Stephanie Boade Silas MD
DeVon Hale MD
Within the last decade, the AIDS epidemic has heightened awareness of several gastrointestinal spore-forming protozoan pathogens. The genera Cryptosporidium, Isospora, and Cyclospora are members of the subclass Coccidia and phylum Apicomplexa; the microsporidia are a group of organisms belonging to the phylum Microspora. The spectrum of disease caused by these protozoans goes beyond gastrointestinal manifestations, and the significance of these protozoan infections is becoming increasingly appreciated in both immunocompromised and immunocompetent hosts.
CRYPTOSPORIDIUM
Essentials of Diagnosis
General Considerations
Transmission of oocysts occurs by person-to-person, animal-to-person, and environmental contacts. Those at risk for person-to-person transmission include household members, sexual partners, health workers, and children in day care. Nineteen percent of household contacts of infected patients have been diagnosed with secondary infection. Household pets, laboratory animals, and farm animals have all been associated with transmission to humans. Nosocomial infection has also been reported.
Waterborne oocyst transmission is considered the most important mode of transmission and is associated with the largest outbreaks. The initial cases of waterborne transmission were described in 1983, in Finnish individuals with diarrhea who had traveled to St. Petersburg (then Leningrad), Russia. Subsequently, other cases of traveler's diarrhea have been attributed to cryptosporidiosis. Many common source outbreaks have been described in Great Britain and the United States, the largest of which occurred in Milwaukee in 1993, affecting 403,000 people. Surface runoff from cow pastures was identified as the contaminating source of oocysts. Unboiled well water has also been implicated in transmission. Cryptosporidiosis related to apple cider has been associated with use of fallen apples in contaminated cow pastures. Oocysts, which survive well in cold, moist environments, are found in 65–87% of surface water. In one study, oocysts were found in 27% of drinking water samples from 66 inspected treatment plants, confirming the resistance of oocysts to current water treatment methods.
The resulting diarrhea is postulated to be either secretory (ie, caused by an unidentified toxin) or osmotic (ie, caused by malabsorption from intestinal villi injury). Histologic findings include atrophy, blunting, fusion, or loss of villi. Crypt hyperplasia is seen, and the lamina propria of the intestine has an inflammatory infiltrate.
The immune response in cryptosporidiosis is also poorly understood. Patients with a variety of immune deficiencies, including both T-cell abnormalities and γ-globulin deficiencies, have prolonged and more severe infections. Acquired immunity can prevent reinfection and limit primary infection, but the mechanism of protection is not entirely understood. Selective antibodies may inhibit an important attachment step. Bovine colostrum containing anti-C parvum antibodies limited infection in immunodeficient patients, but similar colostrum without antibodies did the same in experimental mouse infections. Further, patients with AIDS have been shown to mount an antibody response yet still be unable to clear the infection. This response lends support to other evidence suggesting the importance of adequate CD4 cells in addition to immunoglobulin A antibodies for protection. The presence of particular cytokines may be important in this interaction. Human breast milk does not confer protection from infection.
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BOX 83-1 Cryptosporidiosis Syndromes |
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CLINICAL SYNDROMES
Enteric cryptosporidiosis is the most common clinical presentation in patient populations. In addition, immunocompromised patients may present with cholecystitis or respiratory infections attributed to C parvum (Box 83-1). Asymptomatic infection has also been reported.
Clinical Findings
Up to 10% of AIDS patients with cryptosporidiosis present with cholecystitis. Cryptosporidial cholecystitis has thus far been diagnosed only in AIDS patients.
Clinical Findings
C parvum has rarely been identified in biopsy and lavage specimens of immunodeficient patients who present with dyspnea, hoarseness, wheezing, or cough as well as symptoms of laryngotracheitis and sinusitis. Chest films are generally normal or show modest infiltrates and increased bronchial markings. The respiratory syndrome has not yet been directly associated with C parvum diarrheal illness.
Diagnosis
The majority of diagnoses of C parvum infection are made by identification of the organism in stool specimens (Table 83-1). Stool should be examined while it is fresh or fixed in 10% formalin or polyvinyl alcohol. Oocysts can be identified by light microscopy without specific staining but are more readily seen with modified acid-fast staining. Phase-contrast microscopy reveals birefringent oocysts. Oocysts in duodenal aspirates or respiratory secretions can be similarly identified. Submitting three to four separate stool samples has been recommended for increased diagnostic yield. However, in a study of AIDS inpatients, one stool specimen resulted in 96% sensitivity, increasing to 100% with only two specimens. Orders should specify suspected C parvum because most laboratories do not routinely use acid-fast staining.
Immunofluorescence and enzyme-linked immunosorbent assay techniques are more sensitive and specific methods of diagnosis (Figure 83-1). Sensitivities of 93–100% and specificities of 99–100% have been reported for various direct immunofluorescent-antibody assays. The available enzyme-linked immunosorbent assay techniques have 72–100% sensitivity and 98–100% specificity. The clinical utility of polymerase chain reaction analysis is still developing.
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Table 83-1. Laboratory diagnosis of cryptosporidiosis. |
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Figure 83-1. Cryptosporidium spp. oocysts are more difficult to detect on a routine ova and parasite exam. They are acid-fast positive but are most easily detected with a direct fluorescent antibody stain. This picture compares the size of the C parvum oocyst (4–6 µm) with that of a Giardia cyst (9–12 µm), using a combined-fluorescent-antibody stain (Meriflour stain reproduced with permission from Meridian, Inc.). |
Hematoxylin and eosin staining of intestinal biopsy specimens reveals basophilic organisms on the brush border that appear to project into the lumen because of the intracellular but extracytoplasmic location of the organism. Direct immunofluorescence can also be used on these biopsy specimens, and electron microscopy provides greater detail. However, the invasiveness of biopsy is rarely warranted and is not 100% sensitive.
Serologic antibody identification is available for C parvum infection but is still investigational and not clinically useful for diagnosis due to the persistence of antibody after infection. It is valuable as an epidemiologic tool, however.
Treatment
No reliable treatment is available at this time for cryptosporidiosis (Box 83-2). Supportive care with fluids and antidiarrheal agents can be offered while awaiting resolution in immunocompetent hosts or while addressing the underlying etiology of immunosuppression in compromised hosts. Paromomycin sulfate is currently the drug of choice for C parvum, surpassing spiramycin, for which there are no controlled studies. Paromomycin treatment is well tolerated by patients, but relapse is common even on therapy. For those patients relapsing off of therapy, however, 80% will again have a good response when they are back on paromomycin sulfate. Initial reports on the efficacy of a special lactose-free preparation of azithromycin have been promising. Hyperimmune bovine colostrum has been helpful in decreasing symptoms despite the persistence of oocyst shedding. Bovine transfer factor isolated from infected calf lymph node suspensions has been shown to attenuate symptoms, and octreotide has been similarly helpful. For all of the above therapeutic modalities, large randomized studies are needed for better understanding and documentation of effect.
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BOX 83-2 Treatment of Cryptosporidiosis1 |
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Prognosis
Although cryptosporidiosis is self-limited in immunocompetent patients, patients with AIDS with enteric cryptosporidiosis present with disease, the severity of which is related to the degree of immunosuppression. In one series of patients with AIDS, 29% of cases were transient, 60% chronic, 8% fulminant, and 4% asymptomatic. Mean survival in these patients was 25 weeks.
Prevention & Control
C parvum oocysts are 30-fold more resistant to chlorine than are Giardia cysts. Oocysts are killed in water kept above 65°C for 30 min as well as water boiled for 1 min at any altitude. Oocyst death is also reported in water kept at < 20°C for 30 min, although more recent information suggests greater resistance to freezing than previously thought (Box 83-3).
Enteric precautions and good hygiene should reduce most person-to-person transmission as well as animal-to-human exposures because family contacts account for ≤ 50% of secondary cases. Also, apple juice and cider should be pasteurized or boiled before consumption. No prophylactic regimen for patients with AIDS is yet available, although paromomycin sulfate and azithromycin are being studied. Immunocompromised patients should be instructed to drink bottled or boiled water if water supplies are suspect.
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BOX 83-3 Prevention & Control of Cryptosporidiosis |
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CYCLOSPORA
Essentials of Diagnosis
General Considerations
Cyclospora is a coccidian that had been described as a “large cryptosporidium” or “cyanobacterium-like body” before being confirmed as a member of the phylum Apicomplexa in 1993. The life cycle in humans has not been fully detailed. The organism has been shown to infect jejunal enterocytes. Similar to Isospora, the oocysts are excreted unsporulated, requiring 7–13 days of optimal conditions outside of the host to mature. Each spherical oocyst has a diameter of 8–10 µm and contains two sporocysts, each of which contains two sporozoites. The oocyst is twice the size of C parvum (Figure 83-2).
Cyclospora catayensis infection in humans has been reported worldwide and was first described in Papua New Guinea in 1979. It is endemic in Haiti, Peru, and Nepal, where rainy-season outbreaks of C catayensis infection occur. Outside these areas, infection is usually travel related when not associated with an outbreak. Protective immunity is suggested because infection is more common in children and in non-natives. Infection has been reported in HIV-infected patients, but cyclosporiasis is primarily found in immunocompetent patients. In the United States, Cyclospora oocysts were identified in only 3 of 1042 submitted stools. The low prevalence in patients with AIDS in the United States is attributed to possible misdiagnoses as C parvum, nonuniform use of the acid-fast stain, trimethoprim-sulfamethoxazole (TMP-SMX) prophylaxis in these patients, and the coincident low prevalence in immunocompetent patients in the United States.
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Figure 83-2. C catayensis oocysts have an 8- to 10-µm diameter and can be identified on a wet mount of the routine ova and parasite exam. They tend to have autofluorescent inclusions and will stain acid-fast positive with a Ziehl-Neelsen stain. |
Transmission of Cyclospora has been linked to contaminated water and food sources. The organism is believed to be host specific to humans. In 1990, a Chicago outbreak was related to stagnant water in a storage tank, and in 1994, in Nepal, a British military unit experienced an outbreak related to water from a chlorinated and filtered tank. More recently, cyclosporiasis related to berry consumption has been in the news. In 1995, unwashed strawberries were related to outbreaks in New York and Florida. In 1996, 1465 cyclosporiasis cases in two Canadian provinces and 20 states in the United States were traced to consumption of Guatemalan raspberries. Lettuce and uncooked meat have been reported culprits as well.
The pathogenesis of cyclosporiasis is not well understood. The parasite is found in the upper small bowel and is associated with villous atrophy, crypt hyperplasia, and inflammatory changes similar to those seen in tropical sprue.
Clinical Findings
After an incubation of 2–11 days, immunocompetent patients experience a self-limited, but prolonged, relapsing watery diarrhea lasting an average of 19–43 days, although organism shedding may last ≤ 70 days (Box 83-4). In patients with AIDS, symptoms have been reported for ≤ 4 months. A median of six stools/day is reported. Fatigue, fever, indigestion, abdominal cramps, nausea, and vomiting may occur, as well as weight loss. A prodrome of both myalgia and arthralgia has been reported. Diarrhea may not be the primary complaint, and alternation with constipation is not infrequent. Asymptomatic infection has been reported in a study of immunocompetent Peruvians with documented infection in whom only 11–28% had diarrhea. No specific laboratory findings assist in diagnosis although abnormal D-xylose absorption has been reported.
Diagnosis
Diagnosis is made by identification of the Cyclospora oocyst in fresh or iodine-preserved stool, duodenal aspirates, or small-bowel biopsies. With acid-fast or safranin O staining, oocysts can be seen as 10 µm spheres with clusters of refractile globules. The oocysts autofluoresce blue under UV light with a 365-nm excitation filter (Table 83-2).
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BOX 83-4 Cyclosporiasis Syndromes |
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Table 83-2. Laboratory diagnosis of Cyclospora infection. |
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Treatment
TMP-SMX is the first-line treatment for cyclosporiasis, with symptom resolution noted 2–3 days into therapy (Box 83-5). Immunosuppressed patients may require long-term suppressive therapy owing to an increased relapse rate. In addition to TMP-SMX, metronidazole, tinidazole, and ciprofloxacin hydrochloride have also been reported to stop oocyst excretion.
Prognosis
Infection is generally self-limited in immunocompetent hosts, but prolonged diarrhea with possible symptoms of malabsorption may occur in immunocompromised patients.
Prevention & Control
Reliable means of prevention of Cyclospora spp. infection are not yet known (Box 83-6). Efforts to decrease berry contamination have been directed toward using potable water in all aspects of berry preparation, including water used for insecticide spray and for hand washing by workers. Gamma radiation used on berries is also being studied.
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BOX 83-5 Treatment of Cyclosporiasis |
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BOX 83-6 Prevention & Control of Cyclospora and Isospora spp. and Microsporidia |
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ISOSPORA
Essentials of Diagnosis
General Considerations
In immunocompetent patients, I belli has been identified in cases of chronic traveler's diarrhea and is present in outbreaks of diarrhea in institutional settings such as day care and mental facilities.
Because sporulation can occur only outside the host, transmission may occur through environmental sources such as food and water. A latency state has been postulated based on increased prevalence of infection in Latin American immigrants. In Los Angeles, Latin Americans account for ≤ 20% of AIDS cases but have 80% of I belli infections.
Clinical Findings
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BOX 83-7 Isosporiasis Syndromes |
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Diagnosis
Diagnosis of isosporiasis depends on identification of the oocyst in stool by wet mount with subsequent confirmation using modified acid-fast or trichrome staining. Colonoscopy aspiration or the duodenal string test may be helpful to obtain the necessary sample. Oocysts autofluoresce in UV light when viewed through a 450- to 490-nm excitation filter (Table 83-3).
Treatment
TMP-SMX has been shown to be effective treatment (Box 83-8). Patients with AIDS have a 50% chance of relapse, and low-dose suppressive treatment with TMP-SMX, pyrimethamine-sulfadoxine, or pyrimethamine alone is recommended. Alternative treatments include metronidazole, ciprofloxacin hydrochloride, roxithromycin, and diclazuril.
Prognosis
Untreated isosporiasis in the immunocompromised patient can lead to severe malnutrition and dehydration and may be fatal.
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Table 83-3. Laboratory diagnosis of isosporiasis. |
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Prevention & Control
No proven means of prophylaxis for isosporiasis has yet been established due to unknown details of transmission and the possibility of a latency state (see Box 83-6). The common usage of TMP-SMX for Pneumocystis carinii prophylaxis may indeed also play a role in I belli prophylaxis. Because I belli infection appears to be mediated through water and environmental sources, good fecal-oral hygiene and adequate water treatment are recommended.
MICROSPORIDIA
Essentials of Diagnosis
General Considerations
Microsporidiosis has been found worldwide. Before the AIDS epidemic, only 10 cases had been reported. Subsequently, hundreds of cases have been recognized, mostly in the United States. Between 23% and 33% of patients with AIDS with chronic diarrhea have been diagnosed with microsporidiosis, with Enterocytozoon bieneusi being the most common culprit. Most cases of microsporidiosis occur in male patients with HIV with severe immunosuppression and CD4 counts of < 100. Of 11 cases reported in non-HIV–infected patients, 4 had other forms of immunosuppression such as earlier liver and bone marrow transplants, and 4 had infections of the cornea, a site that is considered to be immunoprivileged. Infection in immunocompetent patients may be under-recognized because of transient infection or milder disease. Self-limited diarrhea has been documented in an immunocompetent traveler, and an asymptomatic carrier state has been identified.
Little is known about transmission of microsporidiosis, and there have been no reports of common-source outbreaks. However, microsporidium spores have been identified in respiratory secretions, urine, stool, and duodenal aspirates, suggesting that person-to-person transmission may occur by fecal-oral contacts, aerosolized secretions, sexual transmission, or direct corneal inoculation. Microsporidia infect insects, fish, snails, and mammals and are also present in surface water, which suggests the zoonotic and environmental transmission of these resistant spores. The spores have been shown to survive > 1 year in water kept at 4°C.
Five genera from the protozoan phylum Microspora have been implicated in human disease: Encephalitozoon (Encephalitozoon hellem and Encephalitozoon cuniculi), Pleistophora, Enterocytozoon (Enterocytozoon bieneusi), Septata (Septata intestinalis), and Nosema, which was recently reclassified as Vittaforma. Unclassified species are placed under a sixth taxon, Microsporidium. The genera and species are separated primarily based on the location of the parasite in relation to the host, that is, whether the parasite is in direct contact with host cytoplasm, separated into a vesicle made by the host, or located in a parasitophorous vacuole.
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BOX 83-8 Treatment of Isosporiasis |
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CLINICAL SYNDROMES IN NON-HIV–INFECTED PATIENTS
Microsporidial infection in non-HIV–infected patients is rare, with < 20 cases reported. Central nervous system, corneal, muscular, enteric, and respiratory infections have all been identified (Box 83-9).
Central nervous system infections have been reported in two immunocompetent children presenting with fever, loss of consciousness, headache, and convulsions, as well as hepatomegaly. E cuniculi was isolated from the cerebrospinal fluid and the urine. Computed tomography of the brain and electroencephalography were normal. Corneal infection has been caused by Nosema spp., Microsporidium ceylonensis, and Microsporidium africanum. Patients have presented with decreased visual acuity, conjunctivitis, and corneal ulcers. Corneal biopsy reveals the organism.
Patients presenting with generalized muscle weakness have been diagnosed with myositis caused by Pleistophora spp. Laboratory tests revealed normal creatine kinase levels, and electromyograms showed myopathy. Muscle biopsies revealed scarring, fibrosis, inflammation, and Pleistophora infiltrate. Self-limited diarrhea caused by E bieneusi has been reported in an immunocompetent host. A patient with chronic myelogenous leukemia who had undergone allogenic bone marrow transplant was recently shown on autopsy to have microsporidial infection along with Candida tropicalis infection within lung sections.
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BOX 83-9 Microsporidiosis in Non-HIV Patients |
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CLINICAL SYNDROMES IN HIV-INFECTED PATIENTS
The most common manifestation of microsporidial infection in HIV-infected patients is enteric disease, but other recognized syndromes include biliary infection, pulmonary infection, keratoconjunctivitis, and myositis (Box 83-10).
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BOX 83-10 Microsporidiosis Syndromes in HIV Patients (Syndrome and Organism) |
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E bieneusi and S intestinalis are the most common etiologic agents identified in patients presenting with chronic diarrhea due to microsporidia. However, there is one case report of E cuniculi identified in intestinal infection.
Clinical Findings
E bieneusi and S intestinalis, often associated with cryptosporidial coinfection, have been identified in patients presenting with symptoms of cholecystitis.
Clinical Findings
E bieneusi has been found in bronchial lavage specimens of patients presenting with cough, dyspnea, wheezing, or sinusitis. Chest x-rays show interstitial infiltrates.
E cuniculi and E hellem as well as S intestinalis have caused corneal infections in HIV-infected patients. Patients present with dry eyes, foreign-body sensation, or ocular pain. Blurred vision, excessive tearing, and photophobia have also been reported. Conjunctival hyperemia may be present, and slit-lamp exam reveals a diffuse, superficial punctate keratopathy. Microsporidial corneal infections have been associated with systemic disease with concomitant bronchiolitis, sinusitis, nephritis, cystitis and ureteritis, hepatitis, and peritonitis.
Pleistophora infection in immunocompromised patients presents similarly to that in immunocompetent patients as already described.
Diagnosis
The small size of the Microsporidium spore makes diagnosis difficult (Table 83-4). The sensitivity and specificity of different identification techniques are not known. Microsporidial spores are gram positive and birefringent, and they have a positive periodic acid-Schiff–staining polar body. Spores can be found in stool, duodenal aspirates, urine, respiratory secretions, and conjunctival scrapings. Diagnostic yield may be enhanced by collecting three stools daily over 3 subsequent days. Spore concentration and sedimentation techniques can increase yield. Weber's modified trichrome, Gram, Giemsa, and chitin-binding fluorochrome stains have all been used successfully to identify spores. Diagnostic yield can be increased by preserving stool in formalin, pretreating in 10% potassium hydroxide, and centrifuging for 5 min before staining with the Weber's modified trichrome stain. Biopsy specimens can be similarly stained, but sensitivity may be decreased due to normal-appearing mucosa. If < 25 spores are identified from a stool specimen per coverslip, the likelihood of identifying organisms by electron microscopy of a biopsy specimen is low. E bieneusi usually involves the distal duodenum and proximal jejunum, and S intestinalis can also involve the colon.
Electron microscopy can more readily identify the small spores and is useful in speciation. Immunofluorescence and polymerase chain reaction techniques are also valuable for speciation.
Treatment
Microsporidial treatment efficacy is anecdotal at this time (Box 83-11). Albendazole has had the best success thus far in alleviating gastrointestinal and biliary symptoms. However, eradication of spores is rare, and relapse occurs 1–2 months after completion of 4 weeks of albendazole. Metronidazole was reported to have a transient clinical response only, and atovaquone has had mixed results. Fumagillin, itraconazole, and fluconazole are currently being studied. Palliative treatment with octreotide and nutrition adjustments such as prescribing low-fat and simple carbohydrate diets may be beneficial.
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Table 83-4. Laboratory diagnosis of microsporidiosis. |
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BOX 83-11 Treatment of Microsporidiosis |
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Topical fumagillin and propamidine isethionate (0.1%) have been effective in treating microsporidial keratopathy. One case report suggests that itraconazole may be beneficial in this realm as well.
Prevention & Control
Control of microsporidial infection will be difficult to address until transmission is better understood (see Box 83-6). Bodily fluid precautions seem warranted to prevent fecal-oral and urinary-oral transmission. Disinfecting, boiling, and autoclaving may also be of benefit. The necessity of respiratory precautions and isolation is uncertain.
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