Volume 2, Number 4—October 1996
PCR Confirmation of Infection with Cyclospora cayetanensis
To the Editor: Cyclospora cayetanensis, formerly known as cyanobacterium-like body, is a variably acid-fast microorganism. Recently, it was classified as a coccidian parasite (1) closely related to the genus Eimeria (2). Humans infected with C. cayetanensis typically have diarrheal illness with a variable number of stools per day and sometimes have nausea and vomiting (3,4). Cyclospora infection has been reported in many parts of the world as clustered or sporadic cases (1,3-5).
Variable success in diagnosing infection with this parasite underscores the need for using (as quality control) molecular methods, which do not rely on the level of expertise of laboratory personnel in microscopy. The key features for diagnosis by light microscopy are size (8mm to 10mm in diameter), internal features of stained and unstained oocysts, and autofluorescence of oocysts (1,6). The definitive diagnosis is understood as visualization of characteristic sporulated oocysts, which contain two sporocysts. However, sporulation typically requires incubating oocysts for up to 2 weeks, and this approach cannot be applied to Formalin or polyvinylalcohol-preserved stool smears.
Sporadic and clustered cases of Cyclospora infections were reported in the United States and Canada during May and June 1996 (5,7). From these outbreaks, more than 900 cases were diagnosed by examining stool specimens under light microscopy (Barbara Herwaldt, pers. comm.). Epidemiologic studies indicated risk for Cyclospora infection from consuming raspberries imported from Guatemala (7). Forty-two stool specimens supplied in 2.5% potassium dichromate from patients with intestinal symptoms were forwarded to the Centers for Disease Control and Prevention to be evaluated by microscopy and by polymerase chain reaction (PCR) amplification. In addition, one well-characterized positive stool specimen from Nepal was provided by John Cross, Armed Forces Research Institute of Medical Sciences, Bangkok, Thailand, to use as the positive control.
Using techniques we developed for diagnosis of other protozoan parasites in stools, we extracted DNA from all stools. The techniques we used employ glass-bead disruption of oocysts in a buffer containing Laureth-12, purification with the RapidPrep Micro Genomic DNA Isolation Kit for Cells and Tissue (Pharmacia Biotech Inc., Piscataway, N.J.), followed by a final purification step employing the QIAquick PCR purification kit protocol (Qiagen, Inc., Chatsworth, Calif.) (8). The glass-bead disruption of oocysts was far more effective than sonication (2) or freeze-thawing techniques (9). We performed nested PCR in all stool specimens by using Relman et al. (2) primers CYCF1E and CYCR2B for the first step of nested amplification and primers CYCF3E and CYCR4B for the second (nested) step of the PCR. These are the only primers described for amplification of Cyclospora DNA. We found optimal conditions for the first step PCR to be denaturation at 94°C for 30 s, annealing at 55°C for 30 s, and extension at 72°C for 90 s, 45 cycles. The same conditions were used for the second step of the nested PCR, but the annealing temperature was 60°C.
By using this approach, we amplified the Cyclospora-specific DNA fragment in 16 (38%) of the 26 (62%) specimens reconfirmed as positive by light microscopy. The 10 specimens negative by PCR but positive by microscopy showed either few or moderate numbers of Cyclospora oocysts. None of the 16 (38%) specimens negative by microscopy generated positive results in the PCR Cyclospora test. Upon further examination by the PCR technique we developed (9), three of these samples were positive for another enteric coccidian, Cryptosporidium parvum.
Preliminary evaluation indicates that the sensitivity of PCR is 62%, and the specificity is 100%. Although the sensitivity of the technique should be evaluated further, these results indicate that PCR can be used to detect Cyclospora. We assessed the sensitivity of this PCR again by using the Nepalese specimen described above. This specimen, which was used as positive control in all reactions, was amplified even when the extracted DNA was diluted at 10-5.
Lastly, a note of caution. As noted by Relman et al. (2) and confirmed by us through GenBank searches, the nested PCR Cyclospora primers cross-amplify other coccidians, especially those belonging to the genus Eimeria (because no molecular data exist for another human coccidian enteric parasite, Isospora belli, potential cross-amplification remains to be determined). This cross-amplification with Eimeria should not present a problem in diagnosing Cyclospora in human stool, as no human infections by Eimeria are known. However, when analyzing food or environmental specimens, this cross-amplification may complicate precise detection of Cyclospora.
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