Retrospective molecular detection and genetic analysis of Cyclospora cayetanensis in stool samples from children with diarrhea in Ningbo City, Zhejiang Province

doi:10.12140/j.issn.1000-7423.2026.04.010

Abstract

Abstract:

Objective To retrospectively detect the presence of Cyclospora cayetanensis infections in stool samples from children with diarrhea in Ningbo City, Zhejiang Province, and to analyze the genetic and evolutionary characteristics of human-derived C. cayetanensis in China. Methods Stool samples from children with diarrhea in Ningbo City that were maintained in our laboratory were tested for C. cayetanensis 18S rRNA gene using nested PCR assay, and positive PCR amplification products were sequenced bidirectionally. Following sequence alignment and analysis, a phylogenetic tree was created using the neighbor joining method in the MEGA 7 software. In addition, the 18S rRNA gene sequences of human-derived C. cayetanensis isolates in China were retrieved in public databases, including NCBI, and the genetic and evolutionary characteristics of these gene sequences were analyzed. Results A total of 227 stool samples from children with diarrhea were included for retrospective testing, and there were 7 samples (3.08%) tested positive for C. cayetanensis. Sequence alignment and phylogenetic analysis demonstrated that six of these positive samples (GenBank accession no.: PZ748322) shared identical nucleotide sequences and exhibited 100% homology with a human-derived reference sequence (GenBank accession no.: KJ569531) from GenBank, and a novel gene sequence (GenBank accession no.: PZ737096) was identified, which shared 99.6% similarity with the sequence KJ569531. In the phylogenetic tree constructed based on the 18S rRNA gene, the gene sequences of these 7 positive samples were clustered with the sequence KJ569531 into the same clade, indicating a highly close genetic relationship. All C. cayetanensis isolates formed a distinct monophyletic clade, which were remarkably separated from outgroup species including Eimeria spp., Babesia microti, and Cryptosporidium parvum, supporting the monophyly of the genus Cyclospora. A total of 62 human-derived C. cayetanensis 18S rRNA gene sequences from China were integrated and screened, ultimately yielding 25 distinct gene sequences, in which 39 nucleotide polymorphic sites were identified. The overall nucleotide diversity (Pi) of the 25 gene sequences was 0.015, indicating that the average probability of a base difference occurring at any site between two random sequences was 1.5%. In addition, compared to other sequences, the new sequence (GenBank accession no.: PZ737096) obtained from this study showed base substitutions C→T at position 404 and A→C at position 406. Conclusion The prevalence of C. cayetanensis infection is high in stool samples from children with diarrhea in Ningbo City, Zhejiang Province, and the locally prevalent C. cayetanensis isolates exhibit genetic sequence differences from previously reported human-derived parasite isolates in China, which supplements the baseline data on genetic variation of C. cayetanensis in Chinese populations.

Key words: Cyclospora cayetanensis, Child, Diarrhea, Nested-PCR

CLC Number:

R382.3

LIU Hua, NI Hongxia, ZHU Na, ZHOU Hao, ZUO Qingqiu, CAO Jianping, ZHENG Bin, YIN Jianhai. Retrospective molecular detection and genetic analysis of Cyclospora cayetanensis in stool samples from children with diarrhea in Ningbo City, Zhejiang Province[J]. CHINESE JOURNAL OF PARASITOLOGY AND PARASITIC DISEASES, 2026, 44(4): 525-531.

Figures/Tables 7

Table 1

Fig. 1

Table 2

Fig. 2

Table 3

Nucleotide variation in 18S rRNA gene of human-derived C. cayetanensis isolates in China

GenBank号 GenBank ID	核苷酸位点 Nucleotide positions
GenBank号 GenBank ID	27	28	29	37	45	52	53	58	59	62	66	67	81	89	90	91	98	100	103	109	114	116	119	125	152	187	193	194	195	201	208	223	329	342	382	404	406	415	426
AF111183	C	T	T	-	C	G	T	C	G	G	T	C	C	G	C	C	C	C	C	C	C	T	A	A	T	T	C	C	C	T	T	G	T	-	G	C	A	A	C
FJ009120	C	T	T	-	C	G	T	C	G	G	T	C	C	C	G	C	C	T	G	C	C	T	A	A	T	T	C	T	T	T	T	G	T	-	G	C	A	A	T
FJ009127	C	T	T	-	C	G	T	C	G	G	T	T	C	C	G	C	C	T	G	C	C	T	A	A	T	T	C	T	T	T	T	G	T	-	G	C	A	A	T
FJ009128	C	T	T	-	C	G	T	T	G	G	T	C	C	C	G	C	C	T	G	C	C	T	A	A	T	T	C	T	T	T	T	G	T	-	G	C	A	A	T
GQ292778	C	T	T	-	C	G	T	C	G	G	T	C	C	C	G	C	C	T	G	C	C	T	A	A	T	T	C	C	T	T	T	G	T	-	G	C	A	A	T
GQ292779	C	T	T	-	C	G	T	C	G	G	T	T	T	C	G	C	C	T	G	C	C	T	A	A	T	T	C	T	T	T	T	G	T	-	G	C	A	A	T
GQ292782	C	T	T	-	C	G	T	C	G	G	T	C	C	C	G	C	C	T	A	C	C	T	A	A	T	T	C	T	T	T	T	G	A	-	G	C	A	A	T
KJ569531	C	T	T	-	C	G	T	C	G	G	T	Y	C	C	G	C	C	T	G	C	C	T	A	A	T	T	C	T	T	T	T	G	T	-	G	C	A	A	C
PZ748322	C	T	T	-	C	G	T	C	G	G	T	Y	C	C	G	C	C	T	G	C	C	T	A	A	T	T	C	T	T	T	T	G	T	-	G	C	A	A	C
KJ569532	C	T	T	-	C	G	T	T	G	G	T	C	C	C	G	C	C	T	G	C	C	T	A	A	T	T	C	T	T	T	T	G	T	-	G	C	A	A	C
KJ569533	C	T	T	-	C	G	T	C	G	G	T	C	C	C	G	C	C	T	G	C	C	T	A	A	T	T	C	T	T	T	T	G	T	-	G	C	A	A	C
KJ569535	C	T	T	-	C	G	T	C	G	G	T	C	C	C	G	C	C	T	G	C	C	T	A	A	T	T	C	C	T	T	T	G	T	-	G	C	A	A	C
KY770758	C	T	T	-	C	G	T	C	G	G	T	T	C	C	G	C	C	T	G	C	C	T	A	A	T	T	C	T	T	T	T	G	T	-	G	C	A	A	C
KY770760	G	C	C	T	G	C	A	C	T	G	A	T	C	A	A	T	T	T	G	T	C	T	A	A	T	T	C	T	C	T	T	A	T	-	G	C	A	A	C
KY770761	G	C	C	T	G	C	A	C	T	G	A	T	C	A	A	T	T	T	G	T	C	T	A	A	T	G	C	T	C	T	T	A	T	-	G	C	A	A	C
KY770762	G	C	C	T	G	C	A	C	T	G	A	T	C	A	A	T	T	T	G	T	C	T	G	A	T	T	C	T	C	T	T	A	T	-	G	C	A	A	C
KY770763	G	T	C	T	G	C	A	C	T	G	A	T	C	A	A	T	T	T	G	T	C	T	A	A	T	T	C	T	C	T	T	A	T	-	G	C	A	A	C
MN893886	C	T	T	-	C	G	T	C	G	G	T	T	C	C	G	C	C	T	G	C	C	T	A	A	T	T	C	T	T	T	T	G	T	-	A	C	A	A	T
MN893888	C	T	T	-	C	G	T	C	G	G	T	T	C	C	G	C	C	T	G	C	G	T	A	A	T	T	C	T	T	G	T	G	T	-	G	C	A	A	T
OP143736	C	T	T	-	C	G	T	C	G	G	T	C	C	C	G	C	C	T	G	C	C	T	A	A	T	T	C	T	T	T	T	G	T	G	G	C	A	G	C
OQ363213	C	T	T	-	C	G	T	C	G	G	T	C	C	C	G	C	C	T	G	C	C	C	A	A	C	T	C	T	T	T	T	G	T	-	G	C	A	A	T
OQ363214	C	T	T	-	C	G	T	C	G	G	T	C	C	C	G	C	C	T	G	C	C	T	A	A	T	T	T	T	T	T	T	G	T	-	G	C	A	A	C
PP593967	C	T	T	-	C	G	T	C	G	G	T	C	C	C	G	C	C	T	G	C	C	T	A	G	T	T	C	T	T	T	C	G	T	-	G	C	A	A	C
PP593968	C	T	T	-	C	G	T	C	G	G	C	C	C	C	G	C	C	T	G	C	C	T	A	A	T	T	C	T	T	T	T	G	T	-	G	C	A	A	C
PP593970	C	T	T	-	C	G	T	C	G	T	T	C	C	C	G	C	C	T	G	C	C	T	A	A	T	T	C	T	T	T	T	G	T	-	G	C	A	A	T
PZ737096	C	T	T	-	C	G	T	C	G	G	T	C	C	G	C	C	C	C	C	C	C	T	A	A	T	T	C	C	C	T	T	G	T	-	G	T	C	A	C

Table 3

Fig. 3

Table 4

Prevalence of C. cayetanensis infection in Chinese populations

地区 Region	检测方法 Detection methods	靶基因 Target gene	人群 Population	样本数 No. examined	阳性样本数（阳性率/%） No. positive (Detection rate/%)	参考文献 Reference
云南 Yunnan	镜检 Microscopic examination		腹泻患者 Diarrhea patients	378	15（4.0）	[5]
安徽 Anhui	镜检 Microscopic examination		幼儿 Children	200	0（0）	[13]
			小学生 Primary School Students	200	1（0.5）
			腹泻患者 Diarrhea patients	178	10（5.62）
			免疫功能低下者 Immunosuppressive patients	32	3（9.38）
浙江 Zhejiang	镜检 Microscopic examination		腹泻患者 Diarrhea patients	860	42（4.88）	[16]
河南 Henan	镜检 Microscopic examination		医院患者 Hospital patients	11 554	81（0.7）	[6]
河南 Henan	镜检 Microscopic examination		医院患者 Hospital patients	6 579	35（0.53）	[20]
河南 Henan	巢式PCR Nested PCR	18S rRNA	医院病人 Hospital patients	6 579	76（1.2）	[17]
江苏 Jiangsu	巢式PCR Nested PCR	18S rRNA	腹泻患者 Diarrhea patients	3 696	7（0.19）	[18]
江苏 Jiangsu	巢式PCR Nested PCR	18S rRNA	腹泻患者 Diarrhea patients	1 388	6（0.43）	[18]
上海 Shanghai	巢式PCR Nested PCR	18S rRNA	医院患者 Hospital patients	291	5（1.7）	[20]
上海 Shanghai	巢式PCR Nested PCR	18S rRNA	腹泻患者 Diarrhea patients	1 645	6（0.36）	[18]
上海 Shanghai	巢式PCR Nested PCR	18S rRNA	腹泻患者 Diarrhea patients	682	5（0.73）	[19]
上海 Shanghai	巢式PCR Nested PCR	18S rRNA	腹泻患者 Diarrhea patients	252	0（0）	[20]
广西 Guangxi	巢式PCR Nested PCR	18S rRNA	实验动物从业人员/村民 Laboratory animal practitioners/Villagers	50	5（10）	[21]
黑龙江 Heilongjiang	巢式PCR Nested PCR	18S rRNA	HIV/AIDS患者 HIV/AIDS patients	601	7（1.2）	[22]
云南 Yunnan	巢式PCR Nested PCR	18S rRNA	村民 Villagers	100	2（2.0）	[23]
浙江 Zhejiang	巢式PCR Nested PCR	18S rRNA	腹泻患者 Diarrhea patients	489	3（0.6）	[24]
浙江 Zhejiang	巢式PCR Nested PCR	18S rRNA	腹泻患者 Diarrhea patients	650	8（1.23）	[19]
浙江 Zhejiang	巢式PCR Nested PCR	18S rRNA	腹泻儿童 Diarrhea children	227	7（3.08）	本研究 This study

Table 4

References 24

[1]	Dubey JP, Khan A, Rosenthal BM. Life cycle and transmission of Cyclospora cayetanensis: knowns and unknowns[J]. Microorganisms, 2022, 10(1): 118.
[2]	Li JQ, Zhang KH, Zhang LX. Cyclospora cayetanensis[J]. Trends Parasitol, 2022, 38(5): 419-420.
[3]	Shields JM, Olson BH. Cyclospora cayetanensis: a review of an emerging parasitic coccidian[J]. Int J Parasitol, 2003, 33(4): 371-391.
[4]	Li JQ, Wang RJ, Chen YC, et al. Cyclospora cayetanensis infection in humans: biological characteristics, clinical features, epidemiology, detection method and treatment[J]. Parasitology, 2020, 147(2): 160-170.
[5]	张炳翔, 俞慧, 张莉莉, 等. 云南省腹泻患者圆孢子虫和隐孢子虫感染情况调查分析[J]. 中国寄生虫学与寄生虫病杂志, 2002, 20(2): 106-108.
	Zhang BX, Yu H, Zhang LL, et al. Prevalence survey on Cyclospora cayetanensis and Cryptosporidium ssp. in diarrhea cases in Yunnan Province[J]. Chin J Parasitol Parasit Dis, 2002, 20(2): 106-108. (in Chinese)
[6]	Zhou Y, Lv B, Wang Q, et al. Prevalence and molecular characterization of Cyclospora cayetanensis, Henan, China[J]. Emerg Infect Dis, 2011, 17(10): 1887-1890.
[7]	Ghosal A, Sardar SK, Das K, et al. Prevalence of Cyclospora cayetanensis in diarrheal patients in eastern India[J]. Jpn J Infect Dis, 2026, 79(4): 129-137.
[8]	Chen YC, Qin ZY, Li JQ, et al. The global prevalence of Cyclospora cayetanensis infection: a systematic review, meta-analysis, and meta-regression[J]. Acta Trop, 2024, 253: 107175.
[9]	Colston JM, Devleesschauwer B, Flynn T, et al. Updated estimates of the global, regional and national burden, and etiology of diarrheal diseases transmissible via food: a systematic review and meta-analytical modelling study for the World Health Organization[J]. medRxiv, 2026: 2026.01.26.26344508.
[10]	Liu H, Shen YJ, Yin JH, et al. Prevalence and genetic characterization of Cryptosporidium, Enterocytozoon, Giardia and Cyclospora in diarrheal outpatients in China[J]. BMC Infect Dis, 2014, 14: 25.
[11]	Li GQ, Xiao SM, Zhou RQ, et al. Molecular characterization of Cyclospora-like organism from dairy cattle[J]. Parasitol Res, 2007, 100(5): 955-961.
[12]	Herwaldt BL, Ackers ML. An outbreak in 1996 of cyclosporiasis associated with imported raspberries[J]. N Engl J Med, 1997, 336(22): 1548-1556.
[13]	Herwaldt BL, Beach MJ. The return of Cyclospora in 1997: another outbreak of cyclosporiasis in North America associated with imported raspberries[J]. Ann Intern Med, 1999, 130(3): 210-220.
[14]	苏庆平, 林秋君, 陈景龙. 我国圆孢子虫病首例报告[J]. 中国人兽共患病杂志, 1995, 11(5): 6.
	Su QP, Lin QJ, Chen JL. The first report of the cyclospore cayetanensis in China[J]. Chin J Zoonoses, 1995, 11(5): 6. (in Chinese)
[15]	王克霞, 李朝品, 王健, 等. 安徽省部分地区不同人群中圆孢子虫感染的初步调查[J]. 中华流行病学杂志, 2003, 24(2): 93.
	Wang KX, Li C/ZP, Wang J, et al. A preliminary survey on Cyclospora infection in different populations in some areas of Anhui Province[J]. Chin J Epidemiol, 2003, 24(2): 93. (in Chinese)
[16]	金永国, 邢文鸾, 吴康为, 等. 温州地区腹泻患者圆孢子虫感染情况调查[J]. 中国寄生虫病防治杂志, 2002, 15(5) : 320-321.
	Jin YG, Xing WL, Wu KW, et al. Investigation of Cyclospora infection in diarrhea patients in the Wenzhou Area[J]. Chin J Parasit Dis Control, 2002, 15(5): 320-321.
[17]	Li JQ, Chang YK, Shi KE, et al. Multilocus sequence typing and clonal population genetic structure of Cyclospora cayetanensis in humans[J]. Parasitology, 2017, 144(14): 1890-1897.
[18]	Jiang YY, Yuan ZY, Liu H, et al. Intestinal protozoan infections in patients with diarrhea: Shanghai municipality, Zhenjiang City, and Danyang City, China, 2011-2015 and 2019-2021[J]. China CDC Weekly, 2022, 4(8): 143-147.
[19]	Liu H, Qin Y, Zuo QQ, et al. Molecular characterization of human pathogenic Enterocytozoon bieneusi, Giardia duodenalis and Cyclospora cayetanensis of diarrheal outpatients in Yangtze River Delta region, China[J]. BMC Microbiol, 2026, 26(1): 99.
[20]	Jiang YY, Yuan ZY, Zang GQ, et al. Cyclospora cayetanensis infections among diarrheal outpatients in Shanghai: a retrospective case study[J]. Front Med, 2018, 12(1): 98-103.
[21]	Li J, Ren YJ, Chen HY, et al. Risk evaluation of pathogenic intestinal protozoa infection among laboratory macaques, animal facility workers, and nearby villagers from one health perspective[J]. Front Vet Sci, 2021, 8: 696568.
[22]	Zhao W, Yao L, Zhuang M, et al. A baseline epidemiological study of the co-infection of enteric protozoans with human immunodeficiency virus among men who have sex with men from Northeast China[J]. PLoS Negl Trop Dis, 2022, 16(9): e0010712.
[23]	王雅雪, 简金花, 刘华, 等. 云南省洱源县人群芽囊原虫和卡耶塔环孢子虫感染情况及其分子特征[J]. 中国寄生虫学与寄生虫病杂志, 2024, 42(2): 242-250.
	Wang YX, Jian JH, Liu H, et al. Infection of Blastocystis and Cyclospora cayetanensis in residents in Eryuan County, Yunnan Province and their molecular characterization[J]. Chin J Parasitol Parasit Dis, 2024, 42(2): 242-250. (in Chinese)
[24]	Liu H, Ni HX, Liu SK, et al. First report on occurrence and genotypes of Enterocytozoon bieneusi, Cryptosporidium spp. and Cyclospora cayetanensis from diarrheal outpatients in Ningbo, Southeast China[J]. Microb Pathogen, 2023, 174: 105952.

引物名称 Primer name	引物序列（5'→3'） Primer sequence (5'→3')	产物长度/bp Amplication length/bp
CYCLF1	AATGTAAAACCCTTCCAGAGTAAC	~1 000
CYCLR1	GCAATAATCTATCCCCATCACG
CYCLF2	AATTCCAGCTCCAATAGTGTAT	~500
CYCLR2	CAGGAGAAGCCAAGGTAGGCRTTT

样本编号 Sample No.	性别 Sex	年龄/月 Age/moth	来源地区 Resource
161	男Male	4	海曙区 Haishu District
180	男 Male	13	余姚市 Yuyao City
204	女 Female	44	余姚市 Yuyao City
208	女 Female	36	余姚市 Yuyao City
215	男 Male	27	余姚市 Yuyao City
222	男 Male	10	余姚市 Yuyao City
225	男 Male	2	慈溪市 Cixi City