胃内尿素酶阳性细菌对幽门螺杆菌感染诊断的影响

Effects of urease-positive bacteria flora on diagnosis of Helicobacter pylori

目的探讨胃内可分解尿素酶菌群对基于尿素酶阳性诊断幽门螺杆菌(Helicobacter.pylori,Hp)感染的影响。方法胃镜下取疑为Hp感染患者的胃黏膜,行快速尿素酶检测(Rapid urease test,RUT)及细菌培养,根据培养物的菌落形态、革兰染色、RUT及Hp特异性16SrRNA基因片段PCR进行Hp的鉴定;提取胃黏膜组织DNA,通过Hp特异性PCR进行Hp感染快速诊断。对Hp阴性(Hp培养或特异性PCR阴性)而RUT阳性胃黏膜培养的非Hp(nonHp)进行常规尿素酶生化试验,选取20株尿素酶阳性non-Hp利用细菌16SrRNA通用引物进行PCR扩增并测序,利用NCBI系统进行序列比对,鉴定细菌种类。结果 606例患者胃黏膜RUT阳性率为58.4%(354/606),Hp培养阳性率29.7%(180/606)。Hp培养阴性胃黏膜特异性PCR阳性113例,Hp感染率为48.35%(293/606)。胃黏膜RUT阳性率高于胃黏膜Hp感染(χ2=1,2.337,P<0.05)。61例RUT阳性Hp培养或特异性PCR阴性的胃黏膜培养出80株non-Hp,其常规尿素酶生化检查均阳性。对其中20株尿素酶阳性non-Hp进行测序鉴定,均为产尿素酶菌。结论胃内存在除Hp外其他细菌,包括尿素酶阳性non-Hp,可造成RUT阳性,干扰Hp感染的实验诊断。

Objective To investigate the effects of urease-positive bacteria in the stomach on the diagnosis of Helicobacter pylori infection. Methods Gastric biopsy samples were collected from patients who experienced stomach discomfort during endoscopy.Biopsy samples were submitted to a rapid urease test（RUT）,bacterial isolation,and PCR amplification of H.pylori-specific 16 SrDNA.H.pylori and isolates other than H.pylori were differentiated using colony morphology,Gram staining,the RUT,and PCR using primers for H.pylori-specific 16 SrDNA.The urease activity of strains other than H.pylori was further identified using the RUT and a conventional urease test.Twenty urease-positive strains other than H.pylori were identified using 16 SrDNA PCR and sequencing with universal primers. Result Six hundred and six gastric biopsy samples tested positive for H.pylori according to the RUT at a rate of 58.4%（354/606）.H.pylori was isolated at a rate of 29.7%（180/606）.One hundred and thirteen biopsy samples tested negative for H.pylori but tested positive according to H.pylori-specific PCR.The rate of H.pylori detection（testing positive for H.pylori or testing positive according to H.pylori-specific PCR）was 48.3%（293/606）.The rate of H.pylori detection by the RUT was higher than the rate of H.pylori isolation（χ^2=12.337,P〈0.05）.Sixty-one biopsy samples tested negative for H.pylori but positive according to the RUT,and 80 strains with urease activity according to the RUT and the conventional urease test were isolated from those samples.All 20 species identified with 16 SrDNA sequencing were urease-positive species.Conclusion There are other bacteria besides H.pylori in the human stomach,including ureasepositive strains.These bacteria may hamper the clinical diagnosis of an H.pylori infection.