Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) for rapid diagnosis of neonatal sepsis

Abstract

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.

Methods: Blood samples from 97 neonates were evaluated for diagnosis of neonatal sepsis using BacT/Alert (automated blood culture) and PCR-RFLP.

Results: 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.

Fig. 1

Representative gel picture of PCR amplification of 16S rRNA gene (996 bp) from different samples. M-1 kb DNA ladder. Lane 1-Positive control; Lane 2-Negative control; Lanes 3-6, different blood samples.

Fig. 2A

PCR-RFLP photograph of 16S rRNA gene amplicon of reference strains. M: 100 bp DNA ladder Lane 1: Staphylococcus aureus; Lane 2: Pseudomonas aeruginosa; Lane 3: Enterococcus faecalis; Lane 4: Klebsiella pneumoniae; Lane 5: Staphylococcus epidermidis; Lane 6: Enterobacter aerogenes; Lane 7: Acinetobacter baumannii; Lane 8: Group-B Streptococcus.

Fig. 2B

Representative gel picture of RFLP pattern of different samples. M-100 bp DNA ladder. (A) Lane-1, Staphylococcus aureus (B) Lane-1, Klebsiella pneumoniae (C) Lane-1, Pseudomonas aeruginosa (C) Lane-2, Enterococcus faecalis.