Molecular diagnostic technology: beyond analytical accuracy

Abstract

Infectious diseases cause high morbidity and mortality, and early, effective antimicrobial therapy improves outcomes. However, conventional microbiology methods like culture are slow and insensitive. Emerging molecular diagnostics—such as multiplex PCR (mPCR), droplet digital PCR (ddPCR), and metagenomic next-generation sequencing (mNGS)—offer rapid, accurate pathogen identification. Challenges persist in result interpretation (e.g., setting positivity thresholds, low positive predictive value) and clinician trust. While molecular diagnostics excel in sensitivity, their real-world impact on specificity and patient prognosis - clinical accuracy -remains limited. Key hurdles include patient selection, timing, result interpretation, and pathogen relevance. Addressing these gaps is critical for standardizing these technologies and maximizing their clinical benefit. 

In the context of infectious diseases, which are associated with significant morbidity and mortality, early and effective anti-infective therapy has been shown to significantly improve the clinical outcomes of affected patients [1]. However, routine microbiology techniques, such as conventional culture, are time-consuming and often yield low sensitivity [2]. Currently, molecular diagnostic technologies are beginning to show promising prospects. The study by Jérémy Contier and colleagues demonstrated that multiplex polymerase chain reaction (mPCR) can effectively reduce the time required to obtain diagnostic results in immunosuppressed patients and decrease exposure to broad-spectrum antibiotics [3]. Similar technologies include droplet digital PCR (ddPCR) and metagenomic next-generation sequencing (mNGS) [4, 5]. These technologies have collectively demonstrated the ability to rapidly identify pathogens with high sensitivity. However, when these technologies were employed in randomized controlled trials, they failed to demonstrate a significant impact on important clinical outcomes of patients. Compared with conventional techniques, patients whose treatments were guided by multiplex PCR did not exhibit improved mortality or reduced lengths of ICU stay [6]. When compared with the use of conventional microbiological tests alone, the combination of metagenomic next-generation sequencing (mNGS) with conventional microbiological tests (CMTs) did not significantly influence clinical improvement within 28 days, 28-day mortality, or lengths of ICU stay among patients with severe community-acquired pneumonia [7]. Moreover, compared with conventional techniques, syndromic PCR did not improve the clinical cure rate in patients with hospital-acquired pneumonia (HAP) and ventilator-associated pneumonia (VAP) [8]. In a study enrolling adults with community-acquired pneumonia who were not immunocompromised, pathogens were identified using both multiplex PCR and traditional techniques in one group, while the other group was tested using traditional techniques alone. The results showed that combining multiplex PCR and serum procalcitonin failed to reduce antibiotic exposure or improve outcomes on Day 28 [9]. It is evident that although molecular diagnostic techniques demonstrate high accuracy in studies focusing on diagnostic precision, they have not been able to improve patient prognosis in clinical applications. This phenomenon brings concerns: Are molecular diagnostic techniques truly effective in diagnosis? Molecular diagnostic technologies may face challenges in interpreting results. Studies have shown that when using mNGS and PCR technologies, it is necessary to set a positivity threshold [4, 5]. Moreover, a low positive predictive value for molecular diagnostic technologies undermines the confidence of clinicians in these diagnostic methods. Surveys have indicated that attitudes towards using molecular diagnostics to inform initial treatment choices and to stop broad-spectrum antibiotics early are nuanced [10]. Guillaume Voiriot pointed out that the use of mPCR testing presents challenges for clinicians, particularly in interpreting results, which are often multi-positive for bacterial microorganisms—especially when performed on sputum [9]. The identification of common microorganisms may not necessarily indicate pathogenicity nor confer benefits to patients. Building on molecular diagnostic technologies and conducting subsequent clinical validation may enhance accuracy and further improve patient outcomes [11]. In our view, multiplex molecular diagnostic technologies face numerous challenges, including identifying the relevant pathogens, selecting the appropriate patients, determining the optimal timing, interpreting the results accurately, and demonstrating clinical outcome benefits. Addressing all these issues is essential and urgent for the further development of standardized procedures.

Abbreviations

PCR

Polymerase chain reaction

mPCR

multiplex polymerase chain reaction

mNGS

metagenomic next-generation sequencing

CMTs

Conventional microbiological tests

HAP

 Hospital-acquired pneumonia

VAP

 Ventilator-associated pneumonia

Author contributions

XQ wrote the first draft of the manuscript. XQ and JL contributed to the final version of the manuscript. All the authors approved the final manuscript.

Data availability

No datasets were generated or analysed during the current study.

Declarations

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.