Clinical Efectiveness of a Multiplex PCR-Based Rapid Diagnostic Method in Bloodstream Infections

Evaluation of the Clinical Efectiveness of a Multiplex PCR-Based Rapid Diagnostic Method in Bloodstream Infections: A Prospective Randomized Controlled Study

Bloodstream infections (BSIs) are associated with high morbidity and mortality, and delays in initiating appropriate antimicrobial therapy significantly worsen clinical outcomes. Conventional culture-based microbiological methods require 24-72 hours to provide definitive pathogen identification and antimicrobial susceptibility results, often leading to prolonged use of broad-spectrum empirical therapy. Rapid multiplex PCR-based diagnostic tests have the potential to shorten diagnostic timelines by identifying pathogens and resistance genes within approximately one hour; however, data on their real-world clinical impact remain limited.

This prospective, randomized, controlled, single-center study aims to evaluate the clinical effectiveness and diagnostic performance of a multiplex PCR-based rapid diagnostic method applied directly to positive blood culture bottles in adult patients with bloodstream infections. A total of 300 patients (≥18 years) with positive blood culture signals will be randomized 1:1 to either a study group or a control group. In the study group, positive blood cultures will be analyzed using both standard microbiological methods and a multiplex PCR panel, while the control group will undergo standard microbiological diagnostics alone.

The primary endpoint is time to optimal antimicrobial therapy (OTT), defined as the time from blood culture collection to initiation of the narrowest-spectrum, guideline-recommended antimicrobial agent active against the identified pathogen. Secondary endpoints include time to effective antimicrobial therapy (ETT), time to pathogen identification, antimicrobial escalation or de-escalation rates, length of hospital stay, total duration of antimicrobial therapy, and 28-day all-cause mortality.

Clinical, demographic, and microbiological data will be collected prospectively, including comorbidity indices and severity scores. Randomization will be stratified by ICU versus ward admission, presence of neutropenia, and Charlson Comorbidity Index to ensure balanced groups. Diagnostic accuracy of the multiplex PCR panel will be assessed by calculating sensitivity, specificity, predictive values, and agreement with standard culture methods.

This study seeks to determine whether rapid multiplex PCR diagnostics can meaningfully improve antimicrobial stewardship and clinical outcomes in patients with bloodstream infections compared with conventional diagnostic workflows.

Bloodstream infections (BSIs) are among the most severe infectious diseases encountered in hospitalized patients and are associated with significant morbidity and mortality. Early initiation of appropriate antimicrobial therapy is one of the most important determinants of survival in these patients. However, timely optimization of antimicrobial treatment is frequently limited by delays inherent to conventional culture-based microbiological diagnostic methods, which typically require 24-72 hours to provide definitive pathogen identification and antimicrobial susceptibility results. As a consequence, clinicians often rely on prolonged broad-spectrum empirical antimicrobial therapy, which contributes to antimicrobial resistance, drug-related adverse events, and increased healthcare costs.

Recent advances in molecular diagnostics have enabled the development of multiplex polymerase chain reaction (PCR)-based assays capable of rapidly detecting common bloodstream pathogens and selected antimicrobial resistance genes directly from positive blood culture bottles. These tests can provide results within approximately one hour, offering the potential to significantly shorten diagnostic timelines and support earlier antimicrobial escalation, de-escalation, or optimization. Despite their increasing availability, there remains limited high-quality evidence regarding the real-world clinical effectiveness of these rapid diagnostic tools, particularly with respect to their impact on antimicrobial stewardship and patient-centered outcomes.

This study is designed as a prospective, randomized, controlled, single-center clinical trial to evaluate the clinical effectiveness and diagnostic performance of a multiplex PCR-based rapid diagnostic panel applied directly to positive blood culture samples in adult patients with bloodstream infections. The study will be conducted in an 810-bed tertiary university hospital and is planned to enroll a total of 300 patients over a 12-month period.

Adult patients (≥18 years of age) with clinical suspicion of bloodstream infection and a positive blood culture signal will be screened for eligibility. Following confirmation of eligibility and informed consent, patients will be randomized in a 1:1 ratio to either the study group or the control group. Randomization will be performed using a computer-generated permuted block randomization method and will be stratified by intensive care unit (ICU) versus ward admission, presence of neutropenia, and Charlson Comorbidity Index category to ensure balanced distribution of baseline risk factors between the study arms.

In the study group, positive blood culture samples will undergo rapid testing using a multiplex PCR-based in vitro diagnostic panel in addition to standard microbiological diagnostic methods. The multiplex PCR assay is designed to detect predefined bacterial and fungal pathogens as well as selected antimicrobial resistance genes directly from positive blood culture bottles. DNA extraction will be performed using a rapid, automated nucleic acid extraction system according to the manufacturer's instructions, followed by real-time PCR amplification and detection. Results of the multiplex PCR assay will be available within approximately one hour and will be promptly reported to the treating clinical team. These results may be used to guide antimicrobial management decisions, including early escalation, de-escalation, or optimization of therapy, in conjunction with clinical judgment and institutional treatment guidelines.

In the control group, positive blood culture samples will be processed using standard microbiological diagnostic methods alone, in accordance with routine clinical practice. Standard diagnostics include Gram staining performed directly from positive blood culture bottles, subculture onto appropriate solid media, organism identification using conventional and automated methods such as MALDI-TOF mass spectrometry, and antimicrobial susceptibility testing using standardized techniques. Results will be reported to the clinical team as they become available through the routine laboratory workflow.

Definitions of Diagnostic and Therapeutic Time Parameters

To allow precise evaluation of diagnostic timelines and antimicrobial treatment processes, the following time-related variables will be prospectively recorded for all enrolled patients:

• Time to pathogen identification (TPI) by standard methods is defined as the time from blood culture collection to identification of the causative microorganism using standard culture-based methods.
• Time to rapid antimicrobial susceptibility testing (RAST) by standard methods is defined as the time from blood culture collection to obtaining the first interpretable antimicrobial susceptibility result from rapid disk diffusion testing applied to the microorganism group determined by Gram staining.
• Time to pathogen and resistance gene identification (PRGI) by multiplex PCR is defined as the time from blood culture collection to initial identification of the pathogen and antimicrobial resistance genes using the multiplex PCR panel.
• Time to antimicrobial susceptibility testing (AST) by standard methods is defined as the time from blood culture collection to completion of definitive antimicrobial susceptibility testing using standard culture techniques, including automated systems, microdilution, or disk diffusion methods.
• Time to effective antimicrobial therapy (ETT) is defined as the time from blood culture collection to administration of the first dose of any antimicrobial agent known to be in vitro active against the isolated microorganism, regardless of antimicrobial spectrum.
• Time to optimal antimicrobial therapy (OTT) is defined as the time from blood culture collection to administration of the first dose of the narrowest-spectrum, guideline-recommended first-line antimicrobial therapy targeting the isolated microorganism. Optimal therapy represents a subset of effective therapy and reflects the most appropriate antimicrobial agent without inclusion of unnecessary broad-spectrum coverage.

Study Endpoints and Outcomes

The primary endpoint of the study is time to optimal antimicrobial therapy (OTT). Secondary endpoints include time to effective antimicrobial therapy (ETT), time to pathogen identification, antimicrobial escalation and de-escalation events, total duration of antimicrobial therapy, length of hospital stay, and 28-day all-cause mortality.

Both effective and optimal antimicrobial therapy classifications will consider all antimicrobial agents administered within 48 hours following blood culture collection and the availability of microbiological and molecular diagnostic results. Discontinuation of antimicrobial therapy for microorganisms determined to represent contamination will be recorded and included in the analysis as time to optimal antimicrobial therapy.

Clinical and demographic data will be collected prospectively for all enrolled patients, including age, sex, hospital location, comorbid conditions, immunosuppressive status, severity of illness scores, presence of intravascular devices, infection source, and source control measures. Time-related variables, including blood culture collection, positive signal detection, diagnostic result availability, and antimicrobial administration, will be recorded with precise timestamps.

Diagnostic performance of the multiplex PCR panel will be evaluated by comparison with standard culture-based methods, which will serve as the reference standard. Sensitivity, specificity, positive predictive value, negative predictive value, and agreement statistics will be calculated for pathogen and resistance gene detection.

All statistical analyses will be performed using standard statistical software. Final analyses will be performed after completion of data collection for all enrolled participants. Continuous variables will be summarized using appropriate descriptive statistics based on data distribution, and categorical variables will be expressed as frequencies and percentages. Comparisons between study groups will be conducted using appropriate parametric or non-parametric tests. Survival analyses will be performed to evaluate 28-day mortality, and multivariable regression models may be used to identify independent predictors of clinical outcomes.

This study aims to provide robust evidence regarding the clinical utility of rapid multiplex PCR diagnostics in bloodstream infections and to determine whether their implementation can lead to earlier optimization of antimicrobial therapy, improved antimicrobial stewardship, and better patient outcomes compared with conventional diagnostic workflows.

Inclusion Criteria:

• Age 18 years or older
• Clinical suspicion of bloodstream infection
• Positive blood culture signal
• Patients managed in hospital wards or intensive care units
• Ability to provide informed consent (patient or legally authorized representative)

Exclusion Criteria:

• Recurrent episode of the same bloodstream infection
• Inadequate or insufficient blood culture sample for analysis
• Refusal or inability to provide informed consent
• Death within the first 24 hours after blood culture collection
• Loss to follow-up during the study period