Background

The rapid and accessible diagnosis of infectious diseases is essential for effective treatment, especially in cases of brain inflammations such as meningitis and encephalitis, where rapid intervention is crucial to preventing complications and decreasing morbidity [2]. For these infections, diagnostic methods need to be highly sensitive, specific, and accessible, ideally matching the point-of-care (POC) parameters: a testing duration of less than 2 h, a cost of less than $10 per test, and suitable for use by untrained personnel. Nucleic acid amplification testing (NAAT) is regarded as the gold standard for molecular diagnostics in this area [3]. PCR-based technologies are among the most commonly used NAAT methods in clinical practice. Recent research indicates that NAATs are essential diagnostic tools [4], particularly in instances where cerebrospinal fluid (CSF) cultures yield negative results [5], as these culturing methods become significantly limited upon antibiotic treatment.

Among NAAT techniques, isothermal amplification has emerged as particularly well-suited for POC diagnostics as it eliminates the need for complex lab equipment [6]. Isothermal amplification eliminates the need for thermal cycling and utilizes specialized enzymes with strand displacement activity to amplify target DNA or RNA sequences at a constant temperature. Of these techniques, loop-mediated isothermal amplification (LAMP) is the most widely used due to its simplicity, rapidity, and high sensitivity. However, despite its advantages, LAMP is prone to nonspecific amplification, which may be acceptable for screening purposes but is unsuitable for guiding therapeutic decisions. Thus, in general practice, LAMP often requires an additional PCR test to confirm primary results [7,8]. A novel method, dual-priming isothermal amplification (DAMP), was developed to overcome the limitations of LAMP. DAMP incorporates a dual-priming mechanism using specialized primers to enhance specificity and reduce false-positive results. While LAMP uses six primers to target eight distinct regions of the target sequence, with inner primers spanning 40–60 nucleotides to facilitate self-priming strand extension, DAMP targets six distinct regions using six primers, with inner primers designed to span less than 40 nucleotides [9]. Additionally, DAMP incorporates pairing-competition primers, which enhance amplification efficiency by balancing self-priming and pairing-priming strand extensions, making it a promising tool for rapid, accurate detection of meningitis in both laboratory and POC settings [9].

A critical aspect of introducing NAAT-based methods into clinical practice is the detection of amplification products. The detection can be achieved through direct or indirect methods. Various visualization techniques can use fluorescent compounds (e.g., DNA intercalating) or naked-eye detection [10]. Although naked eye readouts do not require any specialized tools, their dynamic ranges are often limited, and personnel should have the skills to distinguish subtle changes in turbidity or color. On the other hand, fluorescent detection is preferred for its high sensitivity and quantitative capabilities [11]. Previously, we evaluated three fluorescent dyes for their effectiveness in detecting double-stranded DNA (dsDNA) amplification products: dsGreen, Thioflavin T, and Brilliant Green. Each dye was assessed based on its sample-to-background (S/B) ratio, which is crucial for minimizing background noise and ensuring accurate detection [1]. Brilliant Green was identified as the optimal dye due to its high fluorescence intensity, affordability, and superior S/B ratio. The performance of this dye, combined with the DAMP reaction, provides a highly sensitive and specific assay for detecting the four primary bacterial pathogens responsible for meningitis: Haemophilus influenzae, Neisseria meningitidis, Streptococcus pneumoniae, and Klebsiella pneumoniae. These pathogens were selected as the leading causes of bacterial meningitis in individuals beyond the neonatal stage.

The developed assay was evaluated using bacterial culture and clinical cerebrospinal fluid (CSF) samples. It demonstrated both high specificity and sensitivity. Its versatility allows it to function as a standalone diagnostic tool or to be integrated into POC devices, offering a reliable, rapid, and accessible solution for the diagnosis of meningitis.