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Diagnosis of neonatal sepsis by broad range 16S real-time PCR
Örebro University, School of Health and Medical Sciences.
Clinical Research Centre, Örebro University Hospital.
Women’s and Children’s Health, Uppsala University, Uppsala.
Örebro University, School of Health and Medical Sciences.
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(English)Manuscript (preprint) (Other academic)
Abstract [en]

Context: The standard diagnostic test (blood culture) for suspected neonatal sepsis has limitations in sensitivity, specificity and 16 S polymerase chain reaction has been suggested as a new diagnostic tool for neonatal sepsis.Objective:To develop and evaluate a new real-time polymerase chain reaction (PCR) method for detection of bacterial DNA in blood samples collected from infants with suspected neonatal sepsis. Primary outcome was the sensitivity, specificity, and positive and negative predictive value of the 16 S real-time PCR assay as compared with blood culture.Design: Prospective study of diagnostic test.Setting: Two Swedish Level III neonatal intensive care units.Patients: 317 infants < 3 months of age subjected to blood culture as decided by the attending neonatologist.Main outcome measures: Sensitivity and specificity of the studied PCR method was the main outcome, with simultaneously collected blood culture acting as the gold standard. Detailed case studies was performed in all cases with conflicting results, to verify if PCR could detect pathogens in culture negative sepsis.Results: The material comprised 368 samples from 317 infants. When compared with blood culture, the assay yielded a sensitivity of 79%, a specificity of 90%, a positive predictive value of 59%, and a negative predictive value of 96%. PCR detected 29/35 (83%) of the Coagulase negative staphylococci samples and 15/21 (71%) of the remaining cultures. In five samples, PCR (but not blood culture) could detect a pathogen that was present in blood culture more than 24 hours earlier.Conclusions: This study presents an evaluation of a new real-time PCR technique that can detect culture-positive sepsis, and suggests that PCR has the potential to detect bacteria in culture-negative samples even if collected after the initiation of intravenous antibiotics.

National Category
Medical and Health Sciences Pediatrics
Research subject
Pediatrics; Medicine
Identifiers
URN: urn:nbn:se:oru:diva-12732OAI: oai:DiVA.org:oru-12732DiVA, id: diva2:379332
Note

Andreas Ohlins is also affiliated with Department of Pediatrics, Örebro University Hospital

Available from: 2010-12-17 Created: 2010-12-17 Last updated: 2017-10-17Bibliographically approved
In thesis
1. Aspects on early diagnosis of neonatal sepsis
Open this publication in new window or tab >>Aspects on early diagnosis of neonatal sepsis
2010 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

This thesis presents four studies, all designed to improve the problematic diagnostic situation concerning infants with suspected sepsis. Study I included 401 neonates with suspected sepsis. Nine signs of sepsis and C-reactive protein were prospectively recorded and logistic regression was used to assess associations between these signs and a subsequently confirmed diagnosis of sepsis. C-reactive protein and five of the clinical signs were statistically significantly associated with a positive bloodculture. When the material was stratified by gestational age, differences between premature and full term infants were detected.Studies II and III were prospective studies that used samples collected from neonates with suspected sepsis to evaluate a novel real-timepolymerase chain reaction (PCR) method. The results where compared with simultaneously collected blood cultures. Study II used plasma samples and resulted in a sensitivity of 42% and specificity of 95%. In study III, the protocol was improved and adapted to whole blood samples which resulted in a sensitivity of 79% and specificity of 90%. Both protocols included species-specific probes and study III indicated that PCR has the potential to detect bacteria in culture-negative sepsis.Staphylococcus epidermidis is the most common pathogen in neonatal sepsis, but there is still a lack of typing methods suitable for large materials of S. epidermidis. In Study IV we therefore evaluated a new S. epidermidisgenotyping method based on PCR for the repeat regions of four genes thatencode for cell wall anchoring proteins. The method was applied to 49well-defined neonatal blood isolates of S. epidermidis. The combination ofsdrF and aap seemed to be optimal, resulting in a diversity index of 0.92.Conclusions

• Bradycardia, apnoea, low blood pressure, feeding intolerance and distended abdomen are obvious early signs of neonatal sepsis. Premature and full-term infants differ in terms of the signs they display in neonatal sepsis.

• Blood is superior to plasma for developing PCR methods for bacterial DNA detection. The PCR method described in study III can detect neonatal bacteraemia, but it can be further improved before it is used in routine care.

• There has been a lack of useful typing methods for S. epidermidis.We can now present PCR of the genes for the cell wall anchoring proteins sdrF and aap as a novel and feasible approach when there is a need to type a large number of S. epidermidis isolates.

Place, publisher, year, edition, pages
Örebro: Örebro university, 2010. p. 77
Series
Örebro Studies in Medicine, ISSN 1652-4063 ; 49
National Category
Medical and Health Sciences Pediatrics
Research subject
Pediatrics
Identifiers
urn:nbn:se:oru:diva-11928 (URN)978-91-7668-770-3 (ISBN)
Public defence
2010-12-10, Wilandersalen, Universitetssjukhuset Örebro, Fakultetsgatan 1, Örebro, 09:00
Opponent
Supervisors
Available from: 2010-09-27 Created: 2010-09-27 Last updated: 2017-10-17Bibliographically approved

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Ohlin, AndreasSchollin, Jens

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