Rohit Anusha, Maiti Biswajit, Shenoy Shalini, Karunasagar Indrani
Department of Microbiology, College of Fisheries, Mangalore; Faculty of Biomedical Science, Nitte University Centre for Science Education & Research, India.
Indian J Med Res. 2016 Jan;143(1):72-8. doi: 10.4103/0971-5916.178613.
BACKGROUND & OBJECTIVES: The difficulties in diagnosis of neonatal sepsis are due to varied clinical presentation, low sensitivity of blood culture which is considered the gold standard and empirical antibiotic usage affecting the outcome of results. Though polymerase chain reaction (PCR) based detection of bacterial 16S rRNA gene has been reported earlier, this does not provide identification of the causative agent. In this study, we used restriction fragment length polymorphism (RFLP) of amplified 16S rRNA gene to identify the organisms involved in neonatal sepsis and compared the findings with blood culture.
Blood samples from 97 neonates were evaluated for diagnosis of neonatal sepsis using BacT/Alert (automated blood culture) and PCR-RFLP.
Bacterial DNA was detected by 16S rRNA gene PCR in 55 cases, while BacT/Alert culture was positive in 34 cases. Staphylococcus aureus was the most common organism detected with both methods. Klebsiella spp. was isolated from four samples by culture but was detected by PCR-RFLP in five cases while Acinetobacter spp. was isolated from one case but detected in eight cases by PCR-RFLP. The sensitivity of PCR was found to be 82.3 per cent with a negative predictive value of 85.7 per cent. Eighty of the 97 neonates had prior exposure to antibiotics.
INTERPRETATION & CONCLUSIONS: The results of our study demonstrate that PCR-RFLP having a rapid turnaround time may be useful for the early diagnosis of culture negative neonatal sepsis.
新生儿败血症诊断困难，原因在于临床表现多样、被视为金标准的血培养敏感性低以及经验性使用抗生素影响结果。尽管此前已有基于聚合酶链反应（PCR）检测细菌16S rRNA基因的报道，但这无法鉴定病原体。在本研究中，我们利用扩增的16S rRNA基因的限制性片段长度多态性（RFLP）来鉴定新生儿败血症相关病原体，并将结果与血培养结果进行比较。
采用BacT/Alert（自动化血培养）和PCR-RFLP对97例新生儿的血样进行新生儿败血症诊断评估。
16S rRNA基因PCR检测出55例细菌DNA，而BacT/Alert培养有34例呈阳性。两种方法检测到的最常见病原体均为金黄色葡萄球菌。通过培养从4份样本中分离出克雷伯菌属，但PCR-RFLP检测到5例；不动杆菌属通过培养从1例中分离出，但PCR-RFLP检测到8例。发现PCR的敏感性为82.3%，阴性预测值为85.7%。97例新生儿中有80例曾使用过抗生素。
我们的研究结果表明，周转时间短的PCR-RFLP可能有助于血培养阴性的新生儿败血症的早期诊断。
