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Aspects on early diagnosis of neonatal sepsis
Örebro University, School of Health and Medical Sciences. (andreas.ohlin@orebroll.se)
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. , 77 p.
Series
Örebro Studies in Medicine, ISSN 1652-4063 ; 49
National Category
Medical and Health Sciences Pediatrics
Research subject
Pediatrics
Identifiers
URN: urn:nbn:se:oru:diva-11928ISBN: 978-91-7668-770-3 (print)OAI: oai:DiVA.org:oru-11928DiVA: diva2:353420
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: 2011-04-21Bibliographically approved
List of papers
1. Clinical signs and CRP values associated with blood culture results in neonates evaluated for suspected sepsis
Open this publication in new window or tab >>Clinical signs and CRP values associated with blood culture results in neonates evaluated for suspected sepsis
2010 (English)In: Acta Paediatrica, ISSN 0803-5253, E-ISSN 1651-2227, Vol. 99, no 11, 1635-1640 p.Article in journal (Refereed) Published
Abstract [en]

Aim: To identify which clinical signs at presentation are most predictive of sepsis subsequently confirmed by blood culture and to investigate whether the predictive power of the clinical signs varies by gestational age.

Methods: Among 401 newborn infants <28 days of age with suspected sepsis, nine signs of sepsis and C-reactive protein (CRP) values were prospectively recorded. Logistic regression assessed the association of these signs and laboratory values with a subsequently confirmed diagnosis of sepsis by positive blood culture. The analysis was stratified by gestational age with mutual simultaneous adjustment for the signs and sex.

Results: Five of the nine clinical signs (feeding intolerance, distended abdomen, blood pressure, bradycardia and apnoea), along with CRP were statistically significantly associated with a positive blood culture. After simultaneous adjustment for all of the signs, apnoea, hypotension and CRP were independently predictive of positive blood culture. When the material was stratified by gestational age, differences in the association with positive blood culture were found for bradycardia, tachypnea and irritability/seizures.

Conclusion: In this selected population of infants with suspected sepsis, apnoea and hypotension are independently predictive of a confirmed diagnosis, while bradycardia is more predictive among preterm infants and tachypnea among term infants.

Place, publisher, year, edition, pages
Malden, USA: Wiley-Blackwell, 2010
Keyword
Diagnosis, logistic regression, neonatal sepsis, neonatology
National Category
Medical and Health Sciences
Research subject
Medicine
Identifiers
urn:nbn:se:oru:diva-12721 (URN)10.1111/j.1651-2227.2010.01913.x (DOI)000282641600008 ()20560896 (PubMedID)2-s2.0-78649728350 (Scopus ID)
Available from: 2010-12-15 Created: 2010-12-15 Last updated: 2017-02-13Bibliographically approved
2. Real-time PCR of the 16S-rRNA gene in the diagnosis of neonatal bacteraemia
Open this publication in new window or tab >>Real-time PCR of the 16S-rRNA gene in the diagnosis of neonatal bacteraemia
Show others...
2008 (English)In: Acta Paediatrica, ISSN 0803-5253, E-ISSN 1651-2227, Vol. 97, no 10, 1376-1380 p.Article 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
National Category
Pediatrics Medical and Health Sciences Clinical Medicine
Research subject
Pediatrics; Medicine
Identifiers
urn:nbn:se:oru:diva-5149 (URN)10.1111/j.1651-2227.2008.00924.x (DOI)
Available from: 2009-01-29 Created: 2009-01-29 Last updated: 2011-05-11Bibliographically approved
3. Diagnosis of neonatal sepsis by broad range 16S real-time PCR
Open this publication in new window or tab >>Diagnosis of neonatal sepsis by broad range 16S real-time PCR
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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:nbn:se:oru:diva-12732 (URN)
Note

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

Available from: 2010-12-17 Created: 2010-12-17 Last updated: 2016-12-12Bibliographically approved
4. Rapid typing of neonatal Staphylococcus epidermidis isolates using polymerase chain reaction for repeat regions in surface protein genes
Open this publication in new window or tab >>Rapid typing of neonatal Staphylococcus epidermidis isolates using polymerase chain reaction for repeat regions in surface protein genes
Show others...
2010 (English)In: European Journal of Clinical Microbiology and Infectious Diseases, ISSN 0934-9723, E-ISSN 1435-4373, Vol. 29, no 6, 699-704 p.Article in journal (Refereed) Published
Abstract [en]

Staphylococcus epidermidis is a significant pathogen in neonatal sepsis and other nosocomial infections. For further investigations of the colonisation patterns and invasive pathways, typing methods that are applicable on large populations of bacterial isolates are warranted. In the present study, a genotyping method based on polymerase chain reaction (PCR) for the repeat regions of four genes (sdrG, sdrF, aap and sesE) that encode for bacterial surface proteins was developed and applied to a sample of well-characterised neonatal blood isolates of S. epidermidis (n = 49). The PCR products were visualised on agarose gel (sdrG, sdrF and sesE) or by fragment analysis (aap). The discriminatory index (D-index) for genotyping of the different genes was compared to genotyping by pulsed-field gel electrophoresis (PFGE). The highest D-index for the PCR-based typing methods was found for the combination of sdrF, sdrG and aap (D-index 0.94), whereas the optimal two-gene combination (sdrF and aap) resulted in a D-index of 0.92. We conclude that the described method can be used for the genotyping of large populations of S. epidermidis isolates with a sufficient discriminatory capacity, and we suggest that the combination of sdrF and aap is the most suitable to use.

Place, publisher, year, edition, pages
New York, USA: Springer, 2010
National Category
Medical and Health Sciences Infectious Medicine
Research subject
Medicine
Identifiers
urn:nbn:se:oru:diva-12722 (URN)10.1007/s10096-010-0917-z (DOI)000277711900011 ()20383779 (PubMedID)2-s2.0-77952876158 (Scopus ID)
Available from: 2010-12-15 Created: 2010-12-15 Last updated: 2017-03-24Bibliographically approved

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