bloodstream infection is a leading cause of morbidity and mortality in neonatal intensive care units . the microbiological diagnosis of sepsis in newborns ( nbs ) is a difficult task , because blood culture ( bc ) , which is currently the gold standard for diagnosing nbs ( 1 ) has the disadvantage of producing an elevated percentage of false negatives ( 2 ) . previous studies have shown that 47.1% of nbs who begin antimicrobial treatment have negative culture results , and some analysis concluded that sepsis is diagnosed excessively ( 3 ) . in addition , diagnoses based on clinical criteria and experience favor overdiagnosis and antibiotic abuse ( 4 , 5 ) . many evaluations of the available diagnostic tests have attempted to solve the problem described above , but their success has been limited ( 6 ) . unfortunately , the decision to delay treatment until obvious signs and symptoms of sepsis occur , leads to the risk of preventable mortality ( 7 ) . however , few studies have addressed the clinical impact of the use of lc - sf ( a technique of molecular biology for rapid detection of bacterial and fungal infections ) in nbs . in the present study , we aimed to evaluate the accuracy of the lc - sf assay for detection of bacterial and fungal agents compared to bc and crp , using lc  sf like a routine clinical practice in five hospitals . we evaluated the impact of used lc - sf on some outcome variables of nbs with ns . first , the study was performed prospectively in patients admitted to the central south hospital of petroleos mexicanos , the gynecological - obstetrics hospital number 4 of the mexican institute of social security ; the dalinde hospital ( private institution ) ; and the monterrey nuevo leon university hospital , and national institute of perinatology . all patients with clinical sepsis suspiction and those presenting a score of > 8 points on the nosep-1 scale were included in the study . nosep-1 scale is composed by c - reactive protein > 14 mg / dl ( 5 points ) , neutrophils > 50% ( 3 points ) , thrombocytopenia < 150  10/l ( 5 points ) , parenteral nutrition > 14 days ( 6 points ) , fever > 38.2c ( 5 points ) ( 8) ; it was previously validated for our population ( 9 ) . after the nosep-1 punctuation criterion was satisfied , two blood samples were collected from each patient prior to antimicrobial treatment . only one episode per patient was considered ; we did not exclude nbs who had previously used prophylactic or antimicrobial treatment ( figure 1 ) . for the lc - sf test , dna was extracted ( using sterile technique ) from a 1-ml blood sample in a tube containing edta as an anticoagulant . the sample was mechanically lysed using the magna lyser instrument ( roche diagnostics gmbh , germany ) . the septifast prep mgrade kit was used to purify the dna , and the samples were incubated with a protease and a lysis buffer at 56 c to liberate the dna and protect it from dnases . subsequently , the binding buffer was added , and the solution was transferred to a fibreglass column in which the human , bacterial and fungal genomic dna was retained while the remainder of the material was removed by two washes . finally , the nucleic acids were eluted in 300 l of the solution and used as templates for the amplification reaction . the lightcycler septifast test mgrade kit was used for amplification reactions containing taq polymerase and primers / specific probes designed to detect gram - positive bacteria , gram - negative bacteria and yeasts . the purified dna samples and the other reaction components were added to the capillaries ( 100 l ) of the lightcycler mgrade , and the pcr amplification was performed with the lightcycler 2.0 equipment ( roche diagnostics gmbh , germany ) . a melting curve was then generated for the controls and the amplified samples and was analysed automatically by specific identification software ( septifast identification software , roche diagnostics gmbh , germany ) . each 1-ml sample was inoculated in a bact / alert bottle ( biomerieux sa ) and incubated using an automated system made by the same company . the blood cultures were requested as part of the protocol to study children with suspected sepsis . additionally , the impact of lc - sf results on hospitalization days , mortality , mechanical ventilation days and days of antibiotics use were analyzed retrospectively . we compared 86 nbs with result of lc - sf vs 86 nbs collected from clinical records with nosocomial sepsis suspiction with bc results . the demographic characteristics of the patients were analyzed using descriptive statistics , mainly averages and medians . the efficacy of septifast for microbial identification was analyzed by calculating its sensitivity , specificity and predictive values using the blood culture like gold standard . the concordance between the two types of microbial identification tests , blood culture and lc - sf was analyzed by calculating the kappa coefficient . the demographic characteristics of the patients were analyzed using descriptive statistics , mainly averages and medians . the efficacy of septifast for microbial identification was analyzed by calculating its sensitivity , specificity and predictive values using the blood culture like gold standard . the concordance between the two types of microbial identification tests , blood culture and lc - sf was analyzed by calculating the kappa coefficient . a total of 86 samples from 86 nbs with suspiction of nosocomial sepsis were included in the study . fifty nine ( 68.6% ) patients were male and 27 ( 31.3% ) female . among the patients , 43.7% weighed > 1500 g , and 56.3% weighed < 1500 g. a gestational age of more than 35 weeks was reached by 43.7% of the nbs , whereas 56.3% were born at or before 35th week . results of 86 episodes ( paired blood samples tested by blood culture and septifast ) were evaluated . 13/86 ( 15.1% ) blood cultures were positive , and 34/86 ( 39.5% ) were positive with septifast tests ( table 1 ) . the sensitivity of septifast test compared with blood culture was 0.69 , specificity 0.65 , positive predictive value 0.69 and the negative predictive value 0.65 . results for isolates from all episodes are shown in figure 2 , compared with blood culture , septifast showed similar or higher percentages of isolates for all the studied microorganisms , with differences : e. coli ( 20% ) , k. pneumoniae ( 42.8% ) , scn ( 44.4% ) , pneumococcus ( 0% ) , s. bovis ( 0% ) , candida spp , enterobacter spp , e. faecalis and pseudomonas spp ( 100% ) . polymicrobial infection rates were found in 4.6% . with septifast and 0% with blood culture ( table 2 ) . kappa index concordance between lc - sf and bc was 0.21 ; while kappa index concordance between lc - sf and crp was 0.17 ( table 3 ) . the response time was 6 - 8 hours using the lc - sf test and from 4 to 7 days using blood culture detection . hospitalization days , days of antimicrobial use , and number of subjects died diminished meaningful ( table 4 ) . negative results in both , septifast and blood cultures are not considered in the table ( n = 48 ) . the sensitivity of septifast test compared with blood culture was 0.69 , specificity of 0.65 , positive predictive value was 0.69 and the negative predictive value was 0.65 . e coli , escherichia coli ; k pneumon , klebsiella pneumonia ; cns , coagulase - negative staphylococcus ; s bovis , streptococcus bovis ; e faecalis , enterococcus faecalis .   using blood culture , 8 gram - positive and 5 gram - negative bacteria were isolated , whereas dna sequences from 15 gram - positive bacteria , 18 gram - negative bacteria and 5 yeasts were identified using lc - sf . in two cases , lc - sf identified streptococcus pneumoniae , which is a rare infectious agent in our neonatal population . in both cases , the blood culture results confirmed the presence of s. pneumoniae five days after its detection with molecular biology . abbreviations : bc , blood culture ; cf , concordance factor ; ea , expected average ; ki , kappa index;. oa , observed average . the understanding of neonatal sepsis as a disease with a critical public health impact is based on the identification of factors that magnify the problem by favoring long hospital stays and increasing the use of diagnostic and treatment resources ( 10 ) . the high degree of variation in clinical judgement adds to the difficulty of sepsis microbial identification and is a key reason behind overdiagnosis and the indiscriminate use of antimicrobials ( 11 ) . as the golden standard validation technique , bc has poor sensitivity , favors confusion and contributes to the lack of diagnostic specificity that is characteristic of the systemic inflammatory response syndrome ( sirs ) in nbs . the disadvantages of relying on bc to diagnose sepsis are exemplified by the adoption of terminology to justify antibiotic treatment , such as the concept of  sepsis without an isolated germ  applied to a child who fulfills the criteria for sirs but lacks an isolated microorganism . the main consequence of this variability is the abuse of antibiotics in an effort to prevent mortality due to sepsis ( 12 ) . lc - sf is a multiplex real - time pcr assay based on specific probes that detect and identify dna sequences from 25 microorganisms ( primarily gram - positive bacteria , gram - negative bacteria and yeasts ) that represent 90% of the species isolated from septic patients . detection of virus or mycobacterias is not possible with lc - sf ; and the microbial sensitivity to antibiotics and antibiogram is not available . this technique has led to improved rates of detection of the causative bacteria for diseases such as infectious endocarditis , sepsis in neutropenic patients with hematological malignancies , bacteremias and fungemias in specific groups ( 13 - 16 ) . the reported sensitivity levels of 79 - 90.5% , specificity of 87% , negative predictive value of 83% and positive predictive value of 83% compared with blood culture ( 17 - 19 ) are higher than the values from the present study . a meta - analysis by chang et al . concluded that the septifast assay had a sensitivity of 75% and a specificity of 92% for detecting bacteremia or fungemia ( 20 ) . nonetheless , the identification capacity of septifast was greater than that of blood culture both in the overall data analysis and in the analyses of individual microorganisms , especially yeasts and pseudomonas . the early detection of microorganisms using a technique based on the identification of nucleic acids will promote the prompt and appropriate use of antimicrobials , which decreases mortality . the prompt detection of the causative agent is crucial for the proper treatment of an infection first , we did not examine the possibility of contamination during the identification process , a factor that impacts the efficacy of most assays according to previous studies ( 21 ) , because the conditions for inclusion in our study ensured that none of the isolates were considered contaminated . coagulase - negative staphylococci were considered possible agents of sepsis in these children because these bacteria are frequently isolated in mexican population ( 22 ) . additional support for the absence of contamination in the present work is found in another study that evaluated lc - sf and reported no cases of contamination ; those researchers applied the standardised criteria for sirs developed by a consensus of the american college of chest physicians / society of critical care medicine ( accp / sccm ) , which ensures compliance with practices that minimise contamination ( 17 ) . previously an algorithm for interpretation of bc and lc - sf results was reported by lucignano b et al . there were criteries to consider a pathogen and a contaminant ; these criteries were the same as in our research ( 23 ) . second , some authors , such as lodes ( 17 ) have reported that the septifast test detects polymicrobial infection at rates as high as 11.4% , a higher ( 4.6% ) rate than that was found in our study . this discrepancy might be explained by the lodes study s examination of surgical patients from an intensive care unit , where the polymicrobial infection rate is high . unlike other studies , our findings influenced the treatments administered to our subjects , which substantiates our results . as a prime example , the identification of positive fungal lc - sf results led to specific patient treatments , which resulted in clinical improvement . therefore , analyzing the follow - up care of our subjects enabled a more complete evaluation of the usefulness of the lc - sf test in terms of the length of hospitalisation , use of antimicrobials and duration of recovery . microbial identification in neonates is a relatively understudied topic that has generated some controversy because of possible incorrect decision making due to false positives . therefore , the present work provides valuable support for the use of multiplex pcr as an identification method that can be performed together with bc , has the advantage of a 6 hour response time and allows the use of more targeted antimicrobial therapy . similar to scenarios involving patients with negative blood culture results , when patients have negative lc - sf results but solid clinical data and a high suspicion of sirs , the clinical criteria will continue to be the mainstay for diagnosis and treatment . the initiation of antimicrobial treatment based on suspicion will most likely not decrease . we predict that the only difference associated with lc - sf - based diagnosis will be a higher percentage of positive test results ( compared with bc ) and a more rapid response time leading to decision - making based on more solid evidence , which is the ultimate objective of auxiliary discriminatory tools . it is highly unlikely that septifast ( or another biomarker ) will replace clinical criteria for diagnosis .