多通道分子信标聚合酶链反应在新生儿侵袭性念珠菌感染快速诊断中的应用

目的初步建立新生儿侵袭性真菌感染(IFI)常见念珠菌的多通道分子信标荧光聚合酶链反应(MQ-PCR)检测体系,实现新生儿IFI病原体的早期检出,并探讨患儿不同部位念珠菌检出率的差异。方法收集2018年1月至2019年4月首都医科大学附属北京妇产医院收治的34例临床诊断为脓毒败血症并疑似IFI新生儿的血液、深部痰液和咽拭子标本;常规行血培养及微生物鉴定,并应用MQPCR体系对3类标本进行念珠菌检测,比较两种方法念珠菌阳性检出率和检测时长。结果本研究MQPCR体系检测白色念珠菌、近平滑念珠菌、光滑念珠菌和热带念珠菌的检出下限均为50 FU/ml,线性检测范围为5×10^1~1×10^5 CFU/ml,且MQ-PCR体系所检出细菌与其他14种常见致病性细菌或DNA序列同源性较高的真菌无交叉反应。9例(9/34、26.5%)患儿经血培养念珠菌阳性确定为IFI;此9例IFI患儿同一血液样本、自身不同部位(深部痰液、咽拭子)样本行MQ-PCR均检出念珠菌阳性,且检出菌种与血培养完全一致。比较血标本两种方法念珠菌阳性检出率:血培养法为26.5%(9/34),MQ-PCR为32.4%(11/34),差异无统计学意义(χ^2=0.5、P=0.5)。血培养法念珠菌阳性检出率(26.5%)和相应的自身不同部位标本MQ-PCR法念珠菌阳性检出率差异均无统计学意义(P均> 0.05),其中咽拭子、深部痰液和血标本中念珠菌阳性检出率分别为41.2%(14/34)、32.4%(11/34)和32.4%(11/34)。MQ-PCR检测血液、深部痰液和咽拭子标本时长分别为3.5(3.2,3.9)h、3.1(2.9,3.2)h和2.7(2.5,2.9)h,较血培养时长94.0(78,105.8)h]显著缩短,差异均有统计学意义(Z=-2.7、P=0.008,Z=-2.5、P=0.013,Z=-2.7、P=0.008)。结论对IFI新生儿多部位标本应用MQ-PCR检测念珠菌,较血培养具备高符合率及快速检出的优点,利于新生儿念珠菌定植及血流感染的早期诊断及动态监测。

Application of multi-channel molecular beacon polymerase chain reaction in rapid diagnosis of neonatal invasive Candida infection

ObjectiveTo preliminarily establish a multi-channel molecular beacon polymerase chain reaction (MQ-PCR) method for early detection of common Candida pathogens associated with invasive fungal infections (IFI) in newborns, and to explore the differences of Candidas detection in different parts of neonates with IFI. MethodsThroat swabs, deep sputum and blood samples from 34 neonates suspected of invasive fungal infection in Neonatal Intensive Care Unit (NICU), Beijing Obstetrics and Gynecology Hospital, Capital Medical University, from January 2018 to April 2019 were collected. Blood culture and strain identification were performed routinely, and Candidas were also detected by MQ-PCR system in three types of samples. The positive detection rate and detection period of the two methods were compared, respectively. ResultsThe lower detection limits of the MQ-PCR system for Candida albicans, Candida parapsilosis, Candida glabrata and Candida tropicalis were all 50 CFU/ml, and the linear detection ranges were all 5 × 10¹-1 × 10⁵ CFU/ml. Bacteria detected by MQ-PCR system did not cross-react with 14 other common pathogenic bacteria or fungi with high homology DNA sequence. IFI was identified in 9 cases (26.47%) among the 34 infants by positive Candidas in blood culture. Meanwhile, the same blood sample and the patient’s own (deep sputum and pharyngeal swab) samples from different parts of the above 9 children with IFI were also positive for Candidas by MQ-PCR, and the detected strains were identical with blood culture. The positive detection rate of Candida in blood samples by the two methods was basically the same (χ² = 0.5, P = 0.5), which were 26.5% (9/34) by the culture method and 32.4% (11/34) by MQ-PCR. Compared with the positive detection rate of Candida in blood samples by blood culture (26.5%), there was no significant difference in different parts of the samples by MQ-PCR (all P > 0.05), which were 41.2% (14/34) in pharyngeal swabs, 32.4% (11/34) in deep sputum, and 32.4% (11/34) in blood. Candida detection period of MQ-PCR in blood 3.5 (3.2, 3.9); Z =-2.7, P = 0.008], deep sputum 3.1 (2.9, 3.2); Z =-2.5, P = 0.013], swabs 2.7 (2.5, 2.9); Z =-2.7, P = 0.008] were significantly shorter than that of Candida blood culture 94.0 (78, 105.8)]. ConclusionsCompared with blood culture, MQ-PCR had the advantages of high coincidence rate and early detection when used to detect common Candidas pathogens in multi-types of specimens from neonatal IFI patients. MQ-PCR was helpful for early diagnosis of bloodstream infection and dynamic monitoring of Candida colonization in neonates.