Page 7: Guidelines for Canadian Drinking Water Quality: Guideline Technical Document - Enteric Protozoa: Giardia and Cryptosporidium
Partie II. Science et considérations techniques
The health effects associated with exposure to Giardia and Cryptosporidium, like those of other pathogens, depend upon features of the host, pathogen and environment. The host's immune status, the (oo)cyst's infectivity and the degree of exposure (i.e., number of (oo)cysts consumed) are all key determinants of infection and illness. Infection with Giardia or Cryptosporidium can result in both acute and chronic health effects, which are discussed in the following sections.
Theoretically, a single cyst is sufficient, at least under some circumstances, to cause infection. However, studies have shown that the ID50 (the dose required for infection to be observed in 50% of the test subjects) is usually more than a single cyst and is dependent on the virulence of the particular strain. Human adult volunteer feeding trials suggest that the ID50 of Giardia is around 50 cysts (Hibler et al., 1987), although some individuals can become infected at a much lower dose (Rendtorff, 1978; Stachan and Kunstýr, 1983). The ID50 of Giardia in humans can also be extrapolated from dose-response curves. Using this approach, the ID50 for Giardia in humans is around 35 cysts (Rose and Gerba, 1991), which is comparable to that reported above. Giardia strains that are well adapted to their hosts (e.g., by serial passage) can frequently infect with lower numbers of cysts (Hibler et al., 1987). For example, Rendtorff (1978) reported an ID50 of 19 cysts when using human-source cysts in volunteers.
The prepatent period (time between ingestion of cysts and excretion of new cysts) for giardiasis is 6-16 days (Rendtorff, 1978; Stachan and Kunstýr, 1983; Nash et al., 1987), although this can vary, depending on the strain. Research with animal models has shown that smaller inocula result in longer prepatent periods but do not influence the resulting parasite burden (Belosevic and Faubert, 1983).
The specific mechanisms by which Giardia causes illness are not well understood, and no specific virulence factors have been identified. Some suggest that Giardia causes mechanical irritation or mucosal injury by attaching to the brush border of the intestinal tract. Others have proposed that Giardia attachment results in repopulation of the intestinal epithelium by relatively immature enterocytes with reduced absorptive capacities (leading to diarrhoea).
The host-parasite relationship is complex, and Giardia has been shown to be versatile in the expression of antigens (Nash, 1994), so universal lasting immunity is improbable. Humoral immune response is revealed by increased levels of circulating antibodies (immunoglobulin G [IgG] and immunoglobulin M [IgM]) and secretion of antibodies (immunoglobulin A [IgA]) in milk, saliva and possibly intestinal mucus. These antibodies may play a role in eliminating disease (Heyworth, 1988), but lasting immunity has not been demonstrated. Very little is known about cellular immunity, but spontaneous killing of trophozoites by human peripheral blood monocytes has been described (denHollander et al., 1988).
Typically, Giardia is non-invasive and results in asymptomatic infections. Based on U.S. data, 24% of individuals will develop symptomatic illness after infection with Giardia (Macler and Regli, 1993). Symptomatic giardiasis can result in nausea, anorexia, an uneasiness in the upper intestine, malaise and occasionally low-grade fever or chills. The onset of diarrhoea is usually sudden and explosive, with watery and foul-smelling stools (Wolfe, 1984). The acute phase of the infection commonly resolves spontaneously, and organisms generally disappear from the faeces. Assemblage A has been associated with mild, intermittent diarrhoea, whereas assemblage B has been linked to severe, acute or persistent diarrhoea (Homan and Mank, 2001; Read et al., 2002). Giardia infection can also lead to lactase deficiency (i.e., lactose intolerance) and a general malabsorptive syndrome. Some patients become asymptomatic cyst passers for a period of time and have no further clinical manifestations. Other patients, particularly children, suffer recurring bouts of the disease, which may persist for months or years (Lengerich et al., 1994). In the United States, an estimated 4600 persons per year are hospitalized for severe giardiasis, a rate similar to that of shigellosis (Lengerich et al., 1994). The median length of hospital stay is 4 days.
Giardiasis can be treated using a number of drugs, including metronidazole, quinacrine, furazolidone, tinidazole, ornidazole, nitazoxanide and nimorazole. Olson et al. (1994) showed that potential for a vaccine exists, but infections and symptoms are only attenuated, and prevention of infection is not feasible at this time.
Although human cryptosporidiosis is not well understood, dose-response information has become available through human volunteer feeding trials involving immunocompetent individuals. As is the case for Giardia and other pathogens, a single organism can potentially cause infection, although studies have shown that more than one organism is generally required (DuPont et al., 1995; Okhuysen et al., 1998, 2002; Chappell et al., 1999, 2006). Together, these studies suggest that the ID50 of Cryptosporidium is somewhere between 80 and 1000 oocysts (DuPont et al., 1995; Chappell et al., 1999, 2006; Okhuysen et al., 2002),indicating that Cryptosporidium isolates can differ significantly in their infectivity and ability to cause symptomatic illness. The TAMU isolate of C. parvum (originally isolated from a foal), for example, was shown to have an ID50 of 9 oocysts and an illness attack rate of 86%, compared with the UCP isolates of C. parvum (isolated from a calf), which had an ID50 of 1042 oocysts and an illness attack rate of 59% (Okhuysen et al., 1999). In contrast, the Iowa and Moredun isolates of C. parvum had an ID50 of 132 and approximately 300 oocysts, respectively, whereas illness attack rates were similar (i.e., 55-65%) (DuPont et al., 1995; Okhuysen et al., 2002). Based on a meta-analysis of these feeding studies, the ID50s of the TAMU, UCP and Iowa isolates were estimated to be 12.1, 2066 and 132 oocysts, respectively (Messner et al., 2001). The genetic basis for these differences is not known, although a number of virulence factors have been identified (Okhuysen and Chappell, 2002). In a separate meta-analysis using the TAMU, UCP and Iowa human study data, the probability of infection from ingesting a single infectious oocyst was estimated to range from 4% to 16% (U.S. EPA, 2006a). This estimate is supported by outbreak data, including observations made during the 1993 Milwaukee outbreak (Gupta and Haas, 2004).
The prepatent period for cryptosporidiosis is 4-9 days (Ma et al., 1985; DuPont et al., 1995; Okhuysen et al., 1999, 2002), although this can vary, depending on the isolate.
Infections of Cryptosporidium spp. in the human intestine are known to cause at least transient damage to the mucosa, including villous atrophy and lengthening of the crypt (Tzipori, 1983); however, the molecular mechanisms by which Cryptosporidium causes this damage are unknown. Several molecules are thought to mediate its mobility, attachment and invasion of host cells, including glycoproteins, lectins and other protein complexes, antigens and ligands (Okhuysen and Chappell, 2002; Tzipori and Ward, 2002). Most of the pathological data available have come from AIDS patients, and the presence of other opportunistic pathogens has made assessment of damage attributable to Cryptosporidium spp. difficult.
The primary mechanism of host defence appears to be cellular immunity (McDonald et al., 2000; Lean et al., 2002; Riggs, 2002), although humoral immunity is also known to be involved (Riggs, 2002; Okhuysen et al., 2004; Priest et al., 2006). Studies using animal models have demonstrated the importance of helper (CD4+) T cells, interferon gamma (IFN-γ) and interleukin 12 (IL-12) in recovery from cryptosporidiosis (Riggs, 2002). Antibody responses against certain glycoproteins involved in Cryptosporidium adhesion have been demonstrated (Riggs, 2002).
It is not clear whether prior exposure to Cryptosporidium provides protection against future infections or disease. Okhuysen et al. (1998) reported that initial exposure to Cryptosporidium was inadequate to protect against future bouts of cryptosporidiosis. Although the rates of diarrhoea were similar after each of the exposures, the severity of diarrhoea was lower after re-exposure. Chappell et al. (1999) reported that volunteers with pre-existing C. parvum antibodies (suggesting previous infection) exhibited a greater resistance to infection, as demonstrated by a significant increase in the ID50, compared with those who were antibody negative. However, in contrast to the earlier findings (Okhuysen et al., 1998), the severity of diarrhoea (defined by the number of episodes and duration of the illness) was greater among the subjects presumed previously infected.
Individuals infected with Cryptosporidium are more likely to develop symptomatic illness than those infected with Giardia (Macler and Regli, 1993; Okhuysen et al., 1998, 1999). The most common symptom associated with cryptosporidiosis is diarrhoea, characterized by very watery, non-bloody stools. The volume of diarrhoea can be extreme, with 3 L/day being common in immunocompetent hosts and with reports of up to 17 L/day in immunocompromised patients (Navin and Juranek, 1984). This symptom can be accompanied by cramping, nausea, vomiting (particularly in children), low-grade fever (below 39°C), anorexia and dehydration. Extraintestinal cryptosporidiosis (i.e., in the lungs, middle ear, pancreas, etc.) and death have been reported, primarily among persons with AIDS (Farthing, 2000; Mercado et al., 2007), but are considered rare.
The duration of infection is dependent on the condition of the immune system (Juranek, 1995) and can be broken down into three categories: 1) immunocompetent individuals who clear the infection in 7-14 days, 2) AIDS patients or others with severely weakened immune systems (i.e., individuals with CD4 cell counts <180 cells/mm3) who in most reported cases never completely clear the infection (it may develop into an infection with long bouts of remission followed by mild symptoms) and 3) individuals who are immunosuppressed following chemotherapy, short-term depression or illness (e.g., chickenpox) or malnutrition. In cases where the immunosuppression is not AIDS related, the infection usually clears (no oocyst excretion, and symptoms disappear) within 10-15 days of the onset of symptoms. However, there have been reported cases involving children in which the infection has persisted for up to 30 days. The sensitivity of diagnosis of cryptosporidiosis by stool examination is low--so low that oocyst excreters may be counted as negative prematurely. The application of more sensitive and rapid diagnostic tools, such as immunochromatographical lateral-flow assays, will help to reduce the number of false negatives (Cacciò and Pozio, 2006). Immunocompetent individuals usually carry the infection for a maximum of 30 days. With the exception of AIDS cases, individuals may continue to pass oocysts for up to 24 days. In an outbreak in a daycare facility, children shed oocysts for up to 5 weeks (Stehr-Green et al., 1987). The reported rate of asymptomatic infection is believed to be low, but a report on an outbreak at a daycare facility in Philadelphia, Pennsylvania, concluded that up to 11% of the children were asymptomatic (Alpert et al., 1986), and Ungar (1994) discussed three separate studies in daycare centres where the asymptomatic infection rate ranged from 67% to 100%. It has been suggested that many of these asymptomatic cases were mild cases that were incorrectly diagnosed (Navin and Juranek, 1984).
Nitazoxanide is the only drug approved for treatment of cryptosporidiosis in children and adults (Fox and Saravolatz, 2005), although more than 200 drugs have been tested both in vitro and in vivo (Tzipori, 1983; O'Donoghue, 1995; Armson et al., 2003; Cacciò and Pozio, 2006). This can be explained, in part, by the fact that most inhibitors target biochemical pathways resident in the apicoplast (plastid-derived organelle) (Wiesner and Seeber, 2005), a structure that C. parvum (Abrahamsen et al., 2004) and C. hominis (Xu et al., 2004) lack. Some progress has been reported with furazolidone in reducing the symptoms of immunocompetent patients. Spiramycin has apparently been used with some success in Chile and the United States, but at this time it is not licensed for general use by the U.S. Food and Drug Administration (Janoff and Reller, 1987).
Analysis of the complete genome sequence of Cryptosporidium may help to identify virulence determinants and mechanisms of pathogenesis, thereby facilitating the development of antimicrobials (Umejiego et al., 2004), vaccines (Wyatt et al., 2005; Boulter-Bitzer et al., 2007) and immunotherapies (Crabb, 1998; Enriquez and Riggs, 1998; Schaefer et al., 2000; Takashima et al., 2003) against Cryptosporidium.
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