Multiplex qPCR for the early detection of sepsis pathogens and its impact on antimicrobial therapy in critically ill patients
-
Pooja Pandey
,
Raunak Bir
,
Kuhu Chatterjee
,
Nikhil Verma
,
Yasha Mukim
,
Komal Grover
,
Shefali Sharma
,
Priya Sehgal
,
Swati Chamoli
,
Rajiv M Gupta
,
Anil K Pandey
Abstract
Background and Objectives: Sepsis is a life threatening condition caused by a dysregulated host response to infection and is associated with high morbidity and mortality worldwide. Early bacterial detection and therapy with antibiotics improve outcomes. We compared multiplex quantitative PCR (qPCR) to traditional blood culture for early pathogen detection in critically ill patients with suspected sepsis.
Materials and Methods: This prospective observational study included 200 critically ill ICU patients with suspected sepsis. Multiplex qPCR using the TRUPCR® Sepsis Panel was compared with conventional blood culture for pathogen detection. To assess sensitivity, specificity, PPV, and NPV, blood culture was used as the reference standard. Mortality, ICU stay, and antibiotic therapy time were studied. Multivariable logistic regression was adjusted for baseline severity (SOFA, APACHE II), septic shock, and antibiotic exposure.
Results: Multiplex qPCR significantly reduced the time to initiation of appropriate antibiotic therapy (5.2 vs 8.3 hours, p<0.001). The assay demonstrated higher sensitivity compared with blood culture for pathogen detection. qPCR positivity was associated with shorter ICU stay and lower mortality; however, these associations were interpreted after adjustment for baseline illness severity. Escherichia coli, Klebsiella pneumoniae, and Staphylococcus aureus were the most frequently detected pathogens, and several antimicrobial resistance genes including blaCTX-M, blaNDM, and mecA were identified.
Conclusion: Multiplex qPCR can detect infections early and optimize antimicrobials for sepsis. These findings should be cautiously evaluated and corroborated in larger multicentre trials due to reduced specificity and observational nature.
1. Singer M, Deutschman CS, Seymour CW, Shankar-Hari M, Annane D, Bauer M, et al. The Third International Consensus Definitions for Sepsis and Septic Shock (Sepsis-3). JAMA 2016; 315: 801-810.
2. Kumar A, Roberts D, Wood KE, Light B, Parrillo JE, Sharma S, et al. Duration of hypotension before initiation of effective antimicrobial therapy is the critical determinant of survival in human septic shock. Crit Care Med 2006; 34: 1589-1596.
3. Lamy B, Dargère S, Arendrup MC, Parienti JJ, Tattevin P. How to Optimize the Use of Blood Cultures for the Diagnosis of Bloodstream Infections? A State-of-the Art. Front Microbiol 2016; 7: 697.
4. Dark P, Blackwood B, Gates S, McAuley D, Perkins GD, McMullan R, et al. 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 2015; 41: 21-33.
5. Blaschke AJ, Heyrend C, Byington CL, Fisher MA, Barker E, Garrone NF, et al. Rapid identification of pathogens from positive blood cultures by multiplex polymerase chain reaction using the FilmArray system. Diagn Microbiol Infect Dis 2012; 74: 349-355.
6. Mylonakis E, Clancy CJ, Ostrosky-Zeichner L, Garey KW, Alangaden GJ, Vazquez JA, et al. T2 magnetic resonance assay for the rapid diagnosis of candidemia in whole blood: a clinical trial. Clin Infect Dis 2015; 60: 892-899.
7. Timbrook TT, Morton JB, McConeghy KW, Caffrey AR, Mylonakis E, LaPlante KL. The Effect of Molecular Rapid Diagnostic Testing on Clinical Outcomes in Bloodstream Infections: A Systematic Review and Meta-analysis. Clin Infect Dis 2017; 64: 15-23.
8. Sambrook J, Russell DW )2001). Molecular Cloning: A Laboratory Manual. 3rd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.
9. Rio DC, Ares M Jr, Hannon GJ, Nilsen TW. Purification of RNA using TRIzol (TRI reagent). Cold Spring Harb Protoc 2010; 2010(6): pdb.prot5439.
10. Pourajam S, Kalantari E, Talebzadeh H, Mellali H, Sami R, Soltaninejad F, et al. Secondary Bacterial Infection and Clinical Characteristics in Patients With COVID-19 Admitted to Two Intensive Care Units of an Academic Hospital in Iran During the First Wave of the Pandemic. Front Cell Infect Microbiol 2022; 12: 784130.
11. Clinical and Laboratory Standards Institute (CLSI). Performance Standards for Antimicrobial Susceptibility Testing. 34th ed. CLSI supplement M100. Wayne, PA. Clinical and Laboratory Standards Institute. 2024.
12. European Committee on Antimicrobial Susceptibility Testing (EUCAST). EUCAST setting breakpoints [Internet]. Accessed on: 26 May 2026. Available from: EUCAST https://www.eucast.org/clinical_breakpoints
13. Martin GS, Mannino DM, Eaton S, Moss M. The epidemiology of sepsis in the United States from 1979 through 2000. N Engl J Med 2003; 348: 1546-1554.
14. Mikkelsen ME, Miltiades AN, Gaieski DF, Goyal M, Fuchs BD, Shah CV, et al. Serum lactate is associated with mortality in severe sepsis independent of organ failure and shock. Crit Care Med 2009; 37: 1670-1677.
15. Shankar-Hari M, Phillips GS, Levy ML, Seymour CW, Liu VX, Deutschman CS, et al. Developing a New Definition and Assessing New Clinical Criteria for Septic Shock: For the Third International Consensus Definitions for Sepsis and Septic Shock (Sepsis-3). JAMA 2016; 315: 775-787.
16. Eid R, Zahar JR, Ait Ali C, Mizrahi A, Ibrahim R, Banh E, et al. Bloodstream Infections: Comparison of Diagnostic Methods and Therapeutic Consequences between a Hospital in a Resource-Limited Setting and Two French Hospitals. Microorganisms 2023; 11: 2136.
17. Opota O, Croxatto A, Prod'hom G, Greub G. Blood culture-based diagnosis of bacteraemia: state of the art. Clin Microbiol Infect 2015; 21: 313-322.
18. Hu B, Tao Y, Shao Z, Zheng Y, Zhang R, Yang X, et al. A Comparison of Blood Pathogen Detection Among Droplet Digital PCR, Metagenomic Next-Generation Sequencing, and Blood Culture in Critically Ill Patients With Suspected Bloodstream Infections. Front Microbiol 2021; 12: 641202.
19. Dinç F, Akalin H, Özakin C, Sinirtaş M, Kebabçi N, Işçimen R, et al. Comparison of blood culture and multiplex real-time PCR for the diagnosis of nosocomial sepsis. Minerva Anestesiol 2016; 82: 301-309.
20. Abelenda-Alonso G, Calatayud L, Rombauts A, Meije Y, Oriol I, Sopena N, et al. Multiplex real-time PCR in non-invasive respiratory samples to reduce antibiotic use in community-acquired pneumonia: a randomised trial. Nat Commun 2024; 15: 7098.
21. Tumbarello M, Viale P, Viscoli C, Trecarichi EM, Tumietto F, Marchese A, et al. Predictors of mortality in bloodstream infections caused by Klebsiella pneumoniae carbapenemase-producing K. pneumoniae: importance of combination therapy. Clin Infect Dis 2012; 55: 943-950.
22. Roberts RR, Hota B, Ahmad I, Scott RD 2nd, Foster SD, Abbasi F, et al. Hospital and societal costs of antimicrobial-resistant infections in a Chicago teaching hospital: implications for antibiotic stewardship. Clin Infect Dis 2009; 49: 1175-1184.
23. Klevens RM, Morrison MA, Nadle J, Petit S, Gershman K, Ray S, et al. Invasive methicillin-resistant Staphylococcus aureus infections in the United States. JAMA 2007; 298: 1763-1771.
24. Mathers AJ, Peirano G, Pitout JD. The role of epidemic resistance plasmids and international high-risk clones in the spread of multidrug-resistant Enterobacteriaceae. Clin Microbiol Rev 2015; 28: 565-591.
25. Nordmann P, Naas T, Poirel L. Global spread of Carbapenemase-producing Enterobacteriaceae. Emerg Infect Dis 2011; 17: 1791-1798.
26. Rice LB. Federal funding for the study of antimicrobial resistance in nosocomial pathogens: no ESKAPE. J Infect Dis 2008; 197: 1079-1081.
27. Mulvey MR, Simor AE. Antimicrobial resistance in hospitals: how concerned should we be? CMAJ 2009; 180: 408-415.
28. Lockhart SR, Etienne KA, Vallabhaneni S, Farooqi J, Chowdhary A, Govender NP, et al. Simultaneous emergence of multidrug-resistant Candida auris on 3 continents confirmed by whole-genome sequencing and epidemiological analyses. Clin Infect Dis 2017; 64: 134-140.
29. Chowdhary A, Sharma C, Meis JF. Candida auris: A rapidly emerging cause of hospital-acquired multidrug-resistant fungal infections globally. PLoS Pathog 2017; 13(5): e1006290.
30. Poole K. Efflux pumps as antimicrobial resistance mechanisms. Ann Med 2007; 39: 162-176.
31. Drawz SM, Bonomo RA. Three decades of β-lactamase inhibitors. Clin Microbiol Rev 2010; 23: 160-201. doi:10.1128/CMR.00037-09.
32. Zhang Y, Hu A, Andini N, Yang S. A 'culture' shift: Application of molecular techniques for diagnosing polymicrobial infections. Biotechnol Adv 2019; 37: 476-490.
33. Vincent JL, Brealey D, Libert N, Abidi NE, O'Dwyer M, Zacharowski K, et al. Rapid Diagnosis of Infection in the Critically Ill, a Multicenter Study of Molecular Detection in Bloodstream Infections, Pneumonia, and Sterile Site Infections. Crit Care Med 2015; 43: 2283-2291.
34. Lamy B, Sundqvist M, Idelevich EA; ESCMID Study Group for Bloodstream Infections, Endocarditis and Sepsis (ESGBIES). Bloodstream infections - Standard and progress in pathogen diagnostics. Clin Microbiol Infect 2020; 26: 142-150.
35. Perez KK, Olsen RJ, Musick WL, Cernoch PL, Davis JR, Peterson LE, et al. Integrating rapid diagnostics and antimicrobial stewardship improves outcomes in patients with antibiotic-resistant Gram-negative bacteremia. J Infect 2014; 69: 216-225.
36. Bauer KA, Perez KK, Forrest GN, Goff DA. Review of rapid diagnostic tests used by antimicrobial stewardship programs. Clin Infect Dis 2014; 59(Suppl 3): S134-45.
37. Smith RD, Zhan M, Zhang S, Leekha S, Harris A, Doi Y, et al. Comparison of three rapid diagnostic tests for bloodstream infections using Benefit-risk Evaluation Framework (BED-FRAME). J Clin Microbiol 2024; 62(1): e0109623.
2. Kumar A, Roberts D, Wood KE, Light B, Parrillo JE, Sharma S, et al. Duration of hypotension before initiation of effective antimicrobial therapy is the critical determinant of survival in human septic shock. Crit Care Med 2006; 34: 1589-1596.
3. Lamy B, Dargère S, Arendrup MC, Parienti JJ, Tattevin P. How to Optimize the Use of Blood Cultures for the Diagnosis of Bloodstream Infections? A State-of-the Art. Front Microbiol 2016; 7: 697.
4. Dark P, Blackwood B, Gates S, McAuley D, Perkins GD, McMullan R, et al. 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 2015; 41: 21-33.
5. Blaschke AJ, Heyrend C, Byington CL, Fisher MA, Barker E, Garrone NF, et al. Rapid identification of pathogens from positive blood cultures by multiplex polymerase chain reaction using the FilmArray system. Diagn Microbiol Infect Dis 2012; 74: 349-355.
6. Mylonakis E, Clancy CJ, Ostrosky-Zeichner L, Garey KW, Alangaden GJ, Vazquez JA, et al. T2 magnetic resonance assay for the rapid diagnosis of candidemia in whole blood: a clinical trial. Clin Infect Dis 2015; 60: 892-899.
7. Timbrook TT, Morton JB, McConeghy KW, Caffrey AR, Mylonakis E, LaPlante KL. The Effect of Molecular Rapid Diagnostic Testing on Clinical Outcomes in Bloodstream Infections: A Systematic Review and Meta-analysis. Clin Infect Dis 2017; 64: 15-23.
8. Sambrook J, Russell DW )2001). Molecular Cloning: A Laboratory Manual. 3rd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.
9. Rio DC, Ares M Jr, Hannon GJ, Nilsen TW. Purification of RNA using TRIzol (TRI reagent). Cold Spring Harb Protoc 2010; 2010(6): pdb.prot5439.
10. Pourajam S, Kalantari E, Talebzadeh H, Mellali H, Sami R, Soltaninejad F, et al. Secondary Bacterial Infection and Clinical Characteristics in Patients With COVID-19 Admitted to Two Intensive Care Units of an Academic Hospital in Iran During the First Wave of the Pandemic. Front Cell Infect Microbiol 2022; 12: 784130.
11. Clinical and Laboratory Standards Institute (CLSI). Performance Standards for Antimicrobial Susceptibility Testing. 34th ed. CLSI supplement M100. Wayne, PA. Clinical and Laboratory Standards Institute. 2024.
12. European Committee on Antimicrobial Susceptibility Testing (EUCAST). EUCAST setting breakpoints [Internet]. Accessed on: 26 May 2026. Available from: EUCAST https://www.eucast.org/clinical_breakpoints
13. Martin GS, Mannino DM, Eaton S, Moss M. The epidemiology of sepsis in the United States from 1979 through 2000. N Engl J Med 2003; 348: 1546-1554.
14. Mikkelsen ME, Miltiades AN, Gaieski DF, Goyal M, Fuchs BD, Shah CV, et al. Serum lactate is associated with mortality in severe sepsis independent of organ failure and shock. Crit Care Med 2009; 37: 1670-1677.
15. Shankar-Hari M, Phillips GS, Levy ML, Seymour CW, Liu VX, Deutschman CS, et al. Developing a New Definition and Assessing New Clinical Criteria for Septic Shock: For the Third International Consensus Definitions for Sepsis and Septic Shock (Sepsis-3). JAMA 2016; 315: 775-787.
16. Eid R, Zahar JR, Ait Ali C, Mizrahi A, Ibrahim R, Banh E, et al. Bloodstream Infections: Comparison of Diagnostic Methods and Therapeutic Consequences between a Hospital in a Resource-Limited Setting and Two French Hospitals. Microorganisms 2023; 11: 2136.
17. Opota O, Croxatto A, Prod'hom G, Greub G. Blood culture-based diagnosis of bacteraemia: state of the art. Clin Microbiol Infect 2015; 21: 313-322.
18. Hu B, Tao Y, Shao Z, Zheng Y, Zhang R, Yang X, et al. A Comparison of Blood Pathogen Detection Among Droplet Digital PCR, Metagenomic Next-Generation Sequencing, and Blood Culture in Critically Ill Patients With Suspected Bloodstream Infections. Front Microbiol 2021; 12: 641202.
19. Dinç F, Akalin H, Özakin C, Sinirtaş M, Kebabçi N, Işçimen R, et al. Comparison of blood culture and multiplex real-time PCR for the diagnosis of nosocomial sepsis. Minerva Anestesiol 2016; 82: 301-309.
20. Abelenda-Alonso G, Calatayud L, Rombauts A, Meije Y, Oriol I, Sopena N, et al. Multiplex real-time PCR in non-invasive respiratory samples to reduce antibiotic use in community-acquired pneumonia: a randomised trial. Nat Commun 2024; 15: 7098.
21. Tumbarello M, Viale P, Viscoli C, Trecarichi EM, Tumietto F, Marchese A, et al. Predictors of mortality in bloodstream infections caused by Klebsiella pneumoniae carbapenemase-producing K. pneumoniae: importance of combination therapy. Clin Infect Dis 2012; 55: 943-950.
22. Roberts RR, Hota B, Ahmad I, Scott RD 2nd, Foster SD, Abbasi F, et al. Hospital and societal costs of antimicrobial-resistant infections in a Chicago teaching hospital: implications for antibiotic stewardship. Clin Infect Dis 2009; 49: 1175-1184.
23. Klevens RM, Morrison MA, Nadle J, Petit S, Gershman K, Ray S, et al. Invasive methicillin-resistant Staphylococcus aureus infections in the United States. JAMA 2007; 298: 1763-1771.
24. Mathers AJ, Peirano G, Pitout JD. The role of epidemic resistance plasmids and international high-risk clones in the spread of multidrug-resistant Enterobacteriaceae. Clin Microbiol Rev 2015; 28: 565-591.
25. Nordmann P, Naas T, Poirel L. Global spread of Carbapenemase-producing Enterobacteriaceae. Emerg Infect Dis 2011; 17: 1791-1798.
26. Rice LB. Federal funding for the study of antimicrobial resistance in nosocomial pathogens: no ESKAPE. J Infect Dis 2008; 197: 1079-1081.
27. Mulvey MR, Simor AE. Antimicrobial resistance in hospitals: how concerned should we be? CMAJ 2009; 180: 408-415.
28. Lockhart SR, Etienne KA, Vallabhaneni S, Farooqi J, Chowdhary A, Govender NP, et al. Simultaneous emergence of multidrug-resistant Candida auris on 3 continents confirmed by whole-genome sequencing and epidemiological analyses. Clin Infect Dis 2017; 64: 134-140.
29. Chowdhary A, Sharma C, Meis JF. Candida auris: A rapidly emerging cause of hospital-acquired multidrug-resistant fungal infections globally. PLoS Pathog 2017; 13(5): e1006290.
30. Poole K. Efflux pumps as antimicrobial resistance mechanisms. Ann Med 2007; 39: 162-176.
31. Drawz SM, Bonomo RA. Three decades of β-lactamase inhibitors. Clin Microbiol Rev 2010; 23: 160-201. doi:10.1128/CMR.00037-09.
32. Zhang Y, Hu A, Andini N, Yang S. A 'culture' shift: Application of molecular techniques for diagnosing polymicrobial infections. Biotechnol Adv 2019; 37: 476-490.
33. Vincent JL, Brealey D, Libert N, Abidi NE, O'Dwyer M, Zacharowski K, et al. Rapid Diagnosis of Infection in the Critically Ill, a Multicenter Study of Molecular Detection in Bloodstream Infections, Pneumonia, and Sterile Site Infections. Crit Care Med 2015; 43: 2283-2291.
34. Lamy B, Sundqvist M, Idelevich EA; ESCMID Study Group for Bloodstream Infections, Endocarditis and Sepsis (ESGBIES). Bloodstream infections - Standard and progress in pathogen diagnostics. Clin Microbiol Infect 2020; 26: 142-150.
35. Perez KK, Olsen RJ, Musick WL, Cernoch PL, Davis JR, Peterson LE, et al. Integrating rapid diagnostics and antimicrobial stewardship improves outcomes in patients with antibiotic-resistant Gram-negative bacteremia. J Infect 2014; 69: 216-225.
36. Bauer KA, Perez KK, Forrest GN, Goff DA. Review of rapid diagnostic tests used by antimicrobial stewardship programs. Clin Infect Dis 2014; 59(Suppl 3): S134-45.
37. Smith RD, Zhan M, Zhang S, Leekha S, Harris A, Doi Y, et al. Comparison of three rapid diagnostic tests for bloodstream infections using Benefit-risk Evaluation Framework (BED-FRAME). J Clin Microbiol 2024; 62(1): e0109623.
| Files | ||
| Issue | Vol 18 No 3 (2026) | |
| Section | Original Article(s) | |
| DOI | https://doi.org/10.18502/ijm.v18i3.21654 | |
| Keywords | ||
| Sequential organ failure assessment Multiplex quantitative polymerase chain reaction Sepsis Procalcitonin | ||
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This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License. |
How to Cite
1.
Pandey P, Bir R, Chatterjee K, Verma N, Mukim Y, Grover K, Sharma S, Sehgal P, Chamoli S, Gupta R, Pandey A. Multiplex qPCR for the early detection of sepsis pathogens and its impact on antimicrobial therapy in critically ill patients. Iran J Microbiol. 2026;18(3):312-321.
