Uniform staining of Cyclospora oocysts in fecal smears by a modified safranin technique with microwave heating.

Cyclospora,acoccidianprotist,isincreasinglybeingidentifiedasanimportant,newlyemergingparasitethat causes diarrhea,flatulence, fatigue, and abdominal pain leading to weight loss in immunocompetent persons with or without a recent travel history as well as in patients with AIDS. Modified Kinyoun’s acid-fast stain is themostcommonlyusedstaintoidentifytheoocystofthisparasiteinfecalsmears.OocystsofCyclosporastain variably by the modified acid-fast procedure, resulting in the possible misidentification of this parasite. We examined fecal smears stained by six different procedures that included Giemsa, trichrome, chromotrope, Gram-chromotrope, acid-fast, and safranin stains. We report on a safranin-based stain that uniformly stains oocysts ofCyclosporaa brilliant reddish orange, provided that the fecal smears are heated in a microwave oven priortostaining.Thisstainingprocedure,besidesbeingsuperiortoacid-faststaining,isfast,reliable,andeasy to perform in most clinical laboratories. Cyclosporaisincreasinglybeingidentifiedinthefecalsmears of immunocompetent persons with or without international travel histories (3‐8, 10, 12‐16, 18, 22, 24, 25) as well as in patients with human immunodeficiency virus infection or AIDS (4, 11, 19, 21, 23, 25). Cyclospora, a coccidian protist, previously identified as a coccidian or cyanobacterium-like body or as a novel organism, was found in the diarrheic stools ofNepalesecitizens,foreignersresidinginNepal,andtravelers returning from Southeast Asia and Mexico who complained of nausea,vomiting,anorexia,diarrhea,malabsorptionsyndrome, and weight loss (3, 4, 7, 12, 15, 24, 25). Since then, several outbreaks have been attributed to this parasite (5, 6, 16). Ortega et al. (18) were the first to identify the true coccidian nature of the cyanobacterium-like bodies and describe the sporulation of these organisms. They created a new taxon, Cyclosporacayetanensis(18),forthisparasite.Althoughacomplete life cycle of this organism has not been elucidated thus far, a recent electron microscopic study indicates that different stages of its life cycle can be identified in the duodenal biopsy specimens of infected patients. It is also believed that the entire life cycle ofCyclosporamay occur within the enterocytes of the infected patient (23). The stage that is found in the fecal smears of patients infected withCyclosporais the unsporulated oocyst stage, which measures 8 to 10 mm. The oocysts excreted in the feces sporulate outside the host in about 2 weeks, resulting in two sporocysts,eachwithtwosporozoites(18).Thestainmostcommonly used to identify the oocysts in fecal smears is modified Kinyoun’s acid-fast stain, which has also been used extensively for the identification of the oocysts of another coccidian parasite, Cryptosporidium (1, 2, 8). Published reports indicate that the oocysts of Cyclospora are variably acid fast, with staining patterns ranging from no staining or colorless to pink or deep purple (8). This variability in the staining pattern may result in the misidentification of the parasite and may thus hamper timely treatment of the patient, especially now that we know that the parasite can be eradicated with the administration of trimethoprim-sulfamethoxazole (13, 22). While staining fecal smears with the modified acid-fast stain from patients with diarrhea involved in the newestCyclosporaoutbreak, involving patients in as many as 14 states and Canada (5, 6), we also observed tremendous variations in the staining properties of the oocysts of these organisms. Although a number of the oocysts in a given fecal preparation stained pinkish red, a large number of the oocysts did not take up any stain at all. Furthermore, we encountered some slides in which none of the oocysts took up the stain. Therefore, we undertook a study to see if we could identify a staining technique that would uniformlystaingreaterthan90%ofoocystsinthefecalsmear.We used many different types of chemical stains that have been used previously in clinical laboratories for the identification of Cryptosporidiumandmicrosporidiainfecalsamples.Allexcept one of the stains that we tested stained the oocysts poorly. The safranin staining procedure developed by Baxby et al. (2) for identifying Cryptosporidium oocysts in fecal smears stained Cyclospora oocysts pinkish or reddish orange. The safranin methodhaspreviouslybeenusedtostainoocystsofCyclospora (19),butthesensitivityofthetechniquewasreportedtobelow (30%). In our hands, however, approximately 70 to 75% of the oocysts stained pinkish orange, while the rest of the oocysts remained colorless or stained variably when we stained the smears by the safranin procedure of Baxby et al. (2). Our modification of the procedure of Baxby et al. (2) resulted in a technique that uniformly stained almost all of the oocysts of Cyclospora a brilliant reddish orange. Occasionally, however, an oocyst did not take up the stain, but it could easily be distinguished by its collapsed or wrinkled cyst wall.