Background: Delayed diagnosis remains a primary challenge in leprosy control, often resulting in severe physical impairment and significant psychological distress among those affected. The factors contributing to this delay are complex and multifactorial, encompassing both patient-related and healthcare system-related elements. To investigate the patient- and health system-related factors contributing to delayed diagnosis of leprosy in an endemic area of Northeast Brazil. Methods: This cross-sectional study included 247 leprosy patients. Demographic and clinical characteristics, along with data on patient- and health system-related determinants associated with delays in diagnosis, were collected. Univariate and multivariate Poisson regression analyses were performed to examine associations between the outcome (delay in months) and the independent variables. Results: The median time from the first healthcare visit to leprosy diagnosis was 15.0 months (interquartile range: 6.0-36.0). Approximately 17% of participants had grade 2 disability (G2D) at diagnosis. In the multivariate Poisson regression analysis, female sex, residence in rural areas, delayed healthcare seeking after symptom onset, lack of initial suspicion of leprosy, multiple referrals requiring four or more consultations before diagnostic confirmation, and prior misdiagnosis with treatment for another condition were independently associated with prolonged diagnostic delay. Conclusion: This study identified significant delays in leprosy diagnosis in Sergipe, Brazil, which may contribute to the persistently high proportion of new cases with G2D. Both patient- and health system-related factors were associated with longer diagnostic delays. Urgent interventions are warranted to increase disease awareness among the general population and strengthen the capacity of primary healthcare services.

Antibody-free nucleic acid lateral flow assays (AF-NALFA) are an established approach for rapid detection of amplified pathogens DNA but can yield inconsistent signals across targets. Since AF-NALFA depends on dual hybridization of probes to single-stranded amplicons (ssDNA), site-specific thermodynamic (Gibbs free energy-ΔG) at probe-binding regions may be crucial for performance. This study investigated how site-specific-ΔG and sequence complementarity at probe-binding regions determine Test-line signal generation, comparing native and synthetic amplicons and assessing the effects of local secondary structures and mismatches. Asymmetric PCR-generated ssDNA amplicons of Listeria monocytogenes, Mycobacterium leprae, and Leishmania amazonensis were analyzed in silico and tested in AF-NALFA prototypes with gold-labeled thiol probes and biotinylated capture probes. T-line signals were photographed, quantified (ImageJ version 1.4k), and statistically correlated with site-specific-ΔG. While native ssDNA from M. leprae and L. amazonensis failed to produce AF-NALFA T-line signals, L. monocytogenes yielded strong detection. Site-specific-ΔG below −10 kcal/mol correlated with reduced hybridization. Synthetic oligos preserved signals despite structural constraints, whereas ~3–4 mismatches, especially at capture probe regions, markedly impaired T-line intensity. The performance of AF-NALFA depends on the synergism between thermodynamic accessibility, site-specific-ΔG-induced site constraints, and sequence complementarity. Because genomic context affects hybridization, target-specific thermodynamic in silico evaluation is necessary for reliable pathogen DNA detection.