Egyptian Journal of Medical Microbiology, January 2009 Vol. 18, No. 1 Detection of Neonatal Bacteremia Using 16S rRNA Gene Amplification by Polymerase Chain Reaction Sahar M. Fayed, Azza Abo Senna and Hesham Kamel* Department of Pediatric٭ Department of Clinical Pathology, Banha Faculty of Medicine and Emergencies and Critical Care Children Hospital – Cairo University. ABSTRACT The purpose of this study was to evaluate the potential of a broad diagnostic approach based on 16S rRNA gene amplification and sequencing for rapid detection of neonatal bacteremia. Subjects and Methods: Blood samples were collected from fifty neonates admitted to the neonatal intensive care units with suspected sepsis, for paired analysis of bacterial growth using the BACTEC 9050 instrument and for bacterial 16S rRNA gene using a PCR assay with subsequent DNA sequencing for bacterial species identification. Results: The specimens were positive for bacteria in 43 cases (86%) by blood culture and 44 cases (88%) by PCR out of a total of 50 specimens analysed. There was very good agreement between the results of PCR and blood culture for detection of neonatal bacteremia (kappa ═ 0.8). Taking blood culture as a reference method, the sensitivity, specificity, positive and negative predictive values for PCR were 100%, 75%, 95.5% and 100% respectively after exclusion of candida isolate which was detected by blood culture only and not by PCR( the primer being used was 16S r RNA not 18S r RNA needed to identify fungal sepsis). Concerning the time consumed to detect sepsis, blood culture method took more time (up to 5 days) while PCR took less time <4 hour. Our results revealed the ability of DNA sequencing to recognize two pathogens which were negative by culture, one was Klebsiella pneumoniae and the other was Staphylococcus epidermidis. In addition DNA sequencing identified 2 species (one Acinetobacter lwoffii and one Acinetobacter baumannii) that couldn’t be identified by routine conventional biochemical reactions but only by Microbact test. Conclusion: This PCR-based approach is quite useful in detection and identification of neonatal pathogens and has the potential for excellent sensitivity and a shorter turn around time than those of culture based protocols. Key words: Neonatal sepsis; 16S r RNA; DNA sequencing. for early sepsis and/ or with alterations of some INTRODUCTION laboratory tests (i.e white cell count ,total Infections are still one of the most common neutrophil count , Immature / total ( I/T) ratio, causes of hospitalization and mortality in CRP ). However, the sensitivity and specificity children and neonates, sepsis represents the most of each laboratory test are far from 100% (3,4), so important cause of neonatal morbidity and that a large proportion of neonates not really mortality after congenital malformations (1). The infected are treated with broad-spectrum incidence ranges from 1 to 8 cases for every antibiotics. The possibility of having a 100% 1000 infants, but much higher values are found sensitive and specific method for the in preterms, low birthweight newborn infants identification of bacteria in blood , with results and generally, in newborn infants admitted to available in a short period of time , could allow neonatal intensive care units. Mortality from the onset of treatment only in neonates with septicemia has dropped from 40-50 % to 10-20 infection , thus reducing the use of broad- %, but in cases of early - onset fulminant sepsis, spectrum antibiotics, the need of close it is still around 70 % (2). Indeed, a majority of observation of suspected cases and of course, survivors have significant neurological sequelae medical costs . Molecular biology techniques, as a consequence of central nervous system such as polymerase chain reactions (PCR) have involvement, septic shock or hypoxaemia been used as a specific and sensitive method for secondary to severe parenchymal lung disease or diagnosis of different bacterial, viral and persistent pulmonary hypertension. Clinical protozoal infections, and the number of the diagnosis of sepsis is not easy, because diseases for which this diagnostic approach can symptoms and signs are not specific and be used is steadily increasing (5). DNA sequences moreover, a dramatic deterioration of clinical present in all bacteria, such as portions of DNA conditions can supervene very rapidly, even in encoding the 16 S ribosomal RNA (rDNA), have asymptomatic newborn infants. Because the been used to define organisms as bacteria, those preliminary report of blood culture is not sequences have been amplified with PCR using available before 48-72 h, it is customary to start an automated method ,allowing detection of treatment at birth in all neonates with risk factors even small amounts of bacteria and diagnosis of 21 Egyptian Journal of Medical Microbiology, January 2009 Vol. 18, No. 1 sepsis.(6,7) prefilled with blood from infants at their bedsides. Between 0.5 and 1.0 ml of whole The aim of our study has been to determine blood was added per blood culture bottle. The the potential of a broad diagnostic approach bottles were incubated immediately upon receipt based on 16S rRNA gene amplification and in the microbiology laboratory in accordance sequencing for rapid detection and identification with the manufacturer’s recommendations. of pathogens causing neonatal bacteremia. Positive samples were processed for SUBJECTS AND METHODS identification of microorganisms as follow: This study was carried out in Pediateric and Subculture on blood, chocolate and Clinical pathology departments, Benha MacConkey agars. University Hospital and the NICU department Benha Specialized Children Hospital during the identification of the growing colonies by( 9, 10, 11): period from January 2007 to June 2008. • Colony morphology History taking and inclusion criteria: Infants • Gram staining admitted to the NICU for sepsis evaluation were included in the study if the following criteria - For Gram positive bacteria Catalase test, were fulfilled:- culture on mannitol salt agar, Coagulase test (slide and tube tests), Deoxyribonuclease • The presence of one major or two minor risk (DNase) test, Novobiocin susceptibility test factors ( major risk factors were i- premature and CAMP test were done. rupture of membranes PROM >24h , ii premature onset of labour before 37 weeks , - For Gram negative bacteria both iii chorioamnionitis , iv- intrapartum conventional biochemical reactions {Triple maternal fever >38 ◦C – Minor risk factors sugar,lysine iron agar, citrate utilization, were i PROM > 12< 24 h ii- low birthweight urease production, sulphide, indole, motility <1500g , iii- intrapartum maternal fever and oxidase tests (OXOID)} and Microbact >37.5 < 38 ◦C , iv- twin gestation , v- test were used. maternal apgar score at 1 min < 5. Microbact (12A) test (OXOID): The Microbact • Sings of sepsis including, lethargy, (12 A) gram negative system is a standardized irritability, apnea, cyanosis respiratory micro-substrate system designed to simulate distress and poor capillary refill. conventional biochemical substrates used for identification of enterobacteriaceae and common • Positive CRP (> 6 mg/L). miscellaneous gram negative bacilli (oxidase • Immature / total (I / T) ratio ≥ 0.2 (1). negative, nitrate positive, glucose fermenter comprising 15 genera). Organism identification Fifty neonates were included in our study and is based on PH changes and substrate utilization. were subjected to the following: Procedure: was done according to the Blood sampling: Paired blood samples were manufacturer’s instructions. obtained from two different peripheral veins for paired blood cultures, and for PCR . Blood Interpretation: An octal coding system was sample for PCR ( 0.5 ml) was placed in a sterile adapted for Microbact, each group of 3 reactions vacutainer containing EDTA and stored at -70ºC produce a single digit of the code using the till processed. results obtained. The sum of these indices in each group of 3 reactions form the code number Methods: .This code was entered into the Microbact Blood culture processing : Using an automated computer aided identification package for the continuous monitoring blood culture system, identification choices. The percentage figure BACTEC 9050 (Becton Dickinson, Sparks, MD) shown against the organism name is the which use a fluorescent sensor for detecting percentage share of the possibility for that microorganisms and relies primarily on the organism as apart of the total probabilities for all detection of CO2 produced by actively choices. (8) metabolizing microorganisms. The pediatric - Polymerase Chain Reaction: The nucleic acid sample sized, blood culture bottles (Peds Plus, was extracted from the whole EDTA blood and Becton Dickinson ) were sent from the NICU 22 Egyptian Journal of Medical Microbiology, January 2009 Vol. 18, No. 1 each sample was subjected to consensus primer 890 bp and the samples were compared with the mediated PCR method. The primers supplied by controls. (Chem genes,U.S.A), were complementary to DNA sequencing: Using ALFexpress AutoRead 16S r RNA gene sequences ,at base-pair sequencing kits and ALFexpress II DNA positions 459 and 1349 ,respectively. Automated DNA sequencer (Amersham- U 515F ¯5 TGCCAGCAGCCGCGGTAAT ¯3 pharmacia biotech-Amersham, United Kingdom) EU 202950R ¯5 GGGCGGCGTGTACAAGGC -3 which is a fully automated and highly accurate system for DNA analysis .Its sophisticated Nucleic acid extraction: Nucleic acid was detection system relies on laser-induced