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A high leukocyte count and administration of hydrocortisone hamper PCR-based diagnostics for bloodstream infections

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Abstract

Bloodstream infections (BSIs) require an accurate and fast identification of causative pathogens. Molecular diagnostics, in particular polymerase chain reaction (PCR)-based approaches for BSI diagnostics directly from whole blood, suffer from limitations such as inhibition leading to invalid results. In this retrospective study, we analyzed 23 parameters for their potential interference with LightCycler SeptiFast PCR tests (n = 2167) routinely performed at our institution. The overall inhibition rate was 9.1%. Test date, type of ward, procalcitonin levels, high leukocyte counts, and absolute neutrophil count were significantly associated with inhibition. For a subset (n = 448), cut-off values for leukocyte counts of < 5700 cells/μL and ≥ 26,900 cells/μL were significantly associated with a low (5%) and high (67%) inhibition risk. For patients with a moderate to high leukocyte count (5700–26,900 cells/μL), the additional administration of hydrocortisone significantly increased the inhibition risk. Furthermore, freezing of blood samples prior to DNA extraction and SF testing appeared to neutralize inhibitory factors. It remains to be investigated whether other molecular diagnostic tests are susceptible to similar inhibiting parameters.

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Data availability

Data that is supporting our results is available in the electronic supplementary material. The complete data set for this study is available from the corresponding author [DOH] upon reasonable request.

References

  1. Martin GS, Mannino DM, Eaton S, Moss M (2003) The epidemiology of Sepsis in the United States from 1979 through 2000. N Engl J Med 348:1546–1554. https://doi.org/10.1056/NEJMoa022139

    Article  PubMed  Google Scholar 

  2. Goto M, Al-Hasan MN (2013) Overall burden of bloodstream infection and nosocomial bloodstream infection in North America and Europe. Clin Microbiol Infect 19:501–509. https://doi.org/10.1128/JCM.39.2.485-493.2001691.12195

    Article  CAS  PubMed  Google Scholar 

  3. Kumar A, Roberts D, Wood KE, Light B, Parrillo JE, Sharma S et al (2006) Duration of hypotension before initiation of effective antimicrobial therapy is the critical determinant of survival in human septic shock. Crit Care Med 34:1589–1596. https://doi.org/10.1097/01.CCM.0000217961.75225.E9

    Article  PubMed  Google Scholar 

  4. Korber F, Zeller I, Grünstäudl M, Willinger B, Apfalter P, Hirschl AM et al (2017) SeptiFast versus blood culture in clinical routine – a report on 3 years experience. Wien Klin Wochenschr 129:427–434. https://doi.org/10.1007/s00508-017-1181-3

    Article  PubMed  PubMed Central  Google Scholar 

  5. Lamy B, Dargère S, Arendrup MC, Parienti J-J, Tattevin P (2016) How to optimize the use of blood cultures for the diagnosis of bloodstream infections? A state-of-the art. Front Microbiol 7:697. https://doi.org/10.3389/fmicb.2016.00697

    Article  PubMed  PubMed Central  Google Scholar 

  6. Fenollar F, Raoult D (2007) Molecular diagnosis of bloodstream infections caused by non-cultivable bacteria. Int J Antimicrob Agents 30:7–15. https://doi.org/10.1016/j.ijantimicag.2007.06.024

    Article  CAS  Google Scholar 

  7. Lamas CC (2003) Blood culture negative endocarditis: analysis of 63 cases presenting over 25 years. Heart 89:258–262. https://doi.org/10.1136/heart.89.3.258

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Stevenson M, Pandor A, Martyn-St James M, Rafia R, Uttley L, Stevens J, et al. (2016) Sepsis: the LightCycler SeptiFast Test MGRADE®, SepsiTest™ and IRIDICA BAC BSI assay for rapidly identifying bloodstream bacteria and fungi – a systematic review and economic evaluation Health Technol Assess (Rockv) 20:1–246. https://doi.org/10.3310/hta20460

  9. Dark P, Blackwood B, Gates S, McAuley D, Perkins GD, McMullan R et al (2015) Accuracy of LightCycler® SeptiFast for the detection and identification of pathogens in the blood of patients with suspected sepsis: a systematic review and meta-analysis. Intensive Care Med 41:21–33. https://doi.org/10.1007/s00134-014-3553-8

    Article  CAS  PubMed  Google Scholar 

  10. Pilecky M, Schildberger A, Knabl L, Orth-Höller D, Weber V (2019) Influence of antibiotic treatment on the detection of S. aureus in whole blood following pathogen enrichment. BMC Microbiol 19:180. https://doi.org/10.1186/s12866-019-1559-7

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Grif K, Fille M, Würzner R, Weiss G, Lorenz I, Gruber G et al (2012) Rapid detection of bloodstream pathogens by real-time PCR in patients with sepsis. Wien Klin Wochenschr 124:266–270. https://doi.org/10.1007/s00508-012-0159-4

    Article  CAS  PubMed  Google Scholar 

  12. Elges S, Arnold R, Liesenfeld O, Kofla G, Mikolajewska A, Schwartz S et al (2017) Prospective evaluation of the SeptiFAST multiplex real-time PCR assay for surveillance and diagnosis of infections in haematological patients after allogeneic stem cell transplantation compared to routine microbiological assays and an in-house real-time P. Mycoses 60:781–788. https://doi.org/10.1111/myc.12662

    Article  CAS  PubMed  Google Scholar 

  13. Ratzinger F, Tsirkinidou I, Haslacher H, Perkmann T, Schmetterer KG, Mitteregger D et al (2016) Evaluation of the SeptiFast MGRADE test on standard care wards—a cohort study. PLoS One 11:e0151108. https://doi.org/10.1371/journal.pone.0151108

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. van de Groep K, Bos MP, Savelkoul PHM, Rubenjan A, Gazenbeek C, Melchers WJG et al (2018) Development and first evaluation of a novel multiplex real-time PCR on whole blood samples for rapid pathogen identification in critically ill patients with sepsis. Eur J Clin Microbiol Infect Dis 37:1333–1344. https://doi.org/10.1007/s10096-018-3255-1

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Opota O, Jaton K, Greub G (2015) Microbial diagnosis of bloodstream infection: towards molecular diagnosis directly from blood. Clin Microbiol Infect 21:323–331. https://doi.org/10.1016/j.cmi.2015.02.005

    Article  CAS  PubMed  Google Scholar 

  16. Schrader C, Schielke A, Ellerbroek L, Johne R (2012) PCR inhibitors - occurrence, properties and removal. J Appl Microbiol 113:1014–1026. https://doi.org/10.1111/j.1365-2672.2012.05384.x

    Article  CAS  PubMed  Google Scholar 

  17. Lehmann LE, Hunfeld K-P, Emrich T, Haberhausen G, Wissing H, Hoeft A et al (2008) A multiplex real-time PCR assay for rapid detection and differentiation of 25 bacterial and fungal pathogens from whole blood samples. Med Microbiol Immunol 197:313–324. https://doi.org/10.1007/s00430-007-0063-0

    Article  CAS  PubMed  Google Scholar 

  18. Makristathis A, Harrison N, Ratzinger F, Kussmann M, Selitsch B, Forstner C et al (2018) Substantial diagnostic impact of blood culture independent molecular methods in bloodstream infections: superior performance of PCR/ESI-MS. Sci Rep 8:16024. https://doi.org/10.1038/s41598-018-34298-7

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Burkardt H-J Standardization and Quality Control of PCR Analyses. Clin Chem Lab Med 38:87–91. https://doi.org/10.1515/CCLM.2000.014

  20. Westh H, Lisby G, Breysse F, Böddinghaus B, Chomarat M, Gant V et al (2009) Multiplex real-time PCR and blood culture for identification of bloodstream pathogens in patients with suspected sepsis. Clin Microbiol Infect 15:544–551. https://doi.org/10.1111/j.1469-0691.2009.02736.x

    Article  CAS  PubMed  Google Scholar 

  21. Regueiro BJ, Varela-Ledo E, Martinez-Lamas L, Rodriguez-Calvino J, Aguilera A, Santos A et al (2010) Automated extraction improves multiplex molecular detection of infection in septic patients. PLoS One 5:e13387. https://doi.org/10.1371/journal.pone.0013387

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Benjamini Y, Hochberg Y (1995) Controlling the false discovery rate: a practical and powerful approach to multiple testing. J R Stat Soc Ser B 57:289–300

    Google Scholar 

  23. Quinlan R (1993) C4.5: programs for machine learning. San Francisco, CA, United States: Morgan Kaufmann Publishers Inc.; 1993

  24. Molina JM, Córdoba J, Ramírez P, Gobernado M (2008) Detección automática de bacterias y hongos en sangre. Enferm Infecc Microbiol Clin 26:75–80. https://doi.org/10.1016/S0213-005X(08)76544-3

    Article  PubMed  PubMed Central  Google Scholar 

  25. Al-Soud WA, Radstrom P (2001) Purification and characterization of PCR-inhibitory components in blood cells. J Clin Microbiol.:485–93. https://doi.org/10.1128/JCM.39.2.485-493.2001

  26. Keh D, Boehnke T, Weber-Cartens S, Schulz C, Ahlers O, Bercker S et al (2003) Immunologic and hemodynamic effects of “low-dose” hydrocortisone in septic shock. Am J Respir Crit Care Med 167:512–520. https://doi.org/10.1164/rccm.200205-446OC

    Article  PubMed  Google Scholar 

  27. Olnes MJ, Kotliarov Y, Biancotto A, Cheung F, Chen J, Shi R et al (2016) Effects of systemically administered hydrocortisone on the human immunome. Sci Rep 6:23002. https://doi.org/10.1038/srep23002

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Maxime V, Fitting C, Annane D, Cavaillon J-M (2005) Corticoids normalize leukocyte production of macrophage migration inhibitory factor in septic shock. J Infect Dis 191:138–144. https://doi.org/10.1086/426401

    Article  CAS  PubMed  Google Scholar 

  29. Laviolle B, Annane D, Fougerou C, Bellissant E (2012) Gluco- and mineralocorticoid biological effects of a 7-day treatment with low doses of hydrocortisone and fludrocortisone in septic shock. Intensive Care Med 38:1306–1314. https://doi.org/10.1007/s00134-012-2585-1

    Article  CAS  PubMed  Google Scholar 

  30. Katsenos CS, Antonopoulou AN, Apostolidou EN, Ioakeimidou A, Kalpakou GT, Papanikolaou MN et al (2014) Early administration of hydrocortisone replacement after the advent of septic shock. Crit Care Med 42:1651–1657. https://doi.org/10.1097/CCM.0000000000000318

    Article  CAS  PubMed  Google Scholar 

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Funding

The current investigation was part of the Bridge project “Advanced Pathogen Detection in Blood Stream Infection” (Project Number: 871243) funded by the Austrian Research Promotion Agency (FFG).

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Author notes

  1. Andreas E. Posch and Dorothea Orth-Höller contributed equally to this work.

Authors and Affiliations

  1. Institute of Hygiene and Medical Microbiology, Medical University of Innsbruck, Schöpfstrasse 41, 6020, Innsbruck, Austria

    Silke Huber, Stefan Fuchs, Wilfried Posch, Ludwig Knabl, Reinhard Würzner & Dorothea Orth-Höller

  2. Ares Genetics GmbH, Karl-Farkas-Gasse 18, 1030, Vienna, Austria

    Johannes Weinberger & Andreas E. Posch

  3. Center for Biomedical Technology, Department for Biomedical Research, Danube University Krems, Krems, Austria

    Matthias Pilecky, Anita Schildberger & Viktoria Weber

  4. Department of General and Surgical Critical Care Medicine, Medical University of Innsbruck, Innsbruck, Austria

    Ingo Lorenz

  5. Christian Doppler Laboratory for Innovative Therapy Approaches in Sepsis, Danube University Krems, Krems, Austria

    Viktoria Weber

Authors
  1. Silke Huber
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  2. Johannes Weinberger
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Contributions

Silke Huber: methodology, validation, formal analysis, investigation, data curation, writing original draft, visualization, interpretation of data, writing: reviewing and editing

Johannes Weinberger: methodology, validation, formal analysis, data curation, visualization, interpretation of data, writing: reviewing and editing

Matthias Pilecky: conceptualization, funding acquisition, writing: reviewing and editing

Ingo Lorenz: conceptualization, writing: reviewing and editing

Anita Schildberger: project administration, funding acquisition, writing: reviewing and editing

Viktoria Weber: conceptualization, funding acquisition, writing: reviewing and editing

Stefan Fuchs: visualization, writing: reviewing and editing

Wilfried Posch: writing: reviewing and editing

Ludwig Knabl: writing: reviewing and editing

Reinhard Würzner: writing: reviewing and editing

Dorothea Orth-Höller: conceptualization, methodology, supervision, funding acquisition, writing–original draft, interpretation of data, writing: reviewing and editing

Andreas E. Posch: methodology, validation, formal analysis, data curation, visualization, interpretation of data, writing: reviewing and editing

Corresponding authors

Correspondence to Andreas E. Posch or Dorothea Orth-Höller.

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Conflict of interest

The authors have no conflict of interest to declare.

Ethical approval

This study was conducted retrospectively from data obtained for clinical purposes. In advance, it was clarified in an extensive consultation with the ethics committee of the Medical University of Innsbruck, Austria, that our study did not need ethical approval.

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Huber, S., Weinberger, J., Pilecky, M. et al. A high leukocyte count and administration of hydrocortisone hamper PCR-based diagnostics for bloodstream infections. Eur J Clin Microbiol Infect Dis 40, 1441–1449 (2021). https://doi.org/10.1007/s10096-020-04126-w

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  • DOI: https://doi.org/10.1007/s10096-020-04126-w

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