Early Diagnosis of Neonatal Sepsis: A Molecular Approach and Detection of Diagnostic Markers Versus Conventional Blood Culture

3 Abstract: Neonatal sepsis is one of the major causes of neonatal morbidity and mortality, particularly in developing countries. Diagnosis of neonatal sepsis remains a major challenge, as early signs of sepsis are often non-specific and the laboratory criteria are also not fully reliable. The objective of our study was to evaluate the detection of activation surface markers CD64 on neutrophils and CD69 on lymphocytes and using a broad range PCR assay as diagnostic tools for diagnosis of neonatal sepsis of bacterial etiology in comparison with conventional blood culture method. This study was conducted at Microbiology and Immunology Department and Neonatal Intensive Care Unit (NICU), Menoufia University Hospitals. The study population included 45 neonates, 35 patients suspected of sepsis with a mean age of 12.54±7.17 days and 10 healthy controls with a mean age of 13.80±7.46 days. Patients were screened for sepsis by modified clinical sepsis score and hematological sepsis scoring system (total score 7, score 3 suggestive of sepsis). CBC, semi quantitative CRP, blood culture were done for all neonates. Flow cytometric analysis was done for detection of surface markers CD64 and CD69 on neutrophils and lymphocytes respectively and a broad range PCR assay for detection of bacterial DNA. In the present study, the incidence of culture positive cases (culture proven sepsis) was 26 (74.3%) and culture negative cases (culture unproven sepsis) was 9 (25.7%). Total number of isolates were 26. The most common organism isolated from blood cultures was Klebsiella spp. (26.9%). This study showed high expression of CD64 on neutrophils in patients (71.99±18.65) proven sepsis (79.38±7.69) than culture unproven sepsis (50.63±24.55 ). Neutrophil CD64 showed sensitivity 100%, specificity 66.7%, PPV 89.7% and NPV 100%. The present study showed high statistically significant difference (P =.001) between culture proven sepsis and culture unproven sepsis regarding percentage of expression of CD69 on T-lymphocytes. On evaluation of PCR status in relation to blood culture in studied cases, PCR revealed a 73.1% sensitivity, 100% specificity, 100% positive predictive value and 56.3% negative predictive value. In the present study, CRP results did not differentiate neonatal sepsis patients with bacterial infections from those with no bacterial infections. In conclusion, flow cytometric assessment of neutrophil CD64 may find its role in ruling out bacterial sepsis. Low sensitivity and NPV of broad range PCR were detected in this study reflecting that for this method to replace or supplement blood culture in early diagnosis of bacterial sepsis, it needs to be further developed and improved.