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.

Coordinate expression of multiple proteins in plant cells by exploiting endogenous kex2p-like protease activity.

Simultaneous expression of multiple proteins in plants finds ample applications. Here, we examined the biotechnological application of native kex2p-like protease activity in plants for coordinate expression of multiple secretory proteins from a single transgene encoding a cleavable polyprotein precursor. We expressed a secretory red fluorescent protein (DsRed) or human cytokine (GMCSF), fused to a downstream green fluorescent protein (GFP) by a linker containing putative recognition sites of the kex2p-like protease in tobacco cells and referred to them as RKG and GKG cells, respectively. Our analyses showed that GFP is cleaved off the fusion proteins and secreted into the media by both RKG and GKG cells. The cleaved GFP product displayed the expected fluorescence characteristics. Using GFP immunoprecipitation and fluorescence analysis, the cleaved DsRed product in the RKG cells was found to be functional as well. However, DsRed was not detected in the RKG culture medium, possibly due to its tetramer formation. Cleaved and biologically active GMCSF could also be detected in GKG cell extracts, but secreted GMCSF was found to be only at a low level, likely because of instability of GMCSF protein in the medium. Processing of polyprotein precursors was observed to be similarly effective in tobacco leaf, stem and root tissues. Importantly, we also demonstrated that, via agroinfiltration, polyprotein precursors can be efficiently processed in plant species other than tobacco. Collectively, our results demonstrate the utility of native kex2p-like protease activity for the expression of multiple secretory proteins in plant cells using cleavable polyprotein precursors containing kex2p linker(s).