OPEN Research Support
head

PhD-Student
Samir Badran
Department of Clinical Microbiology, Department og Clinical Diagnostics, University Hopsital of Southern Denmark, Esbjerg


Projekt styring
Projekt status    Open
 
Data indsamlingsdatoer
Start 01.02.2021  
Slut 31.03.2024  
 



Rapid Detection of Pathogens in Blood from Patients with Sepsis using Multiplex Droplet Digital Polymerase Chain Reaction

Short summary

Blood cultures are the cornerstone of diagnosis for detecting the presence of bacteria or fungi in the blood, with an average identification time of 48 hours and failure to detect a pathogen occurring in approximately 50% of patients with sepsis. The aim of this study is to develop and implement a multiplex ddPCR assay as a routine diagnostic tool in the detection and identification of pathogens from whole blood and/or blood culture after 3h of incubation.


Rationale

Sepsis, a dysregulated host response to infection leading to life-threatening organ dysfunction [1], often caused by bloodstream infection (BSI), is a major public health concern worldwide. Sepsis affects more than 48 million people annually, including an estimated 3 million newborns, leading to more than 11 million deaths annually, mainly in a hospital setting [2,3]. This makes it one of the leading causes of death worldwide [4]. In Denmark, the incidence rate is estimated to be 56,145 cases per year with a mortality of 8085, potentially accounting for 15% of all deaths [5,6]. Most sepsis survivors experience additional morbidities, resulting in reduced physical and mental quality of life after diagnosis [7,8]. Up to 32% of patients with sepsis have a rehospitalization episode within 30 days and 60% are readmitted at least once within one year [8]. According to a Danish study by Perner et al [9], more than 50% of sepsis survivors die in the first year following diagnosis. The significant burden of morbidity and mortality from sepsis has a profound impact on patients and their families, and it is a substantial economic burden on health care systems and society [10].

Sepsis is a profound inflammatory response to infections caused by bacterial, viral, fungal, or parasitic pathogens [11]. One of the primary reasons for the high morbidity and mortality rate of sepsis is delay in diagnosis and initiation of antimicrobial therapy-every hour of delay in appropriate antimicrobial treatment increases mortality by 7.6% [12,13]. As many as 80% of sepsis deaths could be prevented with rapid diagnosis and treatment [14]. During BSI, the bacterial load is estimated to be 1-10 CFU/mL [15] or 103 to 104 copies of bacterial DNA/mL [16]. Blood cultures are the cornerstone of microbiological diagnosis of sepsis. Key limitations are low sensitivity and long detection time (24-72 hours), with failure to detect a pathogen occurring in approximately 50% of patients with sepsis [15,17,18]. Gupta et al [19] have shown that sepsis-associated mortality was significantly higher in patients with a negative blood culture (34.6%) compared to patients with a positive blood culture (22.7%). Infection with fastidious microorganisms, antimicrobial treatment prior to blood collection, and low bacterial load all contribute to the occurrence of false-negative blood culture [15,20].

Multiplex real-time quantitative polymerase chain reaction (qPCR) has been increasingly employed in combination with positive blood culture to increase diagnostic sensitivity in patients with sepsis [21]. Multiplex qPCR also facilitates more rapid diagnosis [21-23], as demonstrated for the commercially available Septifast (Roche Diagnostics) [24] and FilmArray Blood Culture ID Panel (BCID; BioFire Diagnostics) [25]. The use of multiplex qPCR demonstrated high concordance with the blood culture technique, with up to 100% specificity and a limit of detection (LOD) ranging from 1 to 10 CFU/reaction [22,24]. It has been reported in some studies that multiplex qPCR detected the presence of a pathogen in 10%-40% of cases that were negative by conventional blood culture [26-28]. However, other studies have shown a reduced sensitivity, ranging from 28%-66%, in comparison with conventional blood cultures [28-30]. It is apparent that there is still a need for techniques to improve the diagnostic yield and reduce the time to diagnosis from blood culture specimens. The combination of nanoliter-sized droplet technology paired with digital polymerase chain reaction (PCR), known as droplet digital PCR (ddPCR), is a novel diagnostic tool that partitions the reaction into up to 20,000 droplets before amplification [31]. This method provides absolute quantification of target sequences and has demonstrated greater sensitivity, reproducibility, precision, and accuracy compared to qPCR [32-34]. For instance, the sensitivity of ddPCR was 6.4 copies/20 μL reaction for plasmid DNA and 5 CFUs/20 μL reaction for bacterial cells as compared to 12 copies/20 μL reaction and 36 CFUs/20 μL reaction using qPCR, respectively [35]. Furthermore, a study by Dong-Ku et al [36] demonstrated that, by using droplet digital detection technology, they were able to detect bacteria at the single-cell level in unprocessed diluted blood.

Recently, ddPCR has been investigated as a novel technique for the detection of pathogens in BSI. Wouters et al [37] demonstrated an overall sensitivity and specificity of 80% and 87%, respectively. Furthermore, they were able to detect Escherichia coli at a 10- to 100-fold lower concentration when compared to qPCR and with a detection limit of approximately 1-2 bacteria. Zhang et al [38] demonstrated similar results, with a detection rate up to 80%-90% of Staphylococcus aureus (S. aureus) and Escherichia coli (E. coli) in blood when using ddPCR; both studies had promising results. In this study, we will investigate ddPCR as a novel technique for sepsis diagnosis from whole blood and blood culture after 3 and 72 hours of incubation. To the best of our knowledge, this would be the first study that involves developing and implementing a multiplex ddPCR assay as a routine diagnostic tool for early detection of the most common sepsis-causing pathogens (ie, S. aureus, Streptococcus pneumoniae, E. coli, Klebsiella pneumonia and Pseudomonas aeruginosa) in patients with sepsis. We believe that the technique will subsequently support clinicians to initiate early and rational antimicrobial treatment by reducing processing time and increasing the detection rate in blood cultures. Improved microbiological diagnosis of sepsis will not only help improve outcomes for sepsis, but also will contribute to improved antimicrobial stewardship and rational antibiotic prescribing.


NOTE: To read the complete protocol, see publication associated with this project (https://www.researchprotocols.org/2021/12/e33746)


Description of the cohort

Patients over 18 years of age admitted to the Department of Emergency Medicine with suspected bloodstream infection or sepsis.


Data and biological material

Biological material will consist of whole blood samples and two set of blood culture bottles, which will be analyzed after 3 hours and 72 hours of incubation. All samples in the clinical phase will be collected as part of routine diagnosis and management of patients. The ddPCR results will not influence patient management decisions.

Blood samples from patients with suspected sepsis will be pseudonymized and only used for method comparison.

Data on antibiotic treatment and routine laboratory analyses will be extracted from clinical records and evaluated after collection and analysis of all blood samples.