Hines Andrea Green, Freifeld Alison, Zhao Xing, Berry Ann Anderson, Willett Lynne, Iwen Peter C, Simonsen Kari A
Adult Infectious Diseases, University of Nebraska Medical Center, Omaha, NE, USA.
Pediatric Infectious Diseases, University of Nebraska Medical Center, Omaha, NE, USA.
BMC Pediatr. 2018 Apr 13;18(1):137. doi: 10.1186/s12887-018-1113-z.
The point prevalence of Clostridium difficile stool shedding in hospitalized infants from two neonatal intensive care units (NICUs) was examined utilizing standard clinical testing compared with duplex PCR to identify toxigenic and non-toxigenic C. difficile strains.
All infants from the two NICUs affiliated with a single academic medical center were eligible for inclusion. Stool collection was blinded to patient characteristics and occurred during a one week period at each NICU and repeated with a second weeklong collection 6 months later to increase sample size. Stools were tested for C. difficile using EIA (GDH/toxin A/B) with samples testing +/+ or +/- subsequently evaluated by Loop-Mediated Isothermal Amplification (LAMP) and by duplex PCR amplification of tcdB and tpi (housekeeping) genes. Cytotoxicity assays were performed on all samples positive for C. difficile by any modality.
Eighty-four stools were collected from unique infants for evaluation. EIA results showed 6+/+ [7.1%], 7 +/- [8.3%], and 71 -/- [84.5%] samples. All 6 EIA +/+ were confirmed as toxigenic C. difficile by LAMP; 6/7 EIA +/- were negative by LAMP with one identified as invalid. Duplex PCR concurred with LAMP in all 6 stools positive for toxigenic C. difficile. PCR identified 2 EIA -/- stools positive for tpi, indicating shedding of non-toxigenic C. difficile. Cytotoxicity assay was positive in 4/6 duplex PCR positive samples and negative for all stools that were EIA +/- but negative by molecular testing.
C. difficile blinded point prevalence in infants from two NICUs was 7.1% by molecular methods; and lower than expected based on historical incidence estimates. In house duplex PCR had excellent concordance with clinically available LAMP and EIA tests, and added detection of non-toxigenic C. difficile strain shedding. Evolving NICU care practices may be influencing the composition of infant gut microbiota and reducing the point prevalence of C. difficile shedding in NICU patient stools.
利用标准临床检测方法并与双重聚合酶链反应（duplex PCR）相比较，对来自两个新生儿重症监护病房（NICU）的住院婴儿艰难梭菌粪便排出的现患率进行了检测，以鉴定产毒和不产毒的艰难梭菌菌株。
隶属于单一学术医疗中心的两个NICU的所有婴儿均符合纳入条件。粪便采集对患者特征保密，在每个NICU的一周期间内进行，6个月后进行为期一周的第二次采集以增加样本量。使用酶免疫分析（EIA，谷氨酸脱氢酶/毒素A/B）检测粪便中的艰难梭菌，对检测结果为+/+或+/-的样本随后通过环介导等温扩增技术（LAMP）以及tcdB和tpi（管家）基因的双重聚合酶链反应扩增进行评估。对通过任何方式检测出艰难梭菌呈阳性的所有样本进行细胞毒性测定。
从不同婴儿收集了84份粪便进行评估。EIA结果显示，6份样本为+/+[7.1%]，7份样本为+/-[8.3%]，71份样本为-/-[84.5%]。所有6份EIA+/+样本经LAMP确认为产毒艰难梭菌；6/7份EIA+/-样本经LAMP检测为阴性，其中1份被鉴定为无效。双重聚合酶链反应在所有6份产毒艰难梭菌呈阳性的粪便中与LAMP结果一致。聚合酶链反应鉴定出2份EIA-/-粪便tpi呈阳性，表明存在不产毒艰难梭菌的排出。细胞毒性测定在4/6份双重聚合酶链反应呈阳性的样本中为阳性，而在所有EIA+/-但分子检测为阴性的粪便中为阴性。
通过分子方法检测，两个NICU婴儿中艰难梭菌的现患率为7.1%；低于基于历史发病率估计的预期值。内部双重聚合酶链反应与临床可用的LAMP和EIA检测具有极好的一致性，并增加了对不产毒艰难梭菌菌株排出的检测。不断发展的NICU护理实践可能正在影响婴儿肠道微生物群的组成，并降低NICU患者粪便中艰难梭菌排出的现患率。
