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Background Organisms referred to as "cyanobacterium-like bodies" have now been identified worldwide in the feces of both immunocompetent and immunocompromised patients with diarrhea. Organisms with a similar appearance have been isolated from Peruvian patients since 1985. From 1988 to 1991 we studied prospectively two cohorts of infants and young children infected with this organism. We now attempt to identify it.
Methods Fecal samples were collected weekly from the children and examined with the use of acid-fast staining and staining with a monoclonal antibody specific for cryptosporidium. Stools positive for cyanobacterium-like bodies were preserved in potassium dichromate and exposed to conditions allowing coccidian sporulation and excystation. Both unsporulated and sporulated oocysts were fixed by freeze-substitution techniques and then examined by electron microscopy.
Results Organisms isolated from the feces of Peruvian patients and two patients from the United States were identified as belonging to the coccidian genus cyclospora, after sporulation and excystation of the oocysts according to standard techniques. Complete sporulation occurred within 5 to 13 days in oocysts maintained in potassium dichromate at 25 or 32 °C. Complete excystation resulted in the liberation of two sporozoites from the two sporocysts within each oocyst (cryptosporidia have four naked sporozoites within each oocyst). The presence of organelles characteristic of coccidian organisms was confirmed by electron microscopy.
Conclusions We have identified organisms of the genus cyclospora that are remarkably similar to cryptosporidia in their morphologic features and the diarrheal disease that they produce in humans. The complete life cycle and epidemiology of this new protozoan parasite remain to be described.
In the largest study of immunocompetent patients, illness was associated with a prolonged but self-limiting diarrhea (lasting a mean [±SD] of 43 ±24 days)8. A pattern of relapsing diarrhea lasting up to four weeks was noted among several physicians at a Chicago hospital who were excreting this organism,6 and epidemiologic investigation indicated that they had been exposed to a contaminated water source. The association of this organism with prolonged illness in patients with the acquired immunodeficiency syndrome (AIDS) is less clear, since few patients with both AIDS and the organism have been studied to date5. In one study a patient died within two weeks after the organism had been detected,4 and in another study intestinal symptoms were reported to clear within two weeks5.
The organisms have been observed in fecal smears after the application of either phenosafranin or modified acid-fast stains, although both are highly variable in their ability to stain the cyst5,7. In addition, the organisms have been reported to show autofluorescence when examined by ultraviolet epifluorescence7. On light microscopy they are nonrefractile spheres 8 to 10 microm in diameter containing a cluster of refractile, membrane-bound globules enclosed within a membrane (morula). A single division of the morula occurred after the organism was cultured in distilled water for 7 days, but no further development followed prolonged cultivation (>7 months)7. Preliminary electron-microscopical examination revealed a spherical object with an outer fibrillar coat and a thick cell wall. The internal cytoplasm, which contained light and dark granules, was bound by cell membrane. In addition, the presence of lamellar structures, suggestive of thylakoid membranes from chloroplasts, and the absence of a nucleus suggested that the organism might be prokaryotic, resembling cyanobacteria5.
This report describes the positive identification of the organism and provides evidence of a new protozoan pathogen that infects humans.
Methods
Sample Collection and Identification of Isolates
Collaborative efforts between investigators of the University of Arizona, the Johns Hopkins University, and Universidad Peruana Cayetano Heredia began in 1985 with the express purpose of defining the epidemiology of C. parvum infections in children living in the slums of Lima, Peru. The initial work focused on the transfer of technological means that would permit easier, more specific identification of cryptosporidium oocysts from the feces of infected children and adults with the use of fluorescence-labeled monoclonal antibodies9. During these studies, fecal samples were also evaluated with a modified acid-fast stain to corroborate the findings of the investigation with oocyst-specific monoclonal antibodies.
From January 1988 to October 1991, two large-scale, prospective cohort studies were conducted in the shantytowns of Lima. The first study was initiated to define the epidemiology of C. parvum in children, and the second to determine the role of breast milk in protecting urban Peruvian children against this infection10. Together, these studies involved weekly examination of feces from 377 children ranging in age from one month to more than two years. Stool samples were examined after both modified acid-fast staining and immunofluorescence reaction with a monoclonal antibody (Merifluor, Meridian Diagnostics, Cincinnati).
Sporulation
Organisms resembling cyanobacterium-like bodies, isolated from the subjects with the heaviest infections in the second study, were preserved in 2.5 percent potassium dichromate for further study. Because the organisms in freshly collected feces bore a strong resemblance to unsporulated coccidian oocysts, sporulation was attempted at different temperatures. Feces preserved in potassium dichromate that were less than two weeks old were layered over a primary discontinuous sucrose gradient (densities, 1.064 and 1.103 g per liter) and centrifuged (1500 x g for 25 minutes)11. Organisms purified by this procedure were then placed in 2.5 percent potassium dichromate in covered petri dishes and incubated in room air at 4, 25, 32, or 37 °C12. Daily changes in the appearance of the organism were monitored by phase-contrast microscopy.
Excystation
Organisms from cultures kept at 25 or 32 °C were mechanically ruptured to liberate sporocysts and then subjected to procedures for coccidian excystation with 0.5 percent trypsin and 1.5 percent sodium taurocholate in phosphate-buffered saline11.
Transmission Electron Microscopy
Unsporulated organisms and organisms that had undergone sporulation for five days at 25 °C also were fixed for electron microscopy according to freeze-substitution techniques13.
Results
Organism Identification and Patient History
In 1985, spherical cyst-like organisms measuring 8 to 10 microm in diameter were observed in the feces of a 65-year-old Peruvian woman presenting with severe chronic diarrhea2. The organisms, shown in Figure 1, had a variably mottled, red appearance on modified acid-fast staining and resembled the oocysts of C. muris; for comparison, oocysts of C. muris from an infected beef cow are shown in Figure 1B, and those of C. parvum from an infected Peruvian child in Figure 1C.
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During the same period a 50-year-old Peruvian woman who was a wholesale meat vendor presented with watery diarrhea and the same acid-fast organisms in her feces. For at least one month she had a pattern of three to five watery bowel movements per day for three or four days, followed by normal bowel movements for two days. Endoscopy was performed as in the other patient, while she was still shedding organisms; the findings were unremarkable. She continued to shed organisms intermittently in her feces for an additional two weeks but had no symptoms. After this time, organisms were no longer detectable in her feces. Informed consent for biopsies was obtained from both patients after the nature and possible consequences of the study had been fully explained to them.
Epidemiologic Investigation
At presentation, 18 percent of the children in the first study (26 subjects) and 6 percent of those in the second (15 subjects) had C. muris-like cysts in their feces. These cysts were easily distinguished from the oocysts of C. parvum (which are 4 to 6 microm in diameter) because they were larger and because they did not react with a monoclonal antibody with 100 percent sensitivity and specificity for C. parvum oocysts14. As in other studies, the reaction of the organisms with the modified acid-fast stain was highly variable: some organisms stained dark red and had a variable number of dark inclusion bodies, whereas others did not stain at all and appeared as transparent spheres. In addition, the organisms showed a fairly strong green autofluorescence when observed under ultraviolet epifluorescence. Six children in the first study and three in the second study each had two episodes of infection with the C. muris-like organism. Of the children subsequently found to be infected with cyclospora species, 28 percent of those in the first study (nine subjects) and 11 percent of those in the second study (two subjects) presented with diarrhea. The mean duration of shedding of organisms was remarkably similar in both studies (22.4 days [range, 7 to 70] vs. 23.3 days [range, 7 to 50]). Since the age at enrollment in the two studies differed, this variable could not be compared. The differences in the features of infection between the new organism and C. parvum are shown in Table 1. Infections with this new organism also showed seasonal tendencies, with peaks between April and June.
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Sporulation of approximately 20 percent of the organisms, which resulted in the formation of two sporocysts similar in appearance to those previously reported,7 was noticed by day 5 in cultures kept at 25 or 32 °C (Figure 2A). Complete sporulation, resulting in the appearance of sporozoites within sporocysts, became evident between days 7 and 13 in cultures. Feces that had been kept at 4 °C in potassium dichromate for four to six months contained organisms that appeared to have already undergone sporulation. Organisms maintained at 37 °C showed a contraction and darkening of the central cell mass after five days in culture.
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Within 15 minutes of exposure to the excysting solution at 37 °C, movement was observed in each sporocyst; within 30 minutes, two crescent-shaped sporozoites measuring 1.2 by 9.0 microm emerged from one of the sporocysts (Figure 2B). Complete excystation, with the liberation of four sporozoites, was observed within 40 minutes of exposure to the excysting solution (Figure 2C and Figure 2D).
Microscopical Findings
The features of both unsporulated (Figure 3A) and sporulated (Figure 3B) organisms observed on electron microscopy indicated that the organisms were coccidian. The immature oocyst had an outer fibrillar coat of 63 nm, beneath which lay a cell wall 50 nm thick. The internal cytoplasmic mass contained light and dark granules similar to those previously found in cyanobacterium-like bodies. The mature oocyst had a fibrillar coat and cell wall similar to that of the unsporulated oocyst. Each sporocyst had a wall 62 nm thick. The sporozoites within the sporocyst had a membrane-bound nucleus and micronemes characteristic of coccidians of the phylum Apicomplexa (Figure 3B).
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The observations made during sporulation and excystation and the characteristic features of the new organism are sufficient evidence to place it within the coccidian genus cyclospora. Organisms belonging to this genus have an oocyst with two sporocysts, each with two sporozoites15. This description, therefore, is very much like that of isosporan parasites of humans except that isosporans are usually larger and have an oocyst with two sporocysts, each with four sporozoites15. Our finding also provides evidence that the organisms recently described as cyanobacterium-like bodies in patients in North, Central, and South America, Caribbean countries, Southeast Asia, and Eastern Europe may in fact be a new coccidian pathogen of the genus cyclospora. This conclusion is also supported by the descriptions in these reports of the organism's characteristics on evaluation with modified acid-fast stains, its autofluorescence under ultraviolet epifluorescence, and its replication in culture; the clinical features of infection; and the resistance of the infection to conventional antimicrobial therapy. More important, organisms similar to cyanobacterium-like bodies that were isolated from the feces of two patients with diarrhea in Los Angeles and Chicago in May and July 1992, respectively, and sent to us for further studies also have been confirmed as cyclospora. Our initial report, which speculated that this organism was a flagellate,2 is obviously incorrect. In all likelihood the organisms referred to in that abstract2 were immature cysts of Giardia intestinalis, another common parasite of children in the shantytowns of Peru.
What is now apparent from the published reports about the cyanobacterium-like bodies and the C. muris-like organism is that persons of all ages can become infected, although rates of diarrhea may prove to be lower among those from zones where these pathogens are endemic. This will have to be confirmed by conducting epidemiologic studies in those areas. The fact that infection with cyclospora occurs at the same time of year as does infection with cryptosporidium in Peru implies that the acquisition of infection may be associated with similar risk factors.
Organisms of the genus cyclospora have previously been found only in reptiles,16 myriapods,17 insectivores,18,19,20,21,22,23 and one murine host24. The oocysts reported in those animals, however, are sufficiently different in size from those reported in humans to warrant regarding the human parasite as a new species. The complete life cycle and epidemiologic features of this new parasite remain unknown. The difference in sporulation time that we noted during the present study (7 to 13 days) may merely reflect the age of the sample when sporulation was initiated.
Except for prolonged intermittent diarrhea, the clinical presentation of patients infected with this organism was quite similar to the presentation of those infected with C. parvum. Because the new organism is acid-fast like the oocyst of cryptosporidium, we strongly recommend that all laboratories screening for the latter parasite provide precise measurements of oocysts. In developing countries, where size discriminations are often not made, it is quite possible that many cases of diarrhea reported to be due to cryptosporidium might actually have been due to cyclospora. Because the cyclospora organism also has been seen in patients with AIDS and intractable diarrhea, this infection should be carefully distinguished from cryptosporidiosis. There is obviously a great deal more to learn about this emerging pathogen.
Supported in part by Thrasher Research Funds (2798-5 and 2803-7).
We are indebted to the personnel of Asociacion Benefica PRISMA and the entire staff of the parasitology laboratory at Universidad Peruana Cayetano Heredia for their support; to Dr. Jorge Naranjo and Dr. Elba Miranda for their contributions; to Dr. John Gilkey for his outstanding technical assistance with the freeze-substitution electron miscroscopy; and to Lynn Garcia, Dr. Frank Kocka, and Dr. Becky Wurtz for the samples from the patients in Los Angeles and Chicago.
Source Information
From the Department of Veterinary Science, University of Arizona, Tucson (Y.R.O., C.R.S., V.A.C.); the Department of International Health, Johns Hopkins University, Baltimore (R.H.G.); and the Departamento de Parasitologia, Universidad Peruana Cayetano Heredia, and the Asociacion Benefica PRISMA, both in Lima, Peru (R.H.G., F.D.).
Address reprint requests to Dr. Sterling at the Department of Veterinary Science, University of Arizona, Bldg. 90, Rm. 201, Tucson, AZ 85721.
References
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Related Letters:
Cyanobacterium-Like Cyclospora Species
Hoge C. W., Shlim D. R., Echeverria P., Heyworth M. F., Sterling C. R.
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Full Text
N Engl J Med 1993;
329:1504-1505, Nov 11, 1993.
Correspondence
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