Clinical Value Of Droplet Digital PCR In Rapid Diagnosis Of Invasive Fungal Infection In Neonates

BackgroundsIn recent years,invasive fungal infection(IFI)has become an important cause of nosocomial infection in neonatal intensive care unit(NICU).IFI is still an important cause of death and morbidity of premature infants.The clinical manifestation of fungal infection in neonates is lack of specificity and it is easy to delay diagnosis and treatment.The rapid progress of the disease often leads to delays in the beginning of treatment of antifungal drugs due to the long waiting time required for the detection of fungal infections.Therefore,early and accurate identification of IFI is important.At present,the golden criteria for mycological diagnosis are still positive or microscopic fungi found in blood or cerebrospinal fluid.It is reported that the positive rate of blood culture of fungi is 8%.The fungal culture took a long time and the time of fungal culture was 3 days.However,the premature infants infected with fungal septicemia lacked the typical symptoms and signs,the disease progressed rapidly,and the best time of diagnosis and treatment was easily missed.In recent years,molecular methods,such as PCR detection,have emerged as a possible alternative to traditional methods for detecting fungal infections.The molecular detection method has higher sensitivity.It allows detection of smallamounts of DNA and is more suitable for newborns.Recently,droplet digital PCR(ddPCR)has been widely used to detect pathogen load in clinical specimens and it can directly quantify the target gene.The specimen sample is divided into thousands of nanometer droplets,which are then amplified through PCR,and the fluorescent signal is detected.To analyze the fluorescent signal,each event(droplet)is defined as a positive or negative event of the target probe.Since the DNA fragment is separated into droplets in a random and independent fashion,the absolute copy number in the original sample can be determined by Poisson algorithm.In recent years,ddPCR technology has been widely used in the study of disease etiology,diagnosis and treatment effect analysis,and the effect is remarkable.The purpose of this study was to establish a method for the detection of fungi based on ddPCR,and to explore the clinical application value of this method in rapid and accurate detection of IFI in NICU.ObjectiveTo evaluate the clinical value of droplet digital PCR(ddPCR)in rapid and accurate diagnosis of invasive fungal infection(IFI)in neonates.MethodsThe highly conserved sequence of fungi 18S RNA was selected as the target sequence,and primers were designed to establish a ddPCR fungal detection system.Blood samples were collected from 83 neonates with high-risk factors for IFI and/or related clinical symptoms in the neonatal intensive care unit(NICU)of a hospital in**,China.Blood culture and ddPCR were used for fungal detection.ResultsThe ddPCR fungal detection system had a specificity of 100%and a sensitivity of 3.2 copies/μL,and had a good reproducibility.Among the 22 blood samples from neonates with a confirmed or clinical diagnosis of IFI,19 were detected positive byddPCR.Among the 61 blood samples from neonates who were suspected of IFI or had no IFI,2 were detected positive by ddPCR.ConclusionsThe ddPCR technique can be used for the detection of neonatal IFI and is a promising tool for the screening and even diagnosis of neonatal IFI.