Loop-Mediated Isothermal Amplification (LAMP): Potential Point-of-Care Testing for Vulvovaginal Candidiasis.

PURPOSE: The aim of this study is to establish a loop-mediated isothermal amplification (LAMP) method for the rapid detection of vulvovaginal candidiasis (VVC). METHODS: We developed and validated a loop-mediated isothermal amplification (LAMP) method for detecting the most common Candida species associated with VVC, including C. albicans, N. glabratus, C. tropicalis, and C. parapsilosis. We evaluated the specificity, sensitivity, positive predictive value (PPV), negative predictive value (NPV), and Kappa value of the LAMP method to detect different Candida species, using the conventional culture method and internal transcribed spacer (ITS) sequencing as gold standards and smear Gram staining and real-time Rolymerase Chain Reaction (PCR) as controls. RESULTS: A total of 202 cases were enrolled, of which 88 were VVC-positive and 114 were negative. Among the 88 positive patients, the fungal culture and ITS sequencing results showed that 67 cases (76.14%) were associated with C. albicans, 13 (14.77%) with N. glabratus, 5 (5.68%) with C. tropicalis, and 3 (3.41%) with other species. Regarding the overall detection rate, the LAMP method presented sensitivity, specificity, PPV, NPV, and Kappa values of 90.91%, 100%, 100%, 93.4%, and 0.919, respectively. Moreover, the LAMP had a specificity of 100% for C. albicans, N. glabratus, and C. tropicalis, with a sensitivity of 94.03%, 100%, and 80%, respectively. Moreover, the microscopy evaluation had the highest sensitivity, while the real-time PCR was less specific for C. albicans than LAMP. In addition, CHROMagar Candida was inferior to LAMP in detecting non-albicans Candida (NAC) species. CONCLUSIONS: Based on the cost-effective, rapid, and inexpensive characteristics of LAMP, coupled with the high sensitivity and specificity of our VVC-associated Candida detection method, we provided a possibility for the point-of-care testing (POCT) of VVC, especially in developing countries and some laboratories with limited resources.

A microfluidic-chip-based system with loop-mediated isothermal amplification for rapid and parallel detection of Trichomonas vaginalis and human papillomavirus.

Cervical cancer is a significant global health issue primarily caused by high-risk types of human papillomavirus (HPV). Recent studies have reported an association between Trichomonas vaginalis (T. vaginalis) infections and HPV infections, highlighting the importance of simultaneously detecting these pathogens for effective cervical cancer risk management. However, current methods for detecting both T. vaginalis and HPV are limited. In this study, we present a novel approach using a microfluidic-chip-based system with loop-mediated isothermal amplification (LAMP) for the rapid and parallel detection of T. vaginalis, HPV16, HPV18, and HPV52 in a reagent-efficient and user-friendly manner. Compared to conventional LAMP assays in tubes, our system exhibits enhanced sensitivity with values of 2.43 × 101, 3.00 × 102, 3.57 × 101, and 3.60 × 102 copies per reaction for T. vaginalis, HPV16, HPV18, and HPV52, respectively. Additionally, we validated the performance of our chip by testing 47 clinical samples, yielding results consistent with the diagnostic methods used by the hospital. Therefore, our system not only offers a promising solution for concurrent diagnosis of T. vaginalis and HPV infections, particularly in resource-limited areas, due to its cost-effectiveness, ease of use, and rapid and accurate detection performance, but can also contribute to future research on the co-infection of these two pathogens. Moreover, the system possesses the capability to simultaneously detect up to 22 different types of pathogens, making it applicable across a wide range of domains such as diagnostics, food safety, and water monitoring.

An Integrated and Multi-Target Nucleic Acid Isothermal Analysis System for Rapid Diagnosis of Vulvovaginal Candidiasis.

Rapid identification of Candida species is significant for the diagnosis of vulvovaginal candidiasis (VVC). An integrated and multi-target system for the rapid, high-specificity, and high-sensitivity detection of four Candida species was developed. The system consists of a rapid sample processing cassette and a rapid nucleic acid analysis device. The cassette could process the Candida species to release nucleic acids in 15 min. The released nucleic acids were analyzed by the device as fast as within 30 min, using the loop-mediated isothermal amplification method. The four Candida species could be simultaneously identified, with each reaction using only 1.41 µL of reaction mixture, which was low cost. The RPT (rapid sample processing and testing) system could detect the four Candida species with high sensitivity (<2 CFU/reaction) and high specificity. The system also processed and analyzed 32 clinical samples, giving the results with high clinical sensitivity and specificity. Hence, the system was a significant and effective platform for the diagnosis of VVC. Furthermore, the period of validity of the reagents and chips used in the system was >90 days, and the system could also be used for the detection of bacteria.

Rapid, Highly Sensitive, and Label-Free Pathogen Assay System Using a Solid-Phase Self-Interference Recombinase Polymerase Amplification Chip and Hyperspectral Interferometry.

Recombinase polymerase amplification (RPA) is a useful pathogen identification method. Several label-free detection methods for RPA amplicons have been developed in recent years. However, these methods still lack sensitivity, specificity, efficiency, or simplicity. In this study, we propose a rapid, highly sensitive, and label-free pathogen assay system based on a solid-phase self-interference RPA chip (SiSA-chip) and hyperspectral interferometry. The SiSA-chips amplify and capture RPA amplicons on the chips, rather than irrelevant amplicons such as primer dimers, and the SiSA-chips are then analysed by hyperspectral interferometry. Optical length increases of SiSA-chips are used to demonstrate RPA detection results, with a limit of detection of 1.90 nm. This assay system can detect as few as six copies of the target 18S rRNA gene of Plasmodium falciparum within 20 min, with a good linear relationship between the detection results and the concentration of target genes (R2 = 0.9903). Single nucleotide polymorphism (SNP) genotyping of the dhfr gene of Plasmodium falciparum is also possible using the SiSA-chip, with as little as 1% of mutant gene distinguished from wild-type loci (m/wt). This system offers a high-efficiency (20 min), high-sensitivity (6 copies/reaction), high-specificity (1% m/wt), and low-cost (∼1/50 of fluorescence assays for RPA) diagnosis method for pathogen DNA identification. Therefore, this system is promising for fast identification of pathogens to help diagnose infectious diseases, including SNP genotyping.