Cryptosporidium parvum (Cryptosporidiosis)

Authors: A. Clinton White, Jr, M.D., Meena H. Seenivasan, M.D.

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Cryptosporidium parvum is a coccidian parasite that affects the intestinal and respiratory epithelium of vertebrates. It is capable of causing disease both in immunocompetent and immunocompromised hosts.

PARASITOLOGY

Life cycle

Cryptosporidium is an intracellular protozoan parasite within the phylum Apicomplexa, group Alveolata. Cryptosporidium parvum causes most of the human infections, although other species such as C. muris, C. felis and C. meleagridis have been reported to cause infection in some individuals (7, 28, 37, 63). Recent evidence suggests that C. parvum includes at least 2 genotypes that differ in host range. Genotype 1 is mainly transmitted from person-to-person. Genotype 2 mainly infects cattle (7, 37). Cryptosporidium is capable of completing both asexual and sexual phase of development in a single host. Humans are infected by ingestion of the oocyst. Excystation occurs in the small intestine with the release of 4 motile sporozoites. The sporozoites indents and invaginates the enterocyte surface to form a parasitoferous vacuole that is confined to the microvillus region. The sporozoites mature asexually to form meronts. Meronts release merozoites that reinfect the intestinal epithelium. Some of the merozoites then differentiate into the sexual forms (gametocytes), which fuse to form the oocysts. The oocysts are then excreted into the feces. Oocysts are thought to rarely excyst within the host, resulting in autoinfection. Autoinfection plays an important role in causing serious and persistent disease in immunocompromised hosts.

Epidemiology

Cryptosporidium occurs worldwide. It is an important cause of diarrhea both in immunocompetent and immunocompromised population. Patients with AIDS are at increased risk. Transmission commonly occurs from person-person by the fecal-oral route, and rarely by fomites. Environmental transmission occurs mainly through contaminated water supply. Cryptosporidial oocysts may be found in all types of water including chlorine treated drinking water (44). The 1993 outbreak in Milwaukee, affected an estimated 403,000 persons and resulted in death of several immunocompromised patients and illness in many previously healthy people (40). Contamination of public water supply occurred due to decrease in the purity of source water and filtration effectiveness (40). Zoonotic transmission from cattle and sheep to humans is known and they serve as reservoirs of cryptosporidial infection. Population at increased risk of cryptosporidial infection includes household contacts and sexual partners of patients, health care workers, day care personnel and travelers to highly endemic regions, and users of communal swimming pools (7).

Clinical Manifestations

Cryptosporidiosis presents with a range of clinical manifestations, which vary with the immune status of the individual.

Immunocompetent Host

Cryptosporidium is confined to the gastrointestinal tract. The three major clinical presentations include asymptomatic carriage, acute diarrhea, and persistent diarrhea that may continue for several weeks. The incubation period is usually a few days. Diarrhea is the commonest complaint. Onset is often abrupt. Watery diarrhea lasts several days to 2 weeks and is often self-limited. In children in developing countries, cryptosporidiosis causes malnutrition and growth retardation.

Immunocompromised Host

In AIDS patients the severity of symptoms depends upon the CD4 count. Four clinical syndromes are recognized and include 1)asymptomatic infection, in which there is no change in bowel movements and the patients pass less than 4 stools/day, 2)transient infection, in which diarrhea lasts for less than 2 months and is followed by complete remission, 3)Chronic diarrhea, lasting 2 months or more, with persistence of parasites in stool and biopsy specimens and 4)fulminant cholera like illness, in which patients pass 2 liters of stool per day, and occurs in AIDS patients with CD4 less than 50 per cubic millimeter. In AIDS patients the stool frequency is often 10 per day, and the patients suffer from severe malabsorption and weight loss (12, 29).

Extraintestinal cryptosporidiosis can involve the lungs, middle ear, biliary tract, pancreas and stomach. Biliary cryptosporidiosis is the commonest extraintestinal manifestation. Patients present with acalculous cholecystitis or sclerosing cholangitis. Diarrhea may or may not accompany the illness.

Laboratory Diagnosis

Diagnosis of Cryptosporidium is primarily based on identification of the oocysts in stool (8). Oocysts stain with a modified acid-fast technique. However, the accuracy of the acid-fast stained smear is highly dependent on technician experience and the time put into the examination. Sensitivity may be poor in light infections (67). Oocysts can also be detected by direct immunofluorescent assays that are commercially available utilizing monoclonal antibodies raised to Cryptosporidium antigens. In addition, enzyme linked immunosorbent assays may be used to detect Cryptosporidium antigens in fecal specimens (18). There is no consensus on the optimal oocyst detection method in fecal samples, although one recent comparison of a modified acid-fast stain and a fluorescein labeled monoclonal antibody technique showed comparability for diarrheal samples but improved detection with the immunofluorescent method for formed specimens (67). Diagnosis of cryptosporidial enteritis can also be made on small intestinal biopsy sections by identification of developmental stages, found individually or in clusters, on the brush border of the mucosal epithelial surfaces. Organisms project into the lumen and appear basophilic with hematoxylin and eosin staining.

Pathogenesis

Cryptosporidium does not infect tissue beyond the superficial surface epithelia. In the intestines it causes villous atrophy, crypt hyperplasia, and infiltration of lymphocytes, neutrophils, plasma cells and macrophages into the lamina propria. While in immunocompetent individuals, infection is confined to the intestine, in immunocompromised patients, Cryptosporidium is found in the entire gastrointestinal tract and within the epithelial cells of the biliary tree, the pancreatic duct and the airways (38). Infection induces increased intestinal permeability and chloride secretion, which are thought to result from the host inflammatory response (8, 24, 37, 51). Control of infection depends on the host cellular immune response and production of cytokines such as interferon gamma (7, 72).

SUSCEPTIBILITY IN VITRO AND IN VIVO

In vitro studies have demonstrated that paromomycin has limited activity against Cryptosporidium with inhibitory concentrations in the range of 100-500 µg/mL (22, 43, 65). Paromomycin is effective in animal models of cryptosporidiosis (2, 50, 64, 66), but usually require doses of 100-500 mg/kg/day. The dose usually used in most human trials, 25-35 mg/kg/d in 2-4 doses, is estimated to result in drug levels in the intestinal lumen at or barely above the amount of drug needed to inhibit the organisms in cell culture (71). Nitazoxanide and its active metabolite tizoxanide are active again C. parvum in vitro and in animal models (19, 23, 62).

ANTIPARASITIC THERAPY

General

The role of antiparasitic therapy in cryptosporidiosis remains controversial. No agent has proven reliably curative in the absence of effective immune response. This poor response to treatment may result from the unique location of the parasite, separated from the lumen by the host membrane but also segregated from the host cytoplasm by the base of the parasitophorous vacuole. Thus, there is limited exposure to drugs in the lumen, serum, and even in the enterocyte cytoplasm (26).

Much of the literature on treatment of cryptosporidiosis is in case reports or uncontrolled trials. Since the disease is self-limited in normal hosts and can be variable in immunocompromised hosts, these data are problematic. The voluminous secretions and altered intestinal absorption caused by cryptosporidiosis may interfere with drug absorption. Furthermore, for the agents tested in clinical trials, most of the trials were not designed to detect partially active agents. Few of the studies of AIDS patients have rigorously excluded patients co-infected with Mycobacteria, microsporidia or cytomegalovirus, all of which are common co-infections which can cause dampen the effects of anti-cryptosporidiial treatments (60, 69). Thus, partially active drugs (which might prove useful in combination or in situations in which the patients immune response can be boosted) may been labeled as ineffective.

Nitazoxanide

Nitazoxanide is a nitrothiazole compound with activity against a broad range of parasites. In a randomized, placebo-controlled trial in apparently immunocompetent children and adults, nitazoxanide significantly decreased the duration of C. parvum-associated diarrhea and oocysts shedding (52). The dose used in the trial was 500 mg bid for 3 days (with doses of 200 mg and 100 mg bid in children). The AIDS clinical trials group (ACTG) conducted a dose-ranging study of Nitazoxanide in AIDS patients with cryptosporidiosis. The study suggested that the drug could be given safely and might be partially effective (10). A larger randomized trial, however, had to be abandoned due to slow patient accrual. A randomized trial in HIV infected patients with cryptosporidiosis conducted in Mexico demonstrated shorter duration of symptoms and cure of oocyst shedding with treatment of patients with CD4 cell counts above 50. However, no effect was noted among patients with CD4 cell counts below 50 (53). Unfortunately, the latter group includes the patients most in need of an effective anti-parasitic therapy. Nitazoxanide is approved for use in some countries and is available in the United States by expanded access from the manufacturer, Romark Laboratories.

Paromomycin

Paromomycin is a non-absorbable aminoglycoside that was approved by the FDA in the 1960s for using in amebiasis. Anti-cryptosporidial activity was initially identified when desperate AIDS patients were treated with every available antiparasitic drugs and some improved when treated with paromomycin (21).

Most of the data on paromomycin in human cryptosporidiosis has been from case series or uncontrolled trials studying patients with AIDS. Among the first 11 published case series including 300 patients, the response rate was reported to be 67% (3, 29). Many of those with initial improvement later relapsed. Three randomized controlled trials have studied paromomycin in AIDS patients with cryptosporidiosis. Kanyok and colleagues presented preliminary data from a small trial in 1993 demonstrating efficacy of paromomycin (34). White and colleagues conducted a small placebo-controlled trial that incorporated quantitation of oocyst excretion. There was a significant reduction in oocyst shedding (about 70%) and decreased stool frequency in those treated with paromomycin (69). However, cures were unusual and many patients subsequently developed biliary involvement. The ACTG also compared paromomycin with placebo in a group of 35 patients (30). On treatment analysis demonstrated no significant difference between groups. However, dropouts only occurred in the placebo arm. Thus, by intent-to-treat analysis with dropouts grouped with failures, the response were similar to those described by White and colleagues with a trend favoring paromomycin over placebo (70). Only limited efforts were made to exclude co-infection with other enteric pathogens. Furthermore, the trial was prematurely terminated due to poor enrollment and thus was not powered to detect this limited response. Thus, the bulk of evidence suggest that paromomycin is partially active against Cryptosporidium parvum.

Since paromomycin levels are likely to vary between individuals and within individuals depending on the amount of intestinal secretions, preclinical testing already suggests limitations with this agent. Dose escalation might improve efficacy. However, a trial incorporating dose escalation demonstrated no further improvement with higher doses (30) and high doses of paromomycin can actually cause malabsorption (36).

Macrolide Antibiotics

Spiramycin is a macrolide antibiotic that is marketed in Europe for treatment of toxoplasmosis and respiratory tract infections. As early as 1983, anecdotes were reported of patients with cryptosporidiosis with improvement or cure after spiramycin therapy. Two controlled trials of spiramycin were performed on children with cryptosporidiosis. Sáenz-Lloren and colleagues reported shorter duration of symptoms and oocyst shedding with spiramycin treatment at 100 mg/kg/d of spiramycin (55). However, this was not confirmed in a second trial using 75 mg/kg/d (73). The ACTG conducted several studies of oral and intravenous spiramycin in adult AIDS patients with cryptosporidiosis. A randomized controlled trial in 75 AIDS patients, spiramycin dosed at 3 million International Units per day in 3 doses was no better than placebo. A trial of intravenous spiramycin at 3 million followed 4.5 million International Units per day noted a significant decrease in oocyst shedding and at least a partial response in 75% of subjects. However, there were high rates of adverse events including drug-associated intestinal injury (68).

Azithromycin is an azalide antibioitic that is FDA approved for respiratory infections as well as treatment and prevention of AIDS-associated Mycobacterium avium complex infection. In vitro and animal studies suggest some activity against Cryptosporidium parvum. Case reports suggest activity in cryptosporidiosis (1, 11, 20, 31, 46, 54). In a multicenter, double blind trial, AIDS patients with cryptosporidiosis were randomly assigned to receive azithromycin 900 mg PO daily or placebo. Overall, there was no significant difference is oocyst shedding, stool frequency or weigh loss between groups (2,61). A post-hoc analysis revealed a significant decrease in oocyst shedding in those with the highest serum levels of azithromycin. A pilot trial of intravenous azithromycin failed to demonstrate any changes in oocyst shedding or stool frequency (2, 15).

Clarithromycin is also active in vitro and in animal studies. The data on use of clarithromycin in cryptosporidiosis are limited to a few case reports. However, the drug may be useful in chemoprophylaxis (see below).

Other Agents

A number of anecdotal reports have noted improvement in chronic cryptosporidiosis in patients treated with oral anti-Cryptosporidium immunoglobulin preparations (25). Two well-controlled trials have examined oral bovine anti-Cryptosporidium Immunoglobulin (BACI) preparations in cryptosporidiosis. In one, volunteers received BACI or placebo before or after being challenged with C. parvum. There was no significant decrease in symptoms or oocyst shedding if BACI was given after challenge (47). In a large randomized, dose-escalating trial of BACI in AIDS patients with chronic cryptosporidiosis, there was no effect of BACI on symptoms. Quantitation of oocyst shedding only demonstrated a modest 50% decrease in oocyst excretion at a dose of 20g/d (16). At higher doses, oocyst excretion decreased, but the immunoglobulin preparation caused diarrhea. Thus, current data do not support a role for this therapy.

Diclazuril and letrazuril are agents that have been used in avian coccidiosis. Initial studies of diclazuril in AIDS patients with cryptosporidiosis demonstrated poor bioavailability. Studies of letrazuril were initially thought to demonstrate marked reduction in oocyst excretion with minimal change in symptoms. Subsequent studies revealed that the treatment mainly interfered with the acid fast staining of oocysts.

Combination Therapy

Since currently available agents are only partially effective, it is logical to try and combine these agents to improve efficacy. Smith and colleagues conducted a pilot open-label study of paromomycin combined with azithromycin in AIDS patients with chronic cryptosporidiosis (60). Overall, there was a marked improvement in oocyst shedding (decreased by over 2 logs). However, few patients were cured. Most of the clinical failures were associated with biliary disease, co-infection with other enteric pathogens (especially CMV), or side effects of the medications. Thus, while unproven, combination therapy merits further study.

Conclusions

In summary, currently available antiparasitic drugs have only limited activity in cryptosporidiosis. Nitazoxanide can hasten cure in immunocompetent patients and patients with HIV with CD4 cell counts above 50. For patients with advanced AIDS and chronic infection, the most important aspect of therapy is treatment with effective anti-retroviral therapy. Since cryptosporidiosis-induced gastrointestinal injury may interfere with anti-retroviral therapy, it is probably prudent to initially include anti-parasitic drugs and anti-motility agents in the treatment regimen. However, this may require combinations of drugs. Paromomycin alone or in combination with a macrolide (such as azithromcyin or spiramycin) is an available treatment that may be useful in this context. However, further studies are needed to design optimal combinations of agents.

Special Situations

Extraintestinal Cryptosporidiosis

There are reports of cases of cryptosporidiosis outside of the intestinal and biliary tract. These patients likely require antiparasitic therapy. There are case reports of patients responding to macrolides or inhaled paromomycin.

Special Groups

Immunocompetent Hosts

Since cryptosporidiosis is a self-limited infection in immunocompetent hosts, the mainstay of therapy is supportive measures including fluid and electrolyte replacement and anti-motility agents. While data are limited, apparently immunocompetent hosts with symptoms lasting more than one week may benefit from anti-parasitic therapy. If it is available, Nitazoxanide at a dose 500 mg PO twice a day should be the treatment of choice (52). Macrolides such as spiramycin and azithromcyin may be used as alternatives.

Immunocompromised Hosts Other Than Patients with AIDS

Patients with leukemia, primary immunodeficiencies, inflammatory bowel disease, and a number of chronic diseases are at increased risk of chronic cryptosporidiosis (33, 39). Case reports suggest that a limited number of patients with uremia or malignancy and chronic cryptosporidiosis have improved after anti-parasitic therapy. Most reports include a macrolide (azithromycin 600 mg per day or spiramycin 750 mg PO 3 to 4 times a day) alone or in combination with paromomycin (25-35 mg/kg/d in 2-3 daily doses). Nitazoxanide alone or with paromomycin and/ or a macrolide may also benefit some patients with chronic infection.

Patients Infected With HIV

For patients with HIV, the key to management of cryptosporidiosis is to reverse their immunosuppression with anti-retroviral therapy. However, anti-parasitic drugs may be very important, especially for treatment of patients with prior anti-retroviral treatment. For patients with relatively intact immune function (e.g. CD4 cell count above 50), nitazoxanide may hasten resolution of cryptosporidiosis (53). However, the dose should be higher than in immunocompetent hosts (1g b. i. d. for 14 days). Recent studies suggest that partially-active anti-parasitic agents such as paromomycin alone or with azithromycin may be effective in the context of anti-retroviral agents (41, 48). Thus, anti-motility agents and anti-parasitic drugs should generally be started before initiation of anti-retroviral therapy.

ADJUNCTIVE THERAPY

Supportive therapy is critically important in cryptosporidiosis. Watery diarrhea results in significant losses of fluids and electrolytes and may contribute to malnutrition. Replacement of fluid and electrolyte losses by either the oral or intravenous route is of paramount importance. Aggressive efforts at oral rehydration should be made with Gatorade, bouillon, or oral rehydration solution that contains glucose, sodium bicarbonate, and potassium. Intravenous therapy may be required to correct losses of bicarbonate, potassium, magnesium, and phosphorus. Nutritional support may also improve responses to therapy (58).

Treatment with antimotility agents is also an important adjunctive therapy. Loperamide or diphenoxylate/atropine will often reduce the stool frequency. Tincture of opiate may decrease transit time in patients who have not responded to loperamide or diphenoxylate/atropine. Octreotide, a synthetic peptide analog of somatostatin is FDA approved for the treatment of tumor-induced secretory diarrhea. Several trials have examined octreotide therapy in AIDS patients with diarrhea (6, 17, 59). Overall, octreotide was not consistently more effective than other oral antidiarrheal agents. Due to its high cost, its use is generally limited to refractory cases. For patients with AIDS and chronic cryptosporidiosis, the key to management is to reverse the immunodeficiency with effective anti-retroviral therapy (5, 14, 27, 42, 45). This should generally take the form of combinations of 3 or more potent anti-retroviral drugs including reverse transcriptase inhibitors with or without protease inhibitors. Patients with severe cryptosporidiosis may not response optimally to anti-retroviral therapy in the setting of profuse watery diarrhea from cryptosporidiosis. Thus, aggressive therapy with anti-motility agents is essential. The role of antiparasitic drugs is controversial in this context, but, as noted above, patients may have a better response to anti-parasitic drugs if they can be treated for HIV as well.

Biliary involvement in cryptosporidiosis usually requires specific interventions. Acalculous cholecystitis should be treated with cholecystectomy. Patients with sclerosing cholangitis can usually be treated by ERCP. Sphincterotomy may result in temporary improvement (4). However, symptoms usually recur unless a stent is placed (9, 29).

ENDPOINTS FOR MONITORING THERAPY

Since the primary clinical manifestation of cryptosporidiosis is diarrhea, resolution of diarrhea should be the main clinical endpoint of therapy. Stool frequency and character (e. g. watery, loose, formed) remain important symptoms. Electrolytes and volume status should also be monitored carefully. In immunocompromised patients, it is also important to follow the patients for symptoms, signs, and laboratory tests suggestive of biliary tract involvement. Right upper quandrant discomfort and/or elevations of alkaline phosphatase should prompt the clinician to obtain imaging studies of the biliary tract (ultrasound, ERCP) to exclude acalculous cholecystis or sclerosing cholangits, which may require surgical therapy. Persistent diarrhea should prompt aggressive testing for possible co-infections, including atypical Mycobacteria, microsporidia, and CMV.

Clinical trials should routinely incorporate quantitative studies on oocyst excretion. Goodgame and colleague have demonstrated that careful quantitation of oocyst shedding correlates well with the level of gastrointestinal dysfunction (24). Since there concentration of oocysts may vary up to a log between stools in an individual, quantitation is better if multiple stools are collected. While clinical laboratories often provide semi-quantitative data on oocyst concentration (1+ to 4+), these data are not adequate to document the effects of partially active agents. Several studies have incorporated careful quantitation (16, 60, 69).

VACCINES

No vaccines against Cryptosporidium are commercially available. Animal studies have demonstrated protection using recombinant DNA vaccines (49, 56, 57). Further studies will be required to see if any of these constructs can prove safe and effective in humans.

PREVENTION

General

Current water purification standards do not uniformly remove viable oocysts. Filtration is particularly important when surface contamination may occur in water sources such as during spring runoff, or in households using well water. If personal-use water filters are utilized, they should be capable of removing particles 1 µm in diameter (35). Boiled or filtered drinking water should be considered by HIV-infected persons with CD4 counts under 200/mm3. HIV-infected persons who travel in developing countries should meticulously avoid drinking tap water and contaminated water resources. At-risk persons should avoid contact with obvious sources of Cryptosporidium oocysts, such as people with diarrhea (especially regarding sexual practices that involve oral exposure to feces), farm animals (particularly cattle), and domestic pets that are either very young (< 6 months), have diarrhea, or have been stray.

Infection Control Measures

Infection control measures are limited by resistance of the Cryptosporidium oocysts to common disinfectant. Enteric precautions with good hygiene, such as hand washing, and proper disposal of contaminated materials, such as diapers, are important.

Antiparasitic Agent Prophylaxis

While cryptosporidiosis is self-limited in immunocompetent individuals, some groups of immunocompromised patients are at high risk of severe disease. These groups include patients with AIDS, especially in developing countries, and some children with congenital immunodeficiencies. At present, there are limited human data on chemoprophylaxis of cryptosporidiosis. Two retrospective studies have examined data on rifabutin, azithromycin, or clarithromycin for prophylaxis of Mycobacterium avium infection asking the question whether the rates of cryptosporidiosis differed between drug-treatment arms. Holmberg and colleagues retrospectively analyzed data from the HIV Outpatient Study cohort comparing the incidence of cryptosporidiosis in those given rifabutin, clarithromycin, or no drug for prevention ofMycobacterium avium complex. There was a dramatic reduction in the incidence of cryptosporidiosis in those treated with rifabutin (hazard ratio 0.15) and a significant reduction in those treated with clarithromycin (hazard ratio 0.25) (32). Fichtenbaum and colleagues performed a cross-protocol analysis of patients enrolled in controlled trials of clarithromycin, rifabutin, both, or no drug for M. avium complex prophylaxis. They also noted a lower incidence of cryptosporidiosis than in groups treated with rifabutin (relative risk 0.42) (13). However, they could not confirm the efficacy of clarithromycin.

While rifabutin (generally dosed at 300 mg PO per day) and clarithromycin (500 mg PO BID) may have prophylactic efficacy, the risk of infection remains small in most patient groups (35). Whether these drugs can help children with congenital immunodeficiencies (such as X-linked immunodeficiency with hyperimmunoglobulin M) remains to be established (39). Instead, precautions aimed at preventing exposure are considered the main prophylactic measure. These include avoiding unfiltered water and fecal-oral exposure to animals, young children, or patients with diarrhea.

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