The study was conducted in 2 urban areas, Zhengzhou and Kaifeng. A total of 11,554 (6,939 male; 4,615 female) child and adult patients at 3 hospitals (Huai River Hospital and 155th Liberation Army Hospital, Kaifeng, and Number One People’s Hospital, Zhengzhou) were enrolled in this study during June 2007–December 2008 and July–October 2009. Only data concerning age, sex, and diarrhea presence or absence were made available to laboratorians. One stool specimen from each patient was examined for Cyclospora
spp. by microscopy of fecal materials that were concentrated by the formalin-ethyl acetate sedimentation method and stained with the modified acid-fast staining technique (1
). We used the χ2
test to compare the frequency of Cyclospora
spp. infection among patients according to age group and sex and by season of the year. Differences were considered significant if p<0.05.
Cyclospora oocysts were detected in 81 (0.70%; 95% confidence interval [CI] 0.70% ± 0.15%) of 11,554 patients by microscopy (). Oocysts were variably stained from light pink to deep purple or remained unstained (, panel A). They measured 8.61 ± 0.32 × 8.64 ± 0.33 µm, with a length/width shape index of 1.01 (n = 55; , panel B), and showed typical blue autofluorescence under an epifluorescence microscope with a 330–380 nm excitation filter (, panel C). Oocysts sporulated at 32°C <13 days in 2.5% potassium dichromate.
Prevalence and distribution of Cyclospora cayetanensis by patient age, sex, and residential area, Henan Province, China, 2009–2010*
Figure 1 Cyclospora cayetanensis oocysts under light microscopy of stool smears stained with the modified acid-fast stain (A), showing differential interference contrast microscopy of wet mount (B), and results of epifluorescence microscopy using a 330–380 (more ...)
Cyclospora oocysts were seen in samples from patients in all age groups, although the age group 7–17 years had the highest detection rate (1.47%, 95% CI ± 0.91%; Pearson correlation >0.05) (). No significant gender difference was found in detection rate; the infection rates for female and male patients were 0.74% (34/4,615, 95% CI ± 0.25%) and 0.68% (47/6,939, 95% CI ± 0.19%), respectively (Pearson correlation >0.05). The overall infection rate of C. cayetanensis was similar between Zhengzhou and Kaifeng: 0.60% (95% CI ± 0.19%) versus 0.79% (95% CI ± 0.22%) (Pearson correlation >0.05; ).
The prevalence of cyclosporiasis was markedly seasonal, occurring only during July through November, with a sharp peak in August (). The occurrence of cyclosporiasis coincided with the rainy season and lagged slightly behind the peaks for mean temperature and precipitation in the year ().
Monthly prevalence of Cyclospora cayetanensis in Kaifeng and Zhengzhou, Henan Province, China, 2009–2010*
Figure 2 Mean monthly rainfall and mean daily average temperature recorded for Zhengzhou and Kaifeng, Henan Province, China, 1995–2008. Data source: www.chinaweatherguide.com.
Among patients in this investigation, 5,533 had records documenting presence or absence of diarrhea at the time of specimen submission. The detection rate of Cyclospora oocysts was significantly higher for patients with diarrhea (2.97% or 12/404; 95% CI 2.97 ± 0.52%) than for patients without diarrhea (0.66% or 34/5,129; 95% CI 0.66 ± 0.22%) (Pearson correlation <0.01).
Genomic DNA was extracted from Cyclospora
oocysts from 35 randomly chosen patients; the oocysts were purified by sucrose gradient centrifugation by using the Mag Extractor-Genome kit (Toyobo Co. Ltd, Osaka, Japan). A nested PCR was used to amplify a 501-bp fragment of the 18S rRNA gene (5
). All 35 microscopy-positive specimens produced the expected PCR product and were sequenced successfully. The C. cayetanensis
identity was established by comparing the sequences obtained with a full sequence (AF111183) of the 18S rRNA gene of C. cayetanensis
from Guatemala and 3 partial sequences (AB368541, AB368542, and AB368543) from Japan. In addition, this comparison revealed the presence of 2 polymorphic sites at nucleotide positions 687 and 696 of the full gene, with a few other inconsistent nucleotide substitutions at other positions. Thus, 3 isolates had a C to T substitution at position 687, and 5 isolates plus AB368542 and AB368543 had a C to T substitution at position 696. Nevertheless, similarities among the 35 C. cayetanensis
isolates and reference sequences were 99.6%–100% at the 18S rRNA locus. Representative sequences of the partial 18S rRNA gene generated were deposited in the GenBank database under accession nos. GQ292774–GQ292782, FJ009120–FJ009129, and EU860998–EU861002.