Xu et al. BMC Infectious Diseases (2020) 20:16 https://doi.org/10.1186/s12879-019-4693-9 RESEARCH ARTICLE Open Access First report of Cryptosporidium viatorum and Cryptosporidium occultus in humans in China, and of the unique novel C. viatorum subtype XVaA3h Ning Xu1,2,3,4,5, Hua Liu1,2,3,4,5, Yanyan Jiang1,2,3,4,5, Jianhai Yin1,2,3,4,5, Zhongying Yuan1,2,3,4,5, Yujuan Shen1,2,3,4,5* and Jianping Cao1,2,3,4,5* Abstract Background: Cryptosporidium is a genus of common intestinal protozoa, members of which cause diarrhea in a wide variety of hosts. Previous studies on Cryptosporidium in China have mainly focused on diarrhea sufferers, children, and immunodeficient individuals such as HIV/AIDS patients. However, the epidemiological characteristics of Cryptosporidium in the population in rural areas remain unclear. Herein, we investigated the prevalence of, and risk factors for, Cryptosporidium in rural areas of Binyang County, Guangxi Zhuang Autonomous Region, China, and genetically characterized the Cryptosporidium isolates we obtained. Methods: From August to December 2016, two villages in Binyang County, Guangxi, were sampled using a random cluster sampling method. Fresh fecal samples were collected from all eligible residents (residence time > 6 months). Molecular characterization of Cryptosporidium was carried out based on its SSU rRNA, gp60, actin and hsp70 gene sequences. Fisher’s exact test were conducted to assess the risk factors for Cryptosporidium infection. Results: A total of 400 fecal samples were collected from 195 males (48.8%) and 205 females (51.2%). Two samples (0.5%) were positive for Cryptosporidium and were identified as C. viatorum and C. occultus respectively. Moreover, a new C. viatorum subtype XVaA3h was identified based on the sequence of the gp 60 gene. Conclusions: To our knowledge, this is the first report of C. viatorum and C. occultus infections in humans in China and of C. viatorum subtype XVaA3h. The findings provide important information on the prevalence of Cryptosporidium in the Chinese population, and expand the range of Cryptosporidium species known to infect people in China. Keywords: Cryptosporidium, Subtype, C. viatorum, C. occultus, Rural area Background with Cryptosporidium oocysts, or humans come into contact Members of the Cryptosporidium genus of intestinal proto- infected individuals or animals, cryptosporidiosis outbreaks zoa infect a wide range of hosts including humans, non- may occur [3]. Cluster outbreaks of cryptosporidiosis re- human primates, birds, amphibians, fish and reptiles [1]. Glo- ported worldwide have represented a great threat to public bal concern was raised following an outbreak of cryptospor- health [3, 4]. Symptoms of human cryptosporidiosis range idiosis in Wisconsin, USA, in 1993, in which 403,000 from self-limiting diarrhea in (mainly) immunocompetent individuals were affected and 100 fatalities were reported [2]. individuals, to persistent diarrhea (especially in children Only small quantities of oocysts are needed to establish in- younger than 5-years-old). Cryptosporidiosis has been de- fection, and once food or drinking water is contaminated scribed as the second ranked causative agent of diarrhea in children in developing countries in southern Africa [5]. * Correspondence: [email protected]; [email protected] However, no effective drugs or vaccines have been devel- 1Key Laboratory of Parasite and Vector Biology, Ministry of Health, Shanghai oped, hence early detection and tracing the source of 200025, China Full list of author information is available at the end of the article © The Author(s). 2020 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Xu et al. BMC Infectious Diseases (2020) 20:16 Page 2 of 11 infection are of great importance for preventing outbreaks of Study design and population cryptosporidiosis. A cross-sectional survey was carried out to investigate Globally, cryptosporidiosis is more endemic in developing the prevalence of, risk factors for, and species/genotype countries than in developed countries [6]. In China, the re- distribution of Cryptosporidium in a rural population in ported human prevalence of Cryptosporidium has ranged Binyang County. The study was conducted in two villages from 0.0 to 16.5% since the first two cases were reported in (A and B) from two towns (village A from town C1, and Jiangsu Province in 1987, and a strong correlation was found village B from town C2) selected at random. A total of 400 between the infection and HIV/AIDS [7–12]. Moreover, individuals were involved in our study. All participants Cryptosporidium was responsible for about 1.4 to 10.4% of were grouped according to gender, age, education level, diarrhea episodes in China [9]. and so on. Males accounted for 48.8% (195/400) and To date, at least 39 species of Cryptosporidium have been females 51.2% (205/400) of participants. The average age identified [13–16], and at least 21 species are considered was 35.7 ± 25.3, ranging from 7 months to 89 years old. zoonotic, among which C. hominis and C. parvum are the two main pathogens causing cryptosporidiosis in humans Table 1 Basic information on participants and assessment of [17]. However, in recent years, with the development of risk factors for Cryptosporidium infection molecular biological technologies, the number of crypto- Variable No. Examined (%) No. Positive p-value sporidiosis cases confirmed to be caused by other Crypto- Gender sporidium species has increased, and some species or Male 195 (48.8) 2 0.237 genotypes are predominant in specific countries or re- gions. For example, studies in Jiangsu and Shanghai, Female 205 (51.2) 0 China, revealed unusually high prevalence of C. andersoni Age in diarrhea patients [17, 18]. C. cuniculus,forwhichthe < 5 years (infants) 61(15.3) 1 0.502 natural host is rabbit, was found in patients with diarrhea 5–12 years (children) 76(19.0) 0 in the UK [19], and C. xiaoi, mainly found in sheep and 13–19 years (youths) 11(2.7) 0 goat, was detected in HIV/AIDS sufferers with clinical 20–49 years (adults) 91(22.7) 0 manifestations including vomiting [20]. To our know- ledge, seven species of Cryptosporidium—C. hominis, C. 50 years up (elderly) 161(40.3) 1 parvum, C. andersoni, C. meleagridis, C. felis, C. canis and Education level C. suis have been identified in humans in China [1]. Primary and below 227 (56.8) 2 0.590 At present, cryptosporidiosis is not included in the Junior middle school 144 (36.0) 0 National Disease Reporting System in China [21], and High school and above 29 (7.2) 0 Cryptosporidium oocyst examination is not performed rou- Drinking water sources tinely on patients with diarrhea or other gastrointestinal symptoms, so the prevalence of Cryptosporidium in China Tap water 222 (55.5) 0 0.203 may be underestimated. The present study aimed to investi- Well or spring water 176 (44.0) 2 gate the prevalence, risk factors and species/genotype distri- Others 2 (0.5) 0 bution of Cryptosporidium among a rural population in Drinking unboiled water China, which are of great relevance to public health. Yes 159 (39.8) 2 0.157 No 241 (60.2) 0 Methods Washing hands before meals Study area Yes 347 (86.8) 2 1.000 Guangxi is situated in southern China, between 22°54′– No 53 (13.2) 0 ′ ′– ′ 26°24 N and 104°26 112°04 E. It borders the Beibu Eating unwashed vegetables and fruits Gulf to the south, and is adjacent to Vietnam in the Yes 179 (44.8) 2 0.200 southwest. Binyang County, which belongs to Nanning City of Guangxi Zhuang Autonomous Region, is located No 221 (55.2) 0 in the south–central part of Guangxi and the northeast Animals raising part of Nanning City. It is high-altitude in the south and Yes 261 (65.3) 2 0.545 low-altitude in the north, surrounded by earthy moun- No 139(34.7) 0 tains to the east, south and west, with an open basin in Diarrhea the middle and a large alluvial plain. It has a subtropical Yes 10 (2.5) 0 1.000 monsoon climate with abundant light and heat, long summer and short winter, and abundant rainfall. No 390 (97.5) 2 Xu et al. BMC Infectious Diseases (2020) 20:16 Page 3 of 11 Participants with primary school education and below ethidium bromide staining. Products of the expected size accounted for 56.8% (227/400), followed by junior high were analyzed using an ABI 3730 DNA Analyzer (Applied school level (36.0%; 144/400), and high school education Biosystems, Foster City, USA) and Big Dye Terminator and above (7.2%; 29/400); 65.3% (261/400) of participants v3.1 Cycle Sequencing Kit (Applied Biosystems). Conti- reported raising animals at home. Basic information on all gExpress software was used for sequence assembly and participants is shown in Table 1. wave peak evaluation. Sequences were searched using the basic local alignment search tool (BLAST) and aligned Sample collection and questionnaire with representative Cryptosporidium sequences. From August to December 2016, 400 fresh fecal samples (>5 g) were collected from villagers in the two selected Phylogenetic analysis villages. Samples were taken from the middle of the stool Phylogenetic trees were constructed using MEGA6.0 to avoid contamination from soil, animal manure or software [26] based on 1000 bootstrap replicates from other human feces, and transported to the laboratory of sequences obtained in this study and representative the local Center for Disease Control within 4 h of collec- Cryptosporidium sequences downloaded from the NCBI tion.