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Introduction: Hospital-acquired infections continue to be a major public health problem, especially among neonates. Large proportions of infants are admitted to neonatal intensive care units (NICUs) and receive potent systemic antibiotics while the diagnostic work-up is still in progress. This study aimed to evaluate the recent methods for diagnosing neonatal sepsis (NS) and compare them to conventional diagnostic work-up. Methodology: The study included 100 neonates divided into three groups: proven early-onset NS, clinical early-onset NS, and negative infectious status. Bacterial DNA was detected in the blood by broad-range 16S rDNA polymerase chain reaction (PCR). Markers for diagnosis of bacterial infection, which includedprocalcitonin (PCT), interleukin-6 (IL-6), and highly sensitive C-reactive protein (hs-CRP), were measured by enzyme-linked immunosorbent assay (ELISA). Results: Blood culture was positive in 25 cases, while PCR for 16S rDNA was positive in 32 cases. Hs-CRP was significantly elevated in 30 patients in group 1, 35 patients in group 2, and 8 patients in group 3. IL-6 was significantly elevated in 28 patients in group 1, 24 patients in group 2, and 9 patients in group 3. PCT was found to be significantly elevated in 29 patients in group 1, 31 patients in group 2, and 2 patients in group 3. Conclusions: The16S rDNA PCR assay was more sensitive than blood culture. The combination of markers (hs-CRP, PCT, and IL-6) is better than single markers to diagnose sepsis. PCT had greater diagnostic value than did hs-CRP and IL-6, while IL-6 was better for diagnosis of neonatal infection. [ABSTRACT FROM AUTHOR]