Aptamer-Based Lateral Flow Assay (ALFA) Without Biotin/Avidin for Trichomonas vaginalis Diagnosis

Trichomonas vaginalis, an anaerobic flagellated parasite, is the causative agent of trichomoniasis, the most prevalent non-viral sexually transmitted infection (STI). The World Health Organization (WHO) estimates 156.3 million new cases annually among adults aged 15–49, with transmission often leading to severe health consequences, including increased risk of HIV acquisition, preterm birth, and cervical dysplasia. Traditional diagnostic methods—such as wet-mount microscopy, culture, and nucleic acid amplification tests (NAATs)—face critical limitations: wet-mount lacks sensitivity (50–60%), culture requires specialized labs, and NAATs are costly and equipment-dependent. This gap underscores the urgent need for a rapid, affordable, and accessible point-of-care test (POCT) aligned with WHO's REASSURED criteria.

Fig.1 Schematic of the biotin/neutravidin-free ALFA for T. vaginalis. (Justo C. A. C., et al., 2025)

Current Landscape of Trichomonas POCTs

Commercially available POCTs for T. vaginalis remain limited. The OSOM Trichomonas Rapid Test, an immunochromatographic assay, offers 10-minute results but relies on antibody-based detection with a limit of detection (LOD) of 2.5 × 10³ cells/mL. The Visby Medical Sexual Health Test integrates automated PCR for multi-pathogen detection but is designed exclusively for vaginal swabs and requires power sources. Both assays fail to address key needs: male sample compatibility, low-cost production, and long-term stability. As of 2025, only two CLIA-waived tests exist, highlighting a critical market gap for robust, user-friendly diagnostics.

Aptamer-Based Lateral Flow Assay (ALFA): A Novel Diagnostic Paradigm

Mechanism of Action

The ALFA leverages DNA aptamers—synthetic oligonucleotides with high target affinity—to enable a biotin/avidin-free sandwich assay. The design employs two aptamers: A6 (95-mer) as a capture probe immobilized on a nitrocellulose membrane via UV cross-linking, and A1_14mer (14-mer) functionalized with thioctic acid-conjugated gold nanoparticles (AuNPs) as a reporter. When a sample containing T. vaginalis is applied, the A1_14mer-AuNP conjugate binds to the parasite, which is then captured by A6 at the test line, forming a visible red band. A control line with polyA oligonucleotide (33A) ensures assay validity.

Key Technological Innovations

Biotin/avidin-free design: Eliminating reliance on biotin-streptavidin systems reduces costs and enhances stability. Thioctic acid-modified aptamers form dithiol linkages with AuNPs, improving conjugate stability and signal intensity compared to thiol-modified counterparts.
Nanoparticle engineering: 20 nm citrate-capped AuNPs were optimized for conjugate preparation, with a shift in absorbance peak from 522 nm to 531 nm confirming functionalization. Larger 40 nm AuNPs caused non-specific signals, emphasizing size-dependent performance.
Buffer system optimization: A running buffer containing 7.5% (w/v) BSA in 1× PBS with 1.5 mM MgCl₂ maximizes signal intensity, while a sample buffer with 1% (v/v) IGEPAL® CA-630 ensures efficient cell lysis without matrix interference from cervicovaginal lavage (CVL) samples.

Analytical Performance Metrics

Sensitivity and Specificity

The ALFA detects as low as 1.6×10⁵ T. vaginalis cells/mL, outperforming antibody-based LFAs for Neisseria gonorrhoeae (10⁶ cells/mL) but slightly lower than its microplate aptamer assay counterpart (3.02×10³ cells/mL). Serial dilution studies confirmed linearity across 2.0×10⁷ to 1.6×10⁵ cells/mL, with signal intensity measured via the Cube LFA reader.

Specificity testing against common vaginal microbes—Candida albicans, Gardnerella vaginalis, Klebsiella pneumoniae, and multiple Lactobacillus species—showed no cross-reactivity, even at high concentrations (e.g., 1.5×10⁸ cells/mL for bacteria). Test line intensities remained near zero for non-target organisms, confirming high selectivity.

Stability and Cost-Effectiveness

The ALFA strip and running buffer demonstrate exceptional stability: stored at 4°C or 37°C for 90 days, the assay retained functionality, with Arrhenius modeling predicting a 1-year shelf-life at 22°C. This stability surpasses many commercial POCTs, which often require refrigeration.

Production costs are remarkably low, totaling < 1€ per test. Key cost drivers include inexpensive materials (nitrocellulose membrane, glass fiber pads) and aptamer synthesis, with the AuNP-aptamer conjugate accounting for ~15% of the total cost.

Clinical Validation and Real-World Applicability

Evaluation in Clinical Samples

Testing with four residual CVL samples from the AVEONS study showed 100% concordance with wet-mount microscopy. Notable findings included:

Detection of T. vaginalis in samples with bacterial vaginosis (Nugent score 8) and healthy vaginal microbiota (Nugent score 2)
Robust performance across vaginal pH ranges (6–7), despite potential interference from mucus or blood 7
Intense test lines in samples with elevated pH (7), possibly reflecting parasite density or metabolic activity

Operational Simplicity

The ALFA requires three user-friendly steps:

Mix CVL with 5× sample buffer (1:5 dilution)
Dilute with running buffer (1:3 ratio)
Dispense 200 μL onto the sample pad and read results in 15 minutes

This workflow aligns with WHO's POCT target product profile, eliminating the need for trained personnel or equipment. Visual interpretation (two red lines for positive, one for negative) ensures accessibility in resource-limited settings.

Future Directions and Market Impact

Ongoing research aims to enhance ALFA performance:

Sensitivity enhancement: Incorporating nucleic acid amplification (e.g., PCR or LAMP) to lower LOD, inspired by the microplate assay's superior sensitivity.
Sample diversity: Validating urine and male genital swab samples, addressing the current gap in male diagnostics.
Manufacturing scalability: Optimizing automated strip production to reduce costs and enable mass distribution.

Conclusion

The aptamer-based ALFA represents a paradigm shift in T. vaginalis diagnostics, combining high sensitivity (1.6×10⁵ cells/mL), specificity, and unprecedented affordability (< 1€/test). Its stability, user-friendly design, and clinical validation position it as a cornerstone for the WHO's 2030 goal of eliminating STIs. By addressing key limitations of existing POCTs, the ALFA holds tremendous potential to improve global access to timely trichomoniasis diagnosis, particularly in low- and middle-income countries, and mitigate the far-reaching health impacts of this neglected STI.

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Reference

Justo, Christine Aubrey C., et al. "Biotin/avidin-free sandwich aptamer-based lateral flow assay (ALFA) for the diagnosis of Trichomonas vaginalis." Sensors & Diagnostics (2025).

This article is for research use only. Do not use in any diagnostic or therapeutic application.