Midan Dina A, Abo El Fotoh Wafaa Moustafa M, El Shalakany Abeer H
a Department of Pediatrics , Faculty of Medicine, Menoufia University , Shebin el kom , Menoufia , Egypt and.
b Department of Clinical Pathology , Faculty of Medicine, Menoufia University , Shebin el kom , Menoufia , Egypt.
J Matern Fetal Neonatal Med. 2017 Jun;30(12):1476-1483. doi: 10.1080/14767058.2016.1219994. Epub 2016 Aug 23.
This study aimed to explore whether 16S rRNA gene amplification by real time PCR and sequencing could serve as genetic-based methods in rapid and accurate diagnosis of neonatal sepsis.
This case control study was conducted on 40 neonates suffering from sepsis like manifestations recruited from the neonatal intensive care unit of Menoufia university hospital over a period of 6 months. Their blood samples were used for paired analysis of bacterial growth using BACTEC 9050 instrument and real time PCR assay with subsequent DNA sequencing for bacterial species identification.
The detection rate of culture proven sepsis was 70%. By using real time 16S r RNA PCR amplification method, the detection of bacteria was improved to 80%. Real time PCR revealed sensitivity, specificity, positive predictive value and negative predictive value of [100%, 66.7%, 87.5% and 100%] respectively. Compared to culture, the 16S rRNA real time PCR demonstrated a high negative value for ruling out neonatal sepsis. There was significant statistical difference between the PCR positive and negative cases as regards the hematological sepsis score. The results demonstrated the ability of DNA sequencing to recognize 4 pathogens which were negative by blood culture. The time consumed to detect sepsis using blood culture was up to 5 days while it took up to 16 h only by PCR and sequencing methods.
16S rRNA gene amplification by real time PCR and sequence analysis could be served as ideal and reliable genetic-based methods to diagnose and rule out sepsis with provision of additional data that cannot be obtained by routine laboratory tests with a shorter turnaround time than those with culture-based protocols.
本研究旨在探讨实时聚合酶链反应（PCR）扩增16S rRNA基因并进行测序是否可作为基于基因的方法用于快速、准确诊断新生儿败血症。
本病例对照研究对从曼努菲亚大学医院新生儿重症监护病房招募的40例有败血症样表现的新生儿进行了为期6个月的研究。采集他们的血样，使用BACTEC 9050仪器进行细菌生长的配对分析，并通过实时PCR检测及后续DNA测序来鉴定细菌种类。
培养证实的败血症检测率为70%。采用实时16S rRNA PCR扩增方法，细菌检测率提高到了80%。实时PCR显示敏感性、特异性、阳性预测值和阴性预测值分别为[100%、66.7%、87.5%和100%]。与培养法相比，16S rRNA实时PCR在排除新生儿败血症方面具有较高的阴性预测值。PCR阳性和阴性病例在血液学败血症评分方面存在显著统计学差异。结果表明DNA测序能够识别4种血培养呈阴性的病原体。使用血培养检测败血症耗时长达5天，而通过PCR和测序方法仅需16小时。
实时PCR扩增16S rRNA基因并进行序列分析可作为诊断和排除败血症的理想且可靠的基于基因的方法，能提供常规实验室检测无法获得的额外数据，且周转时间比基于培养的方法更短。
