肝硬化患者腹水中细菌DNA的检测及临床意义

Clinical significance of bacterial DNA assays in ascites of patients with cirrhosis

目的 利用PCR测序方法检测肝硬化患者腹水中的细菌DNA，探讨肝硬化患者发生细菌移位的高危因素及其后果。方法 在细菌16SrRNA基因保守区设计一对通用引物，对37份肝硬化患者腹水标本进行扩增，腹水中细菌DNA阳性者纯化后经核苷酸测序鉴别细菌种类；同时对患者的临床症状及实验室检查结果进行统计分析。结果 37份腹水标本中有9份获得530bp细菌DNA片段，占24．3％。测序共检测出4种细菌，其中大肠埃希菌6例，表皮葡萄球菌、溶血性链球菌、阴沟肠杆菌各1例；统计分析表明，腹水中细菌DNA阳性和阴性两组肝硬化患者在Child-Pugh积分（12．3±0．8，10．9±1．2）、上消化道出血的发生率（3例，1例）、血清TBil水平[（395．5±216．6）μmol／L，（192．7±206．3）μmol／L]、凝血酶原活动度[（31．3±9．7）％，（50．4±15．1）％]、腹水总蛋白浓度[（4．1±2．8）g／L，（7．9±5．2）g／L]、外周血WBC总数[（12．3±7．5）×10^9／L，（5．3±4．1）×10^9／L]等方面比较，差异有统计学意义（P〈0．05）。结论 PCR测序方法可应用于肝硬化患者腹水中细菌DNA的检测及细菌移位的研究；肝功能损害、腹水调理活性低下和上消化道出血是肝硬化患者发生细菌移位的高危因素和后果。

Objective To detect bacterial DNA in aseites of patients with cirrhosis by polymerase chain reaction（PCR） sequencing and to understand the risk factors for bacterial translocation in patients with cirrhosis. Methods A set of universal primers was designed based on the conservative regions in bacterial 16S ribosomal RNA genes. Thirty-seven samples of ascites of patients with cirrhosis were amplified with PCR. The corresponding bacteria were identified by nucleotide sequencing of purified PCR products. The clinical data of patients were also collected. The main clinical and analytical characteristics of 37 patients were statistically analyzed using SPSS software 11.5. Results Bacterial DNA（530 bp） was detected from 9 of 37 patients（24.3%）. DNA sequencing allowed the identification of Escherichia coli （n = 6）, Staphylococcus epidermidis （n = 1）, Streptococcus hemolyticus （n = 1） and Enterobacter cloacae （n = 1）. There were significant differences in the incidence of upper gastrointestinal hemorrhage （n = 3, n = 1）, serum total bilirubin level[（395. 5±216.6） μmol/L, （192.7±206.3） μmol/L], Child-Pugh score（12.3±0.8, 10.9±1.2）, ascitic total protein[（4. 1±2.8） g/L, （7.9±5.2） g/L] and peripheral blood leucocyte population[（12.3±7.5） ×10^9/L, （5.3±4.1） ×10^9/L] between groups with Bacterial DNA and without Bacterial DNA in ascites （P 〈 0.05）. Conclusions Cirrhotic patients with severe hepatic impairment, deficient ascitic fluid opsonic activity or upper gastrointestinal hemorrhage are predisposed to bacterial translocation.