Cryptosporidiosis and Microsporidiosis
Cryptosporidium parvum (C. parvum), a coccidian protozoan, has been recognized as a human pathogen only since 1976. It received widespread recognition as a disease-producing agent in 1993, when over 400,000 people in Milwaukee Wisconsin developed diarrhea after drinking water contaminated with C. parvum1. Similar, but less spectacular, outbreaks have been reported since then and C. parvum has emerged as the leading cause of waterborne disease outbreaks in the United States, as well as an important opportunistic infection in individuals with AIDS and a significant cause of outbreaks of diarrhea in child day-care centers5. In addition to drinking water, swimming pool water has been identified as a source of cryptosporidium infection. The magnitude of the problem has heightened public concern about the safety of drinking water in the United States. Serious attention has been focused on determining and reducing any existent risks for cryptosporidiosis from community and municipal water supplies by coordinating efforts between public health agencies and water utilities, and by the development of regulatory standards for cryptosporidiosis in drinking water.
C. parvum is widespread in the environment and infects the gastrointestinal tracts of a variety of hosts including fish, reptiles, birds and mammals. Infection begins when oocysts, which are ubiquitous in surface waters such as lakes and rivers, are ingested and encyst in the small intestine. An asexual cycle of development occurs followed by a sexual cycle. In the latter, microgametes fertilize macrogametes with the subsequent development of oocysts and completion of the cycle. Both asexual and sexual cycles can occur in the gastrointestinal tract, and the fully sporulated oocysts are passed in the feces. The fecal-oral route is the mode of human transmission of cryptosporidiosis.
Cryptosporidiosis oocysts are highly resistant to the disinfectants that are commonly employed in municipal water treatment and are incompletely removed by filtration. Small numbers of oocysts have been detected in 17 to 55% of U.S. cities8,9, but there is uncertainty about the parasitic load that is required for successful transmission and, except for immunocompromise, about the risk factors in the recipient that predispose to successful transmission. Additionally, in spite of the use of state-of-the-art technology in water treatment, outbreaks have continued to be reported10. Household transmission has been reported but the findings suggest that the risk of person-to-person transmission is low in the setting of good hygiene and limited exposure to feces.
The compromised immune system of HIV-positive patients with Acquired Immunodeficiency Syndrome (AIDS) permits involvement of the gastrointestinal tract by many microorganisms including C. parvum. Preliminary data from a prospective study of persons with HIV infection in Atlanta suggest that 5 to 10% of these individuals can be expected to develop Cryptosporidium infection annually11. In some instances this may represent a reactivation of a former infection associated with a decrease in CD4 lymphocytes. However, a survey of HIV-infected persons in the Milwaukee outbreak revealed that the attack rate among them was no greater than that of the general population, regardless of CD4 counts. Nonetheless, an increased severity of illness was associated with decreasing CD4 counts. There was a high prevalence of associated biliary tract involvement in the HIV-infected Milwaukee patients, particularly among those with low CD4 counts14. Among those with biliary tract involvement, the 1-year mortality was 83%. Extra-intestinal cryptosporidiosis is more common in AIDS patients than in immunocompetent persons, with the principal sites of involvement being the bile ducts, pancreas, and lungs.
Developmental stages of cryptosporidia can be identified at all levels of the gastrointestinal tract although the jejunum is the most heavily infested site. Parasites can also be found in the colon, duodenum, stomach, gall bladder, and biliary and pancreatic ducts. Histological abnormalities may be absent, but usually there is evidence of an acute enteritis and there may be villous blunting. Cryptosporidium is usually confined to the brush border of the enterocyte and is generally not tissue invasive. However, in cases that are chronic or severe, cellular damage may result both from direct stress caused by the protozoal replication/extrusion process and damage mediated by the inflammatory response. Loss of structural integrity may result in loss of function. Decreased levels of brush border enzymes leads to malabsorption as indicated by impairment of d-xylose absorption and an abnormal Schilling test.
C. parvum is easily identified by light microscopy in biopsy or autopsy tissue stained with hematoxylin and eosin; no special staining techniques are required. Electron microscopy is helpful for confirmation of cryptosporidial infection when the organism cannot be reliably distinguished from fat droplets or other intestinal material.
Intestinal cryptosporidiosis is most commonly diagnosed by finding the characteristic sporulated oocyst with a diameter of four to six (m in the stool. Special stains are required; the most frequently employed is a modification of the acid-fast technique. With this staining technique, the oocyst appears pink to bright red and often contains prominent black granules. Mucosal biopsies for Cryptosporidium are not usually indicated when stool samples are positive. However, even in the presence of known infection with C. parvum, the rate of detection of stool oocysts is relatively low. As might be expected, examination of multiple stool specimens increases sensitivity.
Symptoms of Cryptosporidium infection generally begin within three to 12 (median, five to seven) days after ingestion of oocysts. In immunocompetent individuals cryptosporidiosis usually causes an acute, self-limiting diarrheal disease which lasts an average of five to 10 days. It is often accompanied by nausea, vomiting, abdominal cramps, and low-grade fever. Data from animal studies suggest that as few as two to 10 oocysts may be sufficient to establish infection. In a recent study, Dupont and colleagues infected 29 human volunteers with Cryptosporidium oocysts varying in number from 30 to 1,000,000. Of these, 18 became infected as evidenced by the presence of oocysts in the stool, but only seven (39%) developed gastrointestinal symptoms. The mean dose resulting in infection was 132 oocysts, but one volunteer became infected after 30 oocysts. Patterns of oocyst shedding correlated with the presence of shedding but not with the number of oocysts ingested. Thus, parasitologic confirmation of cryptosporidiosis may be particularly difficult in patients with mild symptoms. Diarrhea may occur intermittently, especially during the two weeks following the initial infection. This characteristic cannot be attributed to re-infection and has important implications concerning the dangers of person-to-person transmission.
In immunocompromised patients and persons with AIDS, diarrhea can be prolonged, voluminous, debilitating, and not infrequently, fatal. Chronic diarrhea, defined as two or more watery stools per day for a period of 30 days, is a particularly serious problem in AIDS patients. Both the World Health Organization (WHO) and the Center for Disease Control (CDC) have recognized this syndrome as the “Diarrhea Wasting Syndrome” (DWS). A significant proportion of these patients have chronic cryptosporidiosis.
The Houston study identified 95 HIV-infected patients (nine children and 86 adults) with cryptosporidiosis over a six-year period. Risk was highest in Hispanics and with a history of male-to-male sexual practices. Diarrhea, weight loss and gastrointestinal discomfort were common complaints at presentation. Among the HIV-infected adults, 20 (23%) developed biliary tract disease. There was a clear relationship of disease severity to biliary tract involvement and low CD4 counts. The biliary disease responded to cholecystectomy or sphincterotomy with stent placement. Despite the morbidity, cryptosporidiosis in this series was rarely the cause of death.
No safe and effective cure is available for cryptosporidiosis. Immunocompetent patients improve without intervention although anti-diarrheal preparations may provide symptomatic relief during the episode of acute diarrhea. In immunocompromised patients, especially those with low CD4 cell counts, paromomycin, in doses of 1.5 to 2.0 gm per day, appears to be the current drug of choice. Forty to 90% of patients are reported to respond to an initial course of treatment with improvement in diarrhea and decreased shedding of organisms, but not with parasitological cure; and relapses are common. A preliminary study with letrazuril has reported improvement in diarrhea in 50% of AIDS-related cryptosporidiosis.
In the absence of effective therapy for cryptosporidiosis, prevention guidelines for cryptosporidia that are based on existing knowledge have been published, and an informational pamphlet on cryptosporidiosis for persons with AIDS has been developed. Additionally, cryptosporidiosis has been recognized as a nationally notifiable disease.
Microsporidia are spore-forming protozoal parasites widely distributed in nature that produce disease in both animals and humans. Their host range is extensive and includes most invertebrates and vertebrates. Microsporidia are obligate intracellular parasites that have no metabolically active stages outside the host cell. There are more than 90 known genera of microsporidia and almost 1000 species have been identified1. Their morphology and biological features suggest that microsporidia are very ancient organisms2.
Only five genera of microsporidia are known to occur in man – Enterocytozoon, Septata. Encephalitozoon, Pleistophora, and Nosema. The potential sources and modes of transmission of human microsporidial infections are uncertain, although the consensus is that the parasite is transmitted through the oral ingestion of feces containing mature spores. The documentation of microsporidial spores in urine and respiratory secretions, in addition to their presence in the stool and duodenal aspirate, suggests a potential role for person-to-person transmission4. Although microsporidiosis has been reported in immunocompetent individuals with traveler’s diarrhea5, the vast majority of cases are seen in the immunocompromised patient with AIDS. Additionally, a few cases have been identified in patients with post-transplant immunosuppression6.
The compromised immune system of HIV-positive patients with Acquired Immunodeficiency Syndrome (AIDS) permits chronic infection of the gastrointestinal tract by many microorganisms which are relatively harmless to the normal individual. The disabling diarrhea which often results from this continual assault contributes significantly to the morbidity and mortality of these patients. Chronic diarrhea, defined as two or more watery stools per day for a period of at least 30 days, leads to malabsorption, weight loss and cachexia. Both the World Health Organization (WHO) and the Center for Disease Control (CDC) have recognized this condition as the “Diarrhea Wasting Syndrome” (DWS).
Two species, Enterocytozoon beineusi (E. bieneusi) and Septata intestinalis (S. intestinalis) are responsible for the vast majority of intestinal microsporidiosis seen in AIDS patients. E. bieneusi has been reported in up to 90% of reported microsporidial infections and as the offending organism in 30% to 50% of all AIDS-related diarrhea where there is an isolatable pathogen. In a number of patients having AIDS-related chronic diarrhea, no etiological agent can be identified.
Unchecked, microsporidial-induced diarrhea is associated with morphological and functional pathology in the intestine. Histological examination of the infected small intestinal mucosa reveals villus atrophy, crypt hypertrophy and decreased mitoses12. At the cellular level, infection is initiated when a spore releases sporozoites which invade and replicate within the enterocyte. The end product is an oocyst or spore which is extruded into the intestinal lumen where it can infect other cells or be excreted and passed to another host. Cellular damage probably results both from direct stress caused by the protozoal replication/extrusion process and damage mediated by the inflammatory response. Loss of structural integrity begets loss of function. Decreased levels of brush border enzymes leads to malabsorption as indicated by impairment of d-xylose absorption and an abnormal Schilling test.
Although the site of microsporidial infections is usually the gastrointestinal tract, microsporidia have been isolated from the eye and ocular adnexa14, respiratory tract15, genitourinary tract16, and central nervous system. In some cases the microsporidiosis is disseminated with multiple organ involvement.
The microsporidial spores seen in human infections are particularly small, measuring one to two microns in diameter, and are difficult to detect because of their size, their location within cells, and their poor response to routine staining techniques. Until recently, the only reliable method for diagnosis was small bowel biopsy using transmission electron microscopy (TEA) for tissue examination. Light microscopy utilizing a modified trichrome stain, Giemsa stain and Uvitex 2B stain are newer techniques which allow for identification of microsporidia in stools and duodenal aspirates. Recent studies suggest that the polymerase chain reaction (PCR) is useful for detecting microsporidia in the stool and in bowel biopsies.
E. bieneusi has been most frequently associated with chronic diarrhea and/or hepatobiliary disease in patients with AIDS. The diarrhea is non-bloody and without fecal leucocytes, but may be profuse and watery and associated with abdominal pain, cramping, nausea, vomiting, anorexia, and weight loss. The patients usually have from three to 10 bowel movements per day. CD4 counts are usually low (<100 cells/mm3). Species of microsporidia have been associated with disseminated disease with involvement of the lungs, kidneys, liver, muscles and the brain. Cholangitis and involvement of the gall bladder is fairly common, but overt jaundice is unusual.
Although reports of deaths directly attributable to E. bieneusi are rare, there is little doubt that the associated malabsorption and secondary wasting produced by the diarrhea contributes to the patient’s demise. Reported mortality rates in patients with E. bieneusi are as high as 56% and even higher in the presence of biliary tract disease9,25. Although less commonly seen than E bieneusi, E. intestinalis (S intestinalis) presents features that are clinically indistinguishable from E. bieneusi.
There is no FDA-approved therapy and no standard of care for the treatment of microsporidiosis, although there have been reports of temporary improvement and partial success with several agents including metronidazole, albendazole, and thalidomide. Virtually all published studies to date have involved severely immunocompromised HIV-infected patients and there are no published blind, placebo-controlled comparative treatment trials. Preliminary reports of a good clinical response to metronidazole could not be confirmed.
Some reports have suggested that treatment with albendazole, a broad-spectrum antiparasitic agent, may lead to improvement of diarrhea and weight gain in some patients even though parasites were still present in biopsy specimens of the small intestine and microsporidial spores were still detected in stool specimens obtained after treatment. Many of the parasites visible on small bowel biopsy appeared damaged under the electron microscope, suggesting the possibility of a direct action of albendazole against the parasite. Dieterich and colleagues28 treated 29 patients with AIDS and diarrhea due to E. bieneusi with 400 mg of oral albendazole twice daily for a month. In the 28 evaluable patients, the mean number of bowel movements decreased from 7.0 to 3.8 per day. No post-treatment biopsy, however, showed clearance of the parasite. A double-blind, placebo-controlled trial of albendazole in patients with AIDS and microsporidiosis is currently underway in five large centers. Patients are randomized to receive either 800 mg of albendazole or placebo twice a day for 28 days followed by 82 days of open-label albendazole. The primary clinical endpoint is the reduction in mean number of bowel movements from baseline by greater than 50%. Stool volume, weight gain, reduced parasite counts, and improved quality of life are secondary endpoints.
Recently, thalidomide has been used in the treatment of microsporidiosis. The rationale for its use was the finding that fecal tumor necrosis factor alpha (TNF-a) is elevated in microsporidiosis and thalidomide is a known anti-TNF-a agent.
Sharpstone and coworkers treated eighteen subjects with chronic diarrhea caused by E. bieneusi that had not responded symptomatically to albendazole and one previously untreated subject with E. intestinal with 100 mg of thalidomide daily at bedtime for one month. Clinical response was assessed by stool frequency and body weight. The histological response was assessed by microscopic determination of villus height/crypt depth ratios, and the immunologic response was assessed by fecal TNF-a levels. Seven subjects with chronic diarrhea due to E. bieneusi had a complete clinical response and three had a partial response to thalidomide. There was a significant decrease in stool frequency from a mean of 5.3 to 3.1 per day (p = 0.001), and weight increased significantly by 1.2 kg (p < 0.02). Thalidomide significantly increased the villus height/crypt depth ratio (p = 0.045), as well as the number of abnormal forms of microsporidia (p < 0.01). Fecal TNF-a levels decreased from 17.9 to 8.9 U/ml (not significant) There was an apparent disruption of all stages of the life cycle of E. intestinalis30.
Reyes-Teran et al performed a double-blind, placebo-controlled clinical trial on 28 adults with advanced HIV disease who had received antiretroviral agents for at least six months, who did not have an active opportunistic infection, but who had lost at least 10% of their body weight in the previous six months. Patients were randomized to receive thalidomide, 100 mg by mouth four times daily (n = 14), or a matching placebo (n = 14) for a period of 12 weeks. Weight gain, the main clinical endpoint, occurred in one patient on placebo and in eight receiving thalidomide (p = .021). Secondary endpoints were the Karnofsky performance status, CD4 cell counts, and HIV viral burden in peripheral blood mononuclear cells (PBMC). The Karnofsy index was significantly higher at the end of the study in the thalidomide-treated patients (p = .003). CD4 and viral burden did not change in either group. Somnolence and erythematous macular skin lesions were significantly more common in the thalidomide-treated group.