| Objective:To understand the infection status,identify isolates and infection risk factors of clonorchis sinensis among the population in some rural areas of Guangxi Zhuang Autonomous Region,China,and to explore the alterations of gut microbiota in C.sinensis-infected individuals,a cross-sectional study was conducted.The results were used to provide evidence for the clonorchiasis prevention and control,and provide a new direction for the diagnosis,treatment and prevention and control of clonorchiosis.Methods:Several villages were selected from Guangxi by multi-stage stratified cluster sampling.Then the demographic information and other relative information were collected by questionnaire survey.Fresh fecal samples from residents over 3 years-old were collected.Eggs of C.sinensis in fecal samples were examined and counted in triplicate using modified Kato-Katz technique.Some C.sinensis egg-positive fecal samples were used to extract DNA.The target gene fragment(internal transcribed spacer 2,ITS2)of C.sinensis was amplified by PCR.The obtained sequences were compared with those deposited in NCBI.The distribution and infection risk factors of C.sinensis was used to analyzed by SPSS 20.0.At the same time,89 fecal samples(47 C.sinensis-positive and 42 C.sinensis-negative)were selected to extract the total bacterial DNA;the v3-v4 sequence of bacterial 16S rRNA was amplified with specific primers;High-throughput sequencing technology was used for sequencing,and the results were analyzed by bioinformatics and statistical analysis.Results:1.The risk factors of C.sinensis infection among residents in some rural areas of GuangxiThe total infection rate of C.sinensis was 20.49%(404/1972).Multivariate logistics regression analysis found that male(aOR=5.79,95%CI:4.16,8.04),Zhuang(aOR=2.50,95%CI:1.70,3.69),farmer(aOR=3.63,95%CI:2.32,5.68),junior high school(aOR=1.63,95%CI:1.17,2.26),college and above(aOR=3.37,95%CI:1.62,7.02),unseparated raw and cooked food cutting boards(aOR=1.57,95%CI:1.13,2.18)and frequent consumption of raw fish(once time a year:aOR=4.73,95%CI:3.40,6.58;once or twice time a month:aOR=13.89,95%CI:9.37,20.58;once time a week:aOR=37.49,95%CI:3.68,382.20)were risk factors,while age(aOR=1.09,95%CI:0.80,1.49)and type of pet(aOR=1.23,95%CI:0.83,1.80)were confound factors.2.C.sinensis infection status and isolation identification in some rural areas of GuangxiThe prevalence of C.sinensis was 43.33%(260/600)in Teng county of Guangxi.Majority of them were with light infection(71.54%,186/260).Tianping Town has the highest infection rate(66.5%,133/200),followed by Jinji Town(37.50%,75/200)and Mengjiang Town(26.00%,52/200).The prevalence in males(54.69%,169/309)was significantly higher than that in females(31.27%,91/291)(χ2=33.47,P<0.001).The highest infection rate appeared in the 30～40 years group(57.6%,76/132),followed by that in 40～50 years old(54.14%,72/133).Individuals who received high school education and above had the highest infection rate(56.8%,25/44),and the lowest among those with primary school education or below(27.13%,51/188).Among the different age groups,the C.sinensis infection rate was significantly different(χ2=59.90,P<0.01),as it was for the different degrees of education(χ2=30.00,P<0.01).156 bp C.sinensis specifical bands was amplified by PCR,which was 100%homology with the ITS2 sequence of a Vietnam Danang isolate(accession number:MF319655)and Guangxi isolate(accession number:KU175246 and KJ137227.1).3.The study on gut microbiota of C.sinensis-infected individuals1)At the level of OTUs,the number of OTUs in the infected group was higher than that in the control group.In the age of 20～60 years old,there were 2 587 bacterial species overlapped between C.sinenesis-infected group and control group,and 1 149 bacterial species overlapped in the age over 60.2)Compared with control group,the simpson diversity of gut microbiota in infection group was significantly increased in the age of 20～60 years old(P<0.05);but,there was no significant change in the a diversity of gut microbiota in the age over 60.3)Compared with control group,the abundance and structure or some gut microbiota were significant changed.We found,in the age of 20～60 years old,the abundance of 19 taxa was significant different between EP1 and EN1(P<0.01);In the age over 60 years old,there were 16 taxa with significant different abundance between EP2 and EN2(P<0.01).In C.sinensis-infected group,the abundance of Bacteroides and Bifidobacterium was significantly reduced.Dore,a potentially pro-inflammatory microbe,was higher in infected subjects than in healthy individuals(P<0.05).What’s more,we found that C.sinensis-infected individuals were with the highest abundace of Variovorax and Agrobacterium.4)There was a correlation between the change of intestinal flora abundance and EPG.In the age of 20～60 years old,Bacteroides(r=-0.37),Parabacteroides(r=-0.37),Oscillospira(r=-0.29),Veillonella(r=-0.28),Paraprevotella(r=-0.26)and Haemophilus(r=-0.24)had a slight negative correlation with EPG;Enterobacter(r=0.21),Trichococcus(r=0.24)and Collinsella(r=0.25).In the age over 60 years old,Bacteroides(r=-0.64),Veillonella(r=-0.53),Bifidobacterium(r=-0.50),Clostridium(r=-0.35),Atopobium(r=0.50),Actinomyces(r=0.55)and Granulicatella(r=0.57).There was a negative correlation between the abundance of bacteroides.There was a negative correlation between the abundance of Bacteroides and Veillonella among the two age groups.Conclusion:1.Binyang county,Guangxi was a overweight epidemic area of clonorchisis.Male,Zhuang,nationality,farmer,junior high school,college and above,unseparated raw and cooked food cutting boards and frequent consumption of raw fish are risk factors for clonorchiasis.Therefore,the prevention and control of C.sinensis in this area should be strengthened,especially for the population with characteristics of risk factors.Strengthen the health education,in order to change some dangerous behaviors of infection C.sinensis through health education,and reduce the infection rate of C.sinensis in this area.2.Teng county,Guangxi was a overweight epidemic area of clonorchisis(43.33%).Prevention and management should be strengthened,especially for the population having a higher C.sinensis infection rate,such as the males and young adults.The PCR results indicated 100%homology between the ITS2 gene fragment of C.sinensis and that of a Vietnam Danang isolate and Guangxi isolate.3.Compared with the control group,there was significant alterations of gut microbiota structure in C.sinensis-infected individuals.The abundance of Bacteroides,Bifidobacterium and Veillonella were significant decreased,while,the abundance of Enterobacter,Enterococcus and Dore were significant increased,which may promote the occurrence and development of clonorchiosis.In addition,we found the highest abundance of the external environment microbiota in(such as Variovorax and Agrobacterium)C.sinenesis-infected group,which may be related to the development of C.sinenesis.In this study,we found the changes of gut microbiota in C.sinensis-infected individuals were correlated with EPG. |
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