Résumé
Early diagnosis and treatment of newborn infants with suspected sepsis are required to reduce sepsis associated mortality and to prevent severe life-threatening complications. Clinical diagnosis is difficult and, blood culture, is often negative in the face of strong clinical indicators of septicemia, on the other hand, molecular diagnostics have proved to be a valuable adjunct for detection of neonatal bacteremia. The purpose of this study was to evaluate the role of l6S rRNA gene sequencing for rapid and sensitive diagnosis of clinically septic neonates with pathogen identification to allow for early and specific treatment of neonatal infections. A prospective study conducted in both NICUs of Kasr Al Aini hospital and Children's hospital, Cairo University over a period of 3 months from April 2008 to June 2008, Fifty neonates undergoing sepsis evaluation were included in this study. Risk factors, clinical manifestations and hematological findings suggestive of sepsis were recorded. Bloodculture and broad range 16S rRNA polymerase chain reaction [PCR] were collected from clinically septic neonates to confirm diagnosis of sepsis. Genotypic identification of bacteria by sequence analysis of the 16S rRNA gene was performed on all samples positive in PCR. Fifty neonates suspected clinically of having sepsis were included in the study. 61 samples were collected as a part of evaluation of infection, 50 samples upon admission and 11 samples as follow-up due to persistence of clinical manifestations. The rate of culture proven sepsis was 19.7% [12/61]. With the molecular method of broad range 16S rRNA PCR the detection of bacteria improved to 29.5% [18/61]. PCR revealed sensitivity, specificity, positive predictive value and negative predictive value of 91.7%, 85.4%. 61% and 97.6% respectively, while the accuracy of this test was 86.7%. so, compared to culture, the 16S rRNA PCR demonstrated a high negative predictive value for ruling out neonatal sepsis. According to the PCR results, hyperthermia, feeding intolerance and abdominal distension were found to be significant among neonates with positive PCR. As for hematological manifestations, we found a significant association between increasing number of abnormal hematological findings and PCR positivity [p=0.006, p for ordinal correlation =0.01]. Odds ratio for having 2 or more abnormal hematological findings and PCR positivity was 3.77 [95% CI=l.2-12.3]. Sequence analysis was done for further identification of organism on all samples positive in PCR. The sequenced PCR was in accordance with blood culture results in 10 samples while one sample showed conflicting results [91% agreement], Seven samples showed no growth by culture but were identified by sequence analysis. The sequencing-based identification of these isolates included: Enterobacter cloacae. Staphylococcus carnosus, Proteus mirabilis, Haemophilus influeuzae, Streptococcus pyogenes and Klebsiella sp. One sample yielded insufficient DNA to be analyzed. Broad range 16S rRNAPCR can he used to rule out sepsis. Both PCR and sequence analysis can provide additional diagnostic data that cannot he obtained with the use of broad-range PCR or routine laboratory tests alone. In neonates with clinically suspected sepsis, a primary decision could be made when the PCR results are completed, and within few hours, genotypic identification from the sequencing could be available to allow for early treatment