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Real-time PCR of the 16S-rRNA gene in the diagnosis of neonatal bacteraemia
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2008 (English)In: Acta Paediatrica, ISSN 0803-5253, E-ISSN 1651-2227, Vol. 97, no 10, p. 1376-1380Article in journal (Refereed) Published
Abstract [en]

OBJECTIVE: To evaluate a real-time PCR assay for the diagnosis of neonatal bacteraemia. PATIENTS AND METHODS: Two hundred ninety-five plasma samples from 288 newborns with suspected neonatal sepsis were collected prospectively for the purpose of polymerase chain reaction (PCR)-based bacterial detection. A real-time PCR targeting the bacterial gene for 16S-rRNA gene combined with four specific probes designed to detect Gram-negative bacteria, Staphylococcus aureus and coagulase-negative staphylococci (CoNS) was developed. All samples positive in the universal PCR were further sequenced for bacterial identification. RESULTS: When applied to a material from 50 patients with positive blood culture and 245 patients with negative blood culture, the universal PCR showed a sensitivity of 42% (28-57), a specificity of 95% (92-97), a positive predictive value of 64% (45-80), and a negative predictive value of 89% (84-92) (95% confidence intervals in brackets). CONCLUSION: A new real-time PCR technique was for the first time applied to a well-defined prospectively and consecutively enrolled material of newborns with suspected sepsis, combining the benefits of real-time PCR with specific probes and sequencing. The method managed to detect bacteraemia with high specificity even though the sensitivity was low. Factors causing the low sensitivity are identified and further strategies to develop the method are described.

Place, publisher, year, edition, pages
Oslo: Taylor & Francis , 2008. Vol. 97, no 10, p. 1376-1380
National Category
Pediatrics Medical and Health Sciences Clinical Medicine
Research subject
Pediatrics; Medicine
Identifiers
URN: urn:nbn:se:oru:diva-5149DOI: 10.1111/j.1651-2227.2008.00924.xOAI: oai:DiVA.org:oru-5149DiVA, id: diva2:158010
Available from: 2009-01-29 Created: 2009-01-29 Last updated: 2017-12-14Bibliographically 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
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Supervisors
Available from: 2010-09-27 Created: 2010-09-27 Last updated: 2017-10-17Bibliographically approved

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Publisher's full texthttp://www.ncbi.nlm.nih.gov/pubmed/18624992?ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_DefaultReportPanel.Pubmed_RVDocSum

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Björkqvist, MariaSchollin, Jens

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