Considering the importance of merozoite surface proteins (MSPs) as vaccine candidates, this study was conducted to investigate the polymorphism and genetic diversity of Plasmodium vivax merozoite surface protein 3-alpha (PvMSP-3α) in Thailand. To analyze genetic diversity, 118 blood samples containing P. vivax were collected from four malaria-endemic areas in western and southern Thailand. The DNA was extracted and amplified for the PvMSP-3α gene using nested PCR. The PCR products were genotyped by PCR-RFLP with Hha I and Alu I restriction enzymes. The combination patterns of Hha I and Alu I RFLP were used to identify allelic variants. Genetic evaluation and phylogenic analysis were performed on 13 sequences, including 10 sequences from our study and 3 sequences from GenBank. The results revealed three major types of PvMSP-3α, 91.5 allelic type A (1.8kb), 5.1 allelic type B (1.5kb), and 3.4 allelic type C (1.2kb), were detected based on PCR product size with different frequencies. Among all PvMSP-3α, 19 allelic subtypes with Hha I RFLP patterns were distinguished and 6 allelic subtypes with Alu I RFLP patterns were identified. Of these samples, 73 (61) and 42 (35.6) samples were defined as monoallelic subtype infection by Hha I and Alu I PCR-RFLP, respectively, whereas 77 (65.3) samples were determined to be mixed-allelic subtype infection by the combination patterns of Hha I and Alu I RFLP. These results strongly indicate that PvMSP-3α gene is highly polymorphic, particularly in blood samples collected from the Thai-Myanmar border area (the western part of Thailand). The combination patterns of Hha I and Alu I RFLP of the PvMSP-3α gene could be considered for use as molecular epidemiologic markers for genotyping P. vivax isolates in Thailand.

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.

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), 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.