two different

studies identified the same ameba strain in the domestic water supply used for preparation

of wash solutions, the lens case, and the cornea of the patient (Kilvington et al., 1990;

Ledee et al., 1996). Compliance with procedures for contact lens care should protect

wearers against infection. Care should be taken to maintain a clean lens case in order

to prevent formation of bacterial films, a potential growth surface for the amebae. There

is evidence of protective IgA antibody levels at the corneal surface of normal individuals,


as compared to non-protective IgG serum levels in keratitis victims (Alizadeh et al.,

2001).

6.2. Transmission of Entamoeba histolytica and Balantidium coli

The situation with parasitic infections caused by E. histolytica and B. coli is somewhat

different in that animal species harbor these organisms, so the potential for zoonotic

transmission exists. Transmission is through fecal contamination of water or food by cysts

passed in stools. Humans appear to be the major source for infecting other humans through

fecal contamination of food or the water supply (Albach and Booden, 1978). Dogs, though

they can be infected with E. histolytica, are an unlikely source of human infection (Barr,

1998). Entamoeba is also found in a variety of non-human primates including baboons;

vervet, Sykes, and Debrazza’s monkeys; and in both black and grey mangabeys (Muriuki

et al., 1998). There is limited opportunity for primate feces to contaminate domestic water

supplies, even in parts of the world where there are large numbers of monkeys. Individuals

at risk include those involved in caring for monkeys (zoos or laboratory facilities,

veterinarians), butchering animals for food, or those keeping the animals as pets. Primates

in zoos and laboratory facilities can be monitored for the presence of Entamoeba and can

be treated to eliminate infection.

Domestic and wild swine represent a reservoir for human Balantidium infections and

several studies have found the incidence of infection to be 100% (Hindsbo et al., 2000;

Nakauchi, 1999). Although the organism is a commensal in the pig colon, it can be

pathogenic in humans. In a survey of 56 Japanese-bred mammalian species, primates and

pigs were positive for Balantidium (Nakauchi, 1999). A chimpanzee in this study was

found to be passing >1200 cysts and trophozoites per gram of feces. In rural or agrarian

areas where swine feces can contaminate the water supply, this would be a major source of

infection to the human population.

6.3. Treatment

There is no optimal anti-microbial therapy for the encephalitides caused by

Acanthamoeba and Balamuthia. Multiple drugs have been used in both these infections

with varying degrees of success, including amphotericin B, azithromycin, fluconazole,

flucytosine, pentamidine isethionate, and sulfa drugs (Schuster and Visvesvara, 2003).

Most Acanthamoeba encephalitis victims are immunodeficient, and succumb to multiple

opportunistic infections rather than to acanthamebiasis per se.

Amebic keratitis is treatable with one or more drugs (Kumar and Lloyd, 2002; Schuster

and Visvesvara, 2003). Chlorhexidine gluconate (a component of germicidal soaps) and

polyhexamethylene biguanide (a disinfectant and swimming pool cleaner) have become

the drugs of choice in treating keratitis cases. These drugs are well-tolerated in the eye,

although there are strains of Acanthamoeba that have shown resistance. Propamidine

isethionate, present in Brolene, an over-the-counter preparation available in Great Britain,

has also been used with some success, but is not as well-tolerated in the eye. In the presence

of these drugs, the amebae are likely to encyst in the corneal stroma and, with premature

cessation of treatment, excyst to give rise once again to trophozoites. Therefore, an


aggressive regimen of initial hourly drug applications should be followed, and

continued over several months at less frequent intervals (Schuster and Visvesvara,

2003).

Of the approximately 100 published cases of balamuthiasis, there are only two known

survivors, following intensive anti-microbial therapy (Deetz et al., 2003). In both cases,

following demonstration of brain lesions by magnetic resonance imaging, the diagnoses

were made by detection of Balamuthia amebae in biopsied brain tissue and Balamuthia

antibodies by immunofluorescence staining. A combination of anti-microbials was used in

treatment, including pentamidine isethionate, flucytosine, fluconazole, sulfadiazine, and a

macrolide antibiotic (azithromycin or clarithromycin). Recovery from GAE also depends

upon early diagnosis, the virulence of the infecting strain, the infectious dose of amebae,

and prompt initiation of treatment.

Few individuals infected with Naegleria meningoencephalitis have survived, in large

part because of the fulminant nature of the disease. Amphotericin B is the drug of choice in

treating victims, but early diagnosis is essential (Seidel et al., 1982).

Amebic dysentery is treatable with metronidazole and prospects for recovery are

excellent (Haque et al., 2003). Treatment, however, is often unavailable in developing

countries, and this is reflected in the disproportionate numbers of deaths from amebiasis in

these regions. Oxy- and chlorotetracycline and carbasone are used in the treatment of

balantidiasis but, for the same reasons as for amebiasis, people still suffer and die from the

disease. A summary of information about diagnostic techniques and anti-microbial therapy

can be found in Table 4.

7. Future trends

7.1. Globalization

With the increase in reciprocal travel between developed and developing nations of the

world, there is a greater likelihood of spread of infections caused by amebae and ciliated

protozoa. Globalization, however, probably would not affect the incidence of diseases

caused by free-living amebae because the potential for infection is already fairly uniform

throughout the world. The incidence of acanthamebiasis and balamuthiasis which occur in

immunocompromised hosts, will reflect the overall health of a population. In parts of the

globe where HIV/AIDS infections are rampant, opportunistic infections, including

the encephalitides, would be expected to have a higher occurrence. But since these

infections represent ‘‘dead ends’’ for the etiologic agent, human-to-human spread is most

unlikely.

Parasitic infections such as amebiasis and balantidiasis can spread, as a result of

globalization, from tropical and subtropical areas to temperate zones of the globe. Persons

infected with the parasites and serving as carriers can bring their parasites to developed

areas of the world. But these diseases reflect the lack of adequate sanitary facilities and

clean drinking water, and are not transmissible where sewage disposal and water quality

control are rigorously practiced. In recent years, sewage-tainted produce (e.g. cantaloupes,

lettuce, and berries) and apple cider has caused outbreaks of bacterial, cryptosporidial, and


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Table 4

Diagnostic techniques, treatment options, and prognoses for recovery from opportunistic and parasitic infections

Disease Available diagnostic procedures Antimicrobial treatment Prognosis for

recovery

Acanthamebiasis H&Ea, immunofluorescence staining; PCRb;

CTc and MRId scans; cultivation from clinical samples

Amphotericin B, azithromycin, fluconazole, flucytosine,

pentamidine, sulfa drugs

Poor

Acanthamoeba

keratitis

Corneal scrapings; cultivation Chlorhexidine, polyhexamethyl biguanide, propamidine Excellent

Balamuthiasis H&E, immunofluorescence; PCR;

CT and MRI scans; cultivation

Pentamidine, fluconazole, flucytosine, sulfadiazine Poor

Naegleriasis Trophic amebae in CSFe; H&E, immunofluorescence

staining; serology; cultivation

Amphotericin B Poor

Amebic dysentery Trophs and/or cysts in stool; ELISAf

for stool antigen; PCR; sonography,

CT and MRI scans for extra-intestinal abscesses

(liver, lung, brain); cultivation

Metronidazole Excellent

Balantidiasis Trophs and/or cysts in stool; cultivation Oxy- and chlortetracycline, carbasone Excellent

a Hematoxylin and eosin.b Polymerase chain reaction.c Computerized tomography.d Magnetic resonance imaging.e Cerebrospinal fluid.f Enzyme-linked immunosorbent assay.

cyclosporidial infections in North America and Europe (Ho et al., 2002; Millard et al.,

1994; Tauxe, 1997). But there have been no reported outbreaks of amebiasis and

balantidiasis as a result of food contamination. Cysts of these parasites are less likely than

bacteria to survive prolonged exposure to environmental stress. Surveys of pigs in Japan

and Denmark, neither of which is tropical or subtropical, have demonstrated a high

prevalence of B. coli. Yet Denmark has had no human cases of balantidiasis (Hindsbo et al.,

2000), emphasizing the effectiveness of sanitary precautions in prevention of human

infections.

7.2. Climate change

The generally accepted notion of global warming has caused concern regarding the

spread of infectious and parasitic diseases, particularly those transmitted by arthropod

vectors. Diseases caused by free-living amebae, amebic dysentery, and balantidiasis, are

only briefly mentioned, if referred to at all, in scenarios for disease spread due to global

warming (Cook, 1992; Patz et al., 2000).

For free-living soil amebae, increasing environmental temperatures might enhance

the growth of thermotolerant species which, in turn, might be better adapted to infect

humans and other mammals. But as noted before, thermotolerance is not necessarily

synonymous with pathogenicity and virulence, and growth at �37 8C, is no guarantee of

pathogencity in the mouse animal model of disease. Acanthamoeba spp., found in both

fresh and seawaters, may proliferate with rising environmental temperature and pose a

threat to swimmers. Since their primary food source is bacteria, the amebae would more

likely favor areas of pollution, with high bacterial counts. But those at risk for

acanthamebiasis are the immunocompromised or immunodeficient, and the disease is rare

in healthy individuals. Measures can be taken to insure that swimming pools and man-

made lakes are adequately chlorinated, but this cannot be accomplished with large bodies

of water.

Increased environmental temperatures would lead to increased use of air conditioning

units in buildings. This, in turn, would provide opportunities for growth of Acanthamoeba

in the cooling towers. As noted previously, Acanthamoeba spp. can serve as hosts for

intracellular Legionella, which eventually cause lysis of the amebae and release of the

bacteria in aerosols. Thus, it is possible that these amebae have been vectors in outbreaks of

legionellosis and its less severe form, Pontiac fever. At particular risk would be patients

in hospitals and residents of homes for the elderly, both with concentrations of

immunoimpaired persons. If properly treated with biocides, proliferation of protozoa and

bacteria in cooling towers can be prevented. For extensive plumbing systems, periodic

flushing with hot water can minimize biofilm formation in the piping that serves as a

growth substrate for bacteria and amebae.

Acanthamoeba keratitis is caused by contamination of contact lenses or the lens

case with bacterial films that would serve as food for the amebae. The source of the

amebae is non-sterile water used in lens care, and it is unlikely that climate change

would affect their presence. More important is compliance with recommendations for

lens care, and not wearing lenses while swimming or relaxing in thermal pools or hot

tubs.


As for N. fowleri, the causal agent of PAM, the organism is already found in warm

waters and, with an increase in warmed lakes and such, they would be expected to extend

their distribution, leading to higher incidence of PAM in both humans and animals. As with

Acanthamoeba, growth of Naegleria can be checked by chlorination of pools and man-

made swimming lakes, but that is not feasible for large bodies of water.

E. histolytica is endemic in many tropical and subtropical regions where climatic

conditions favor survival of cysts in the environment. Cyst viability, however, is not enough

to insure disease transmission and, in those areas of the globe that have adequate facilities

for treating sewage and protecting the water supply from contamination, there would likely

be no major change in the incidence of invasive amebiasis. There might be an increase in

sporadic cases of amebiasis, mostly among travelers returning from areas with sub-

standard water quality, but no generalized explosion of infections. The same would be true

for B. coli, which shares a similar fecal–oral route of transmission.

7.3. Modern technology

Two aspects of human technological progress and their effect on the occurrence of some

of the diseases dealt with in this report are noteworthy. The development and extensive use

of soft and extended-wear contact lenses since the 1980’s have resulted in emergence of

Acanthamoeba keratitis as a significant ophthalmic disease. Prior to that period, the

occurrence of amebic keratitis was a rare event and limited to accidental injury to the

cornea, with subsequent infection by the ameba either from water or soil. With widespread

use of contact lenses and lack of clear guidelines for their care, amebic keratitis reached

epidemic proportions with cases being reported from all over the globe. The problem was

aggravated by the lack of effective anti-microbial agents for treatment. At present, medical

personnel are aware of amebic keratitis, effective therapy is available, and contact lens

users are better informed of the risks of non-compliance with recommendations for proper

lens care.

The extensive use of ventilation systems in otherwise sealed buildings has led to

sporadic outbreaks of legionellosis, associated with the cooling towers that are part of

the air conditioning systems. Acanthamoeba spp. have also been isolated from hospital

plumbing systems where they can serve as host to Legionella spp., releasing bacteria

from sink tap, shower head, and toilet aerosols into an environment having a

disproportionate number of patients with weakened immune systems (La Scola et al.,

2003). Hot tubs and physiotherapy pools, unless scrupulously maintained, can also favor

growth of thermotolerant amebae. Cases of amebic keratitis have been traced back to

contact lens users who wore their lenses while soaking in a hot tub or spa (Samples et al.,

1984). Among 108 patients with amebic keratitis surveyed from the literature, 3.7% had

spent time in a hot tub prior to developing the infection (Lang and von Heimburg-Elliger,

1991).

There was passing concern about possible release of Naegleria amebae in the

misting spray released over the fruit and vegetable displays in many supermarkets in the

United States. Since these displays release cool water, it is unlikely that growth of N.

fowleri and thermophilic amebae would be favored, but other species of Naegleria and


Acanthamoeba might be found. These amebae, however, are not transmitted by an oral

route.

8. Conclusions

Infections caused by opportunistic and pathogenic free-living amebae represent an

emerging group of diseases of humans and other animals recognized within the past few

decades. Generally, they are neither waterborne nor zoontic diseases. Escalating numbers

of immunocompromised patients, the result of the HIV/AIDS epidemic, has led to an

increase in some of these diseases, as has an increasing number of immunosuppressed

patients undergoing organ transplantation and other medical procedures. Because of

technical difficulties in diagnosis, these infections are reported mainly from the developed

world but are likely to go undiagnosed in the developing world.

In contrast, infections by the parasitic protozoa E. histolytica and B. coli were

recognized more than a century ago. They have the potential for zoonotic transmission but

their spread is, particularly for amebic dysentery, person-to-person. In the contemporary

world, amebiasis and balantidiasis remain public health problems chiefly in developing

countries where sewage-contaminated water and food, and lack of available therapy have

allowed them to persist.

Note added in proofAddendum to veterinary parasitology paper (Schuster and Visvesvara)

Two reports concerning the free-living amebae update the literature on Acanthamoeba

and Balamuthia. The number of different sequence types recognized in Acanthamoeba has

increased to 15 (T15) with the report of Hewett et al. (2003).

A 2-year old Great Dane died of Balamuthia encephalitis (Foreman et al., 2004). The

animal was being treated for inflammatory bowel disease with immunosuppressive doses

of prednisone. During the 6-month course of therapy, the dog was taken to swim in a pond

containing stagnant water one to two times a week. Histopathology of brain and kidney

tissues from the dog showed amebic trophozoites and cysts, possibly Acanthamoeba. With

immunostaining, the polymerase chain reaction, and electron microscopy, the amebae were

identified as Balamuthia. This case is of importance because it is the first report in the

literature of balamuthiasis in a dog, and it also raises the possibility that the amebic

infection was acquired from water.

Acknowledgements

We gratefully acknowledge our late colleague and friend Dr. A. Julio Martinez as the

source of several micrographs of amebae used in this paper. Dr. Martinez, who died in

December 2002, was a neuropathologist who was expert in identification of amebae in

brain and other tissues and had done much in the way of increasing awareness of amebic

infections in humans through numerous publications and presentations at meetings. We

thank Henry Bishop (Centers for Disease Control and Prevention) for providing

photomicrographs of B. coli.


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