宏基因组二代测序技术在假体周围感染病原诊断中的应用

目的探讨基于假体周围组织的宏基因组二代测序技术(metagenomic next-generation sequencing,mNGS)在关节置换术后假体周围感染(periprosthetic joint infection,PJI)的诊断效率和价值。方法回顾性分析2019年6月至2020年6月接受髋或膝关节翻修术33例患者的病历资料。诊断为PJI的病例设为感染组共21例,男9例,女12例;年龄(59.14±14.55)岁(范围:28~84岁);膝关节17例,髋关节4例;体质指数(body mass index,BMI)为(23.7±2.8)kg/m²(范围:17.7~29.4 kg/m²)。诊断为假体无菌性松动的病例设为对照组共12例,男4例,女8例;年龄为(53.08±10.05)岁(范围:39~70岁);膝关节4例,髋关节8例;BMI为(25.2±2.9)kg/m²(范围:18.3~31.2 kg/m²)。收集所有病例关节液和组织微生物培养的结果,收集所有病例mNGS检测假体周围组织的结果。比较微生物培养和mNGS检测对膝或髋关节PJI诊断的敏感性和特异性,总结和比较两种技术检出致病菌的种类,比较取样前2周内抗生素的使用对两种技术检出率的影响。结果感染组21例中mNGS共检出13例阳性,微生物培养共检出6例阳性。对照组12例中mNGS仅检出1例阳性,微生物培养结果均为阴性。在PJI的诊断中,mNGS敏感性(61.9%)与微生物培养(28.6%)的差异有统计学意义(χ2=4.71,P=0.03);mNGS特异性(91.7%)与微生物培养(100%)的差异无统计学意义(χ2=1.04,P=0.31)。在2周内有抗生素暴露的PJI病例中,mNGS阳性率(53.8%)明显高于微生物培养(15.4%),差异有统计学意义(χ2=4.25,P=0.04);而在2周内无抗生素暴露的PJI病例中,mNGS阳性率(66.7%)与微生物培养(44.4%)的差异无统计学意义(χ2=0.90,P=0.34)。在致病菌检出上,mNGS共检出9种细菌(金黄色葡萄球菌、科氏葡萄球菌、奥斯陆莫拉菌、痤疮丙酸杆菌、毗邻颗粒链菌、表皮葡萄球菌、结核分枝杆菌、里昂葡萄球菌、脆弱拟杆菌)和2种真菌(烟曲霉菌、近平滑念珠菌);微生物培养共检出3种细菌(金黄色葡萄球菌、卡他莫拉菌、结核分枝杆菌)和1种真菌(近平滑念珠菌)。mNGS和微生物培养均为阳性的患者共5例,3例检出完全一致的致病菌(金黄色葡萄球菌、结核分枝杆菌、近平滑念珠菌),1例部分一致(mNGS检出更多致病菌),1例完全不同。另外,mNGS在3例结核性PJI的诊断中表现出100%的特异性和敏感性,优于微生物培养。结论基于假体周围组织的mNGS检测技术是诊断PJI和确定致病菌的有效手段。取样前抗生素暴露对mNGS技术检出效力的影响小于微生物培养。

Performance of metagenomic next-generation sequencing in diagnosis of prosthetic joint infection

Objective To assess if metagenomic next-generation sequencing(mNGS)of periprosthetic joint tissue can provide an alternative rapid and sensitive tool for the diagnosis of prosthetic joint infection(PJI),especially compared to microbiological culture.Methods A total of 33 eligible patients who underwent revision arthroplasty from June 2019 to June 2020 in orthopedic surgery department of the first affiliated hospital of Zhengzhou University were retrospectively analyzed.Twenty-one patients were included in PJI group according to the American Academy of Musculoskeletal Infection diagnostic criteria,with 17 cases of knee and 4 cases of hip,including 9 cases of male and 12 cases of female,with an average age of 59.14±14.55 years old(range from 28 to 84),and an average BMI of 23.7±2.8 kg/m2(range from 17.7 to 29.4 kg/m2).Twelve patients were included in aseptic loosening group(control group),with 4 cases of knee and 8 cases of hip,including 4 cases of male and 8 cases of female,with an average age of 53.08±10.05 years old(range from 39 to 70),and an average BMI of 25.2±2.9 kg/m2(range from 18.3 to 31.2 kg/m2).Microbiological culture results of synovial fluid and periprosthetic joint tissue and mNGS results of periprosthetic joint tissue were collected.The sensitivity and specificity of mNGS and microbiological culture were calculated and compared.The species of pathogenic microorganismsdetected by the two techniques were summarized.In addition,the impact of antibiotic use on the efficacy of both techniques were compared.Results mNGS detected 13 positive cases and microbiological culture detected 6 positive cases in the PJI group.In the aseptic loosening group,1 case was determined positive by mNGS,and all the microbiological culture results were negative.In the diagnosis of PJI,mNGS showed significantly higher sensitivity than that of culture(61.9%vs 28.6%,χ2=4.71,P=0.03),while no statistical difference was observed in terms of specificity(91.7%vs 100%,χ2=1.04,P=0.31).In the PJI cases with prior exposure to antibiotics within two weeks,the sensitivity of mNGS was significantly higher than that of culture(53.8%vs 15.4%,χ2=4.25,P=0.04).However,there was no significant difference in the sensitivity between mNGS and culture in patients without antibioticsexposure(66.7%vs 44.4%,χ2=0.90,P=0.34).In the detection of pathogenic microorganism,mNGS detected 9 kinds of bacteria(Staphylococcus aureus,Staphylococcus family,Moraxella Oslo,Propionibacterium acnes,Streptococcus acnes,Staphylococcus epidermidis,Mycobacterium tuberculosis,Staphylococcus Lyons,Bacteroides fragilis)and 2 kinds of fungi(Aspergillus fumigatus,Candida parapsilosis),while microbiological culture detected 3 kinds of bacteria(Staphylococcus aureus,Moraxella catarrhalis,Mycobacterium tuberculosis)and one kind of fungi(Candida parapsilosis).mNGS and microbiological culture were both positive in 5 cases,among which 3 cases had completely matched results(Staphylococcus aureus,Mycobacterium tuberculosis,Candida parapsilosis),one case had partly matched results(mNGS detected more bacteria than culture)and one case had totally mismatched results.Additionally,in the diagnosis of the 3 included tuberculous PJI,mNGS showed 100%specificity and sensitivity.Conclusion mNGS of periprosthetic joint tissue is a more powerful tool for diagnosis and pathogen detection of PJI compared to microbiological culture,especially in the diagnosis of tuberculosis PJI.Besides,mNGS is more resistant to antibiotic exposure than culture.