目的 揭示轻度无症状钩虫感染对中老年人群肠道菌群与代谢的影响，为蠕虫-肠道菌群相互作用的研究提供参考。方法 以2022年10月—11月安徽省黄山市祁门县、黄山区乡村土源性线虫病监测点居民检出钩虫感染者（改良加藤厚涂片法钩虫虫卵阳性）为感染组，以钩虫虫卵阴性者为对照组。采集受检者新鲜粪样，提取肠道菌群DNA，扩增16S rRNA基因并测序。计算基于丰度的覆盖估计值（ACE）、PD-wholetree、Chao指数和Shannon指数等多样性指数，采用主坐标分析（PCoA）和不加权算术平均对方法（UPGMA）层次聚类分析的β多样性分析，比较感染组和对照组肠道菌群分布的差异，通过指示值分析筛选两组的指示性菌群。用甲醇-水萃取粪样中的代谢物，进行气相色谱-质谱（GC-MS）和液相色谱-质谱（LC-MS）双平台非靶向测序的代谢组学分析，用正交偏最小二乘方判别分析（OPLS-DA）建立筛选感染组与对照组差异代谢物的模型，制作火山图。根据P值、变量权重值和差异倍数筛选差异代谢产物。通过京都基因与基因组百科全书（KEGG）数据库检索差异代谢物的相关代谢通路，对差异代谢物进行富集分析。两个代谢物之间的线性相关程度采用Pearson相关分析。结果 研究对象共22人，其中感染组11人，对照组11人。感染组平均年龄72.2岁，均为轻度无症状带虫者；对照组平均年龄53.1岁。测序共获得1 755 649条16S rRNA序列，经序列质控和拼接后聚类为1 245条扩增序列变体。共鉴定出15个门22个纲58个目94个科191个属389个菌种。多样性指数分析结果显示，感染组肠道菌群多样性的ACE、PD-wholetree、Chao和Shannon指数分别为188.768、16.533、192.667和5.195，对照组分别为167.829、15.294、157.371和4.898，两组间差异均无统计学意义（t = 1.266、0.952、1.266、0.962，均P > 0.05）。PcoA分析结果显示，感染组与对照组的肠道菌群有不同程度的重叠，但仍可分为两个不同的类群。UPGMA层次聚类分析结果显示，感染组和对照组均先在本组中聚类，再与另一组聚类，两组间仅有1例重叠。多元变量统计分析显示，感染组的优势菌群有7个，分别为普雷沃菌、普氏粪杆菌、变栖克雷伯菌、Parasutteralla、粪球菌、梭菌UCG-014和肠杆菌；对照组的优势菌仅1种，为普通拟杆菌。指示值分析结果显示，感染组指示值居前的菌群依次是普雷沃菌、克雷伯菌、拟杆菌、小杆菌、肠杆菌、粪球菌、UCG-002、粪杆菌；对照组则依次为变形菌门的Clade_Ⅰa和Clade_Ⅲ、布劳特菌、另枝菌、Lachnoclostridium、嗜胆菌和苏黎世杆菌。建立的OPLS-DA模型能有效区分感染组和对照组的粪样代谢物，共鉴定出400个有差异粪样代谢物，其中156个显著上升，244个显著下降。KEGG富集分析结果显示，差异粪样代谢物显著富集在蛋白质的消化与吸收（P = 0.000）、中心碳代谢（P = 0.000）、矿物质吸收（P = 0.000）、氨酰基-tRNA合成（P = 0.000）、神经活性的配体-受体相互作用（P = 0.003）等代谢通路上。粪样代谢物中delta 2-三己胺与(22E)-3β-羟基-5α-胆甾-7,22-二烯酸（r = 0.935，P < 0.01）等15对代谢物呈正相关关系，棕榈酰溶血磷脂酰乙醇胺与氨基戊酸甜菜碱（r = -0.500，P < 0.05）等9对代谢物呈负相关关系。结论 安徽省黄山市乡村中老年人群无症状钩虫感染可影响肠道菌群的组成和代谢，有利于营造有益菌的共生和定植环境。

Objective To reveal the effect of mild asymptomatic hookworm infection on intestinal microflora and metabolome in middle-aged and the elderly people, to provide reference information for study on interaction of helminth-intestinal microflora. Methods In October to November 2022, from the parasite infection survey in residents at the surveillance site for soil-transmitted nematodes infection in Qimen County and Huangshan District of Huangshan City, Anhui Province, the hookworm egg positives detected by modified Kato-Katz thick smear method were assigned in infection group, and the hookworm egg negatives in control group. Fresh fecal samples from the examinees were collected for extraction of intestinal microflora DNA, of which 16S rRNA gene sequence was amplified by PCR and sequenced. The abundance-based coverage estimator (ACE), PD-wholetree, Chao index and Shannon index were calculated. The principal coordinate analysis (PCoA) and β diversity analysis of the unweighted pair group method with arithmetic mean (UPGMA) hierarchical clustering were used to compare the difference in the distribution of intestinal microflora between the infection group and the control group, to screen the indicator flora by indicator value analysis. The metabolites in the fecal samples were extracted with methanol-water extraction method for metabolome analysis by gas chromatography-mass spectrometry (GC-MS) and liquid chromatography-mass spectrometry (LC-MS) for non-targeted sequencing. Orthogonal partial least squares discriminant analysis (OPLS-DA) was used to establish a model for screening differential metabolites between the infection group and the control group, and generate the volcano plot. The differential metabolites were screened based on P-values, variable weight value, and difference multiple. The Kyoto Encyclopedia of Genes and Genomes (KEGG) database was used to search the metabolic pathways of related differential metabolites. Enrichment analysis of differential metabolites was performed, and the degree of linear correlation between two metabolites was analyzed by Pearson correlation analysis. Results A total of 22 subjects were studied, including 11 subjects of positive fecal eggs and 11 subjects of negative control. The average age of the infected group was 72.2 years old, and all of them presented mild asymptomatic hookworm infection. The average age of the control group was 53.1 years old. A total of 1 755 649 16S rRNA sequences were obtained and 1 245 amplicon sequence variants were clustered after quality control and assembly. A total of 389 species from 15 phyla, 22 classes, 58 orders, 94 families, and 191 genera were identified. The results of the diversity index analysis showed that the ACE, PD whole tree, Chao and Shannon indexes of intestinal flora diversity in the infection group were 188.768, 16.533, 192.667 and 5.195, respectively, whereas the control group were 167.829, 15.294, 157.371 and 4.898, respectively. No significant difference was seen between the two groups （t = 1.266, 0.952, 1.266, 0.962, P > 0.05）. The PCoA analysis demonstrated that the intestinal microbiota of the infected group and the control group overlapped to varying degrees but could still be divided into two distinct taxa. Additionally, the UPGMA hierarchical clustering analysis exhibited that both the infection group and the control were clustered in the same group first, and then clustered with the other group, and only one case overlapped. Multivariate statistical analysis showed seven flora dominating in the infection group, including Prevotella, Feacalibacterium, Klebsiella variicola, Parasutteralla, Coprococcus, Clostridium UCG-014 and Enterobacterium; while in the control group, only B. vulgatus was abundant. The results of indicator value analysis revealed the bacteria with the largest indicator value that affect the intestinal environment in the infection group, including Prevotella, Klebsiella, Bacteroides, Dialister, Enterobacterium, Coprococcus, UCG-002 and Faecalibacterium in the infection group; while in the control group, Clade_Ⅰa, Clade_Ⅲ, Blautia, Alistipes, Lachnoclostridium, Bilophila, Turicibacter and Muribaculaceae were dominant. The OPLS-DA model could effectively distinguish the metabolites in fecal samples of the infection group and the control. A total of 400 different metabolites were identified in the two groups, of which 156 were significantly increased and 244 decreased. The KEGG enrichment analysis indicated that the differential fecal metabolites were mainly enriched in protein digestion and absorption (P = 0.000) and central carbon metabolism (P = 0.000). Positive correlations were noted in 15 pairs metabolites i.e. between delta2-THA and (22E)-3 beta-hydroxy-5 alpha-chola-7,22-dien-oic acid (r = 0.935, P < 0.01), and negative correlations were seen in 9 pairs metabolites i.e. between lysoPE and aminovaleric acid betaine (r = -0.500, P < 0.05). Conclusion Asymptomatic hookworm infection in middle-aged and elderly people in rural area of Huangshan City may affect the composition and metabolism of intestinal microflora, which may create a symbiotic and colonization environment for probiotics