Treponema pallidum subspp. pallidum is a spirochaete bacterium that causes syphilis, one of the most common sexually transmitted diseases. Syphilis progresses through four distinct stages, each characterized by specific symptoms, namely primary, secondary, latent, and late (tertiary) syphilis. Serology has been considered the primary diagnostic approach. However, it is plagued by problems such as the limited specificity of nontreponemal tests and the inadequate correlation of treponemal tests with disease activity. In this study, we focused on the development of a loop-mediated isothermal amplification assay utilizing hydroxy naphthol blue (LAMP-HNB) for the diagnosis of T. pallidum subspp. pallidum. Specifically, this study seeks to determine the analytical sensitivity (limit of detection; LOD) and analytical specificity. Four hundred clinical serum samples were analyzed for diagnostic sensitivity, specificity, and predictive value, and each technique’s 95% confidence intervals (95% CI, p < 0.05) were evaluated. The limit of detection for polymerase chain reaction with agarose gel electrophoresis (PCR-AGE), the loop-mediated isothermal amplification assay combined with agarose gel electrophoresis (LAMP-AGE), and LAMP-HNB were 116 pg/µL, 11.6 pg/µL, and 11.6 pg/ µL, respectively. Analytical specificity examinations indicated the absence of cross-reactivity with Leptospira interrogans, Staphylococcus aureus, Enterococcus faecalis, Escherichia coli, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, human immunodeficiency virus (HIV), and healthy human serum in PCR-AGE, LAMP-AGE, and LAMP-HNB. The diagnostic sensitivity, diagnostic specificity, positive predictive value (PPV), and negative predictive value (NPV) for PCR-AGE were 100.00 (100.00)%, 94.50 (94.40–94.60)%, 94.79 (94.69–94.88)%, and 100.00 (100.00)%, respectively. While, for LAMP-AGE and LAMP-HNB, they were 100.00 (100.00)%, 91.00 (90.87–91.13)%, 91.74 (91.63–91.86)%, and 100.00 (100.00)%, respectively. The LAMP-HNB test is simple, rapid, highly sensitive, and highly specific, without requiring expensive equipment. In the future, the LAMP-HNB assay may develop into a single-step diagnostic process, enabling the use as point-of-care testing for the diagnosis, prevention, and management of syphilis infection.

Plasmodium vivax is the most prevalent cause of malaria in Thailand and is predominant in malarial endemic areas worldwide. P. vivax infection is characterized by low parasitemia, latent liver-stage parasites, or asymptomatic infections leading to underreported P. vivax cases. These are significant challenges for controlling and eliminating P. vivax from endemic countries. This study developed and evaluated a dot-blot enzyme-linked immunosorbent assay (ELISA) using PvMSP1-42 recombinant antigen for serological diagnosis based on the detection of antibodies against P. vivax. The optimal PvMSP1-42 concentration and dilutions of anti-human IgG horseradish peroxidase (HRP)-conjugated antiserum were tested on 88 serum samples from P. vivax, Plasmodium falciparum and bacterial infection, including healthy individuals. A cut-off titer of 1:800 produced optimal values for sensitivity and specificity of 90.9 and 98.2%, respectively, with an accuracy of 95.5%. The positive and negative predictive values were 96.8 and 94.7% respectively. The results from microscopic examination and dot-blot ELISA showed strong agreement with the 0.902 kappa index. Thus, the dot-blot ELISA using PvMSP1-42 antigen provided high sensitivity and specificity suitable for serodiagnosis of P. vivax infection. The test is a simple and quick diagnostic assay suitable for field testing as it does not require specific equipment or particular skills.

Background: Venereal syphilis in humans is caused by Trepenoma pallidum subspp. pallidum. A study has shown that 30,302 individuals in Thailand had syphilis in 2020, with a male-to-female ratio of 1:0.8 and the highest incidence rate at ages between fifteen and twenty-four. Methods: This research aimed to develop a loop-mediated isothermal amplification assay using gold nanoparticles (LAMP-AuNPs). Analytical sensitivity, diagnostic specificity, accuracy, and predictive values for each technique are provided. Results: The diagnosis sensitivities of polymerase chain reaction using agarose gel electrophoresis (PCR-AGE), loop-mediated isothermal amplification assay using agarose gel electrophoresis (LAMP-AGE), and LAMP-AuNPs were 116 ng/µL, 11.6 ng/µL, and 11.6 ng/µL, respectively. We evaluated the analytical specificity using PCR and a LAMP-based assay, and there was no cross-reactivity to Leptospira interrogans, Staphylococcus aureus, Enterococcus faecalis, Escherichia coli, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, human immunodeficiency virus (HIV), and healthy humans. After analyzing 400 serum samples of patients suspected of syphilis, the LAMP-AGE and LAMP-AuNPs assays displayed 100% diagnostic sensitivity scores, 91% diagnostic specificity scores, 95.5% accuracy rates, 100% positive predictive values (PPVs), and 91% negative predictive values (NPVs), the positive likelihood ratio (LR+) was 11.11, while the negative likelihood ratio (LR−) was 0. Conversely, for PCR assays displayed 100% diagnostic sensitivity scores, 94.5% diagnostic specificity scores, 97.25% accuracy rates, 100% PPVs, and 94.5% NPVs, LR+ was 18.18, and LR− was 0. Conclusions: The LAMP-AuNPs technique demonstrates rapidity, affordability, and convenience, rendering it well-suited for point-of-care applications in the diagnosis, prevention, and management of pathogenic infections.

Vancomycin-resistant enterococci (VRE), particularly vancomycin-resistant Enterococcus faecium, are important healthcare-associated pathogens, and vanA is a major acquired determinant of high-level glycopeptide resistance. The conventional polymerase chain reaction (PCR) method is reliable for molecular detection of vanA but requires thermal cycling and, in conventional formats, post-amplification analysis. In this study, we developed and evaluated a closed-tube colorimetric loop-mediated isothermal amplification assay using hydroxy naphthol blue (LAMP-HNB) for visual detection of vanA. A total of 130 Enterococcus isolates were tested, comprising 100 previously characterized in our published LAMP–lateral flow dipstick (LAMP-LFD) study (52 vanA-positive and 48 vanA-negative) and an additional 30 (13 vanA-positive and 17 vanA-negative), yielding 65 PCR-positive and 65 PCR-negative isolates overall. Reaction conditions were optimized sequentially, and the final LAMP-HNB protocol took place at 63 °C for 45 min with 4.0 mM MgSO₄ and 5 μM HNB. The visual endpoint was confirmed by 2% agarose gel electrophoresis. LAMP-HNB correctly classified all 65 PCR-positive and all 65 PCR-negative isolates, yielding 100% sensitivity (65/65; exact 95% CI: 94.48–100%), 100% specificity (65/65; 95% CI: 94.48–100%), and 100% overall agreement (130/130; 95% CI: 97.20–100%). The limit of detection was 8.67 × 10³ genome copies/µL (28.52 pg/µL) for LAMP-HNB compared with 8.67 × 10⁴ genome copies/µL (285.2 pg/µL) for conventional PCR, representing a 10-fold lower DNA concentration endpoint under the experimental conditions employed. In the 100-isolate overlapping cohort, LAMP-HNB, the previously reported LAMP-LFD platform, and PCR were concordant. With its 45 min requirement for amplification after DNA preparation, the HNB format provides a rapid, closed-tube visual endpoint without a labeled probe, lateral flow strip, running buffer, or post-amplification hybridization step. These findings support LAMP-HNB as a promising molecular screening approach for vanA-positive enterococci, while larger, fully independent multicenter validation remains necessary before routine implementation.

Carbapenem-resistant Acinetobacter baumannii (CRAB) is a major cause of hospital-acquired infections. Conventional phenotyping for carbapenem resistance requires more than 24 h, which delays clinical decisions and increases the risk of outbreaks. This study aims to develop a lateral flow dipstick (LFD) assay that detects loop-mediated isothermal amplification (LAMP) products targeting the bla_OXA-23-like gene, providing results within 1–1.5 h. Primer specificity was evaluated using seven Gram-positive and nine Gram-negative species commonly present in clinical samples, as well as carbapenem-susceptible A. baumannii and CRAB isolates harboring other resistance genes. The assay detected the bla_OXA-23-like gene at concentrations as low as 1.57 pg/µL, demonstrating ~10-fold greater sensitivity than conventional PCR. The gene was present in 224/268 CRAB samples (83.6%) and absent in 100 carbapenem-susceptible A. baumannii samples. Results were fully concordant with PCR, yielding 100% sensitivity and specificity. The method requires no expensive or complex instrumentation. Future studies should validate the assay across multiple hospitals and clinical specimens to confirm its generalizability and clinical utility. Multiplex LAMP with dual LFD readouts could also enable simultaneous detection of additional carbapenem resistance genes, supporting rapid CRAB surveillance and outbreak control.

Vancomycin-resistant enterococci (VRE) have emerged as major nosocomial pathogens worldwide, primarily due to the acquisition of the vanA gene, which confers high-level resistance to vancomycin. Rapid detection of the vanA gene, which is associated with vancomycin resistance, is crucial for timely clinical management and infection control. Through this research, we aimed to develop an assay combining loop-mediated isothermal amplification and a lateral flow Dipstick (LAMP-LFD) for the rapid detection of the vanA gene in VRE. Genomic DNA was extracted and amplified from 100 clinical urine samples by LAMP targeting vanA under optimized, constant-temperature conditions, with the amplified product then detected using a lateral flow Dipstick assay based on probe pairing. The diagnostic performance of the LAMP-LFD method was evaluated and compared with a conventional PCR reference method. The LAMP-LFD assay successfully detected the vanA gene in all positive samples, with negative samples showing no assay cross-reactivity with 12 species of bacteria commonly found in clinical settings. The developed LAMP–LFD assay showed promising diagnostic performance for detecting the vanA gene in Enterococcus isolates, demonstrating complete agreement with the reference PCR method in this study. Owing to its rapid turnaround time, simplicity, and visual readout, the assay has potential as a molecular screening tool for VRE. However, further validation in larger, multicenter studies and comparison with phenotypic antimicrobial susceptibility testing are required before routine clinical implementation.